bims-novged 2022-09-11 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2022–09–11
ten papers selected by
the Merkel lab, Ludwig-Maximilians University

Preparation of PEI-modified nanoparticles by dopamine self-polymerization for efficient DNA delivery.
Construction of Degradable and Amphiphilic Triblock Polymer Carriers for Effective Delivery of siRNA.
Co-Formulation of Amphiphilic Cationic and Anionic Cyclodextrins Forming Nanoparticles for siRNA Delivery in the Treatment of Acute Myeloid Leukaemia.
Simultaneous quantification of multiple RNA cargoes co-loaded into nanoparticle-based delivery systems.
Delivery and assessment of a CRISPR/nCas9-based genome editing system on in vitro models of mucopolysaccharidoses IVA assisted by magnetite-based nanoparticles.
Pulmonary delivery of liposomes co-loaded with SN38 prodrug and curcumin for the treatment of lung cancer.
A Coarse-to-Fine Pathology Patch Selection for Improving Gene Mutation Prediction in Acute Myeloid Leukemia.
Influence of diverse storage conditions of double-stranded RNA in vitro on the RNA interference efficiency in vivo insect Tribolium castaneum.
Coarse-Grained Simulation of Bottlebrush: From Single-Chain Properties to Self-Assembly.
The Application of Light-Assisted Drying to the Thermal Stabilization of Nucleic Acid Nanoparticles.

Biotechnol Appl Biochem. 2022 Sep 07.

Preparation of PEI-modified nanoparticles by dopamine self-polymerization for efficient DNA delivery.

Liang Liu, Zhaojun Yang, Chaobing Liu, Mengying Wang, Xin Chen.

  Achieving efficient and safe gene delivery is great of significance to promote the development of gene therapy. In this work, a polydopamine (PDA) layer was coated on the surface of Fe3 O4 nanoparticles (NPs) by dopamine (DA) self-polymerization, and then magnetic Fe3 O4 NPs were prepared by the Michael addition between amino groups in polyethyleneimine (PEI) and PDA. The prepared Fe3 O4 NPs (named Fe3 O4 @PDA@PEI) were characterized by FTIR, atomic force microscopy (AFM) and scanning electron microscope (SEM). As an efficient and safe gene carrier, the potential of Fe3 O4 @PDA@PEI was evaluated by agarose gel electrophoresis, MTT assay, fluorescence microscopy, flow cytometry. The results shows that the Fe3 O4 @PDA@PEI NPs is stable hydrophilic nanoparticles with a particle size of 50-150 nm. It can efficiently condense DNA at low N/P ratios and protect it from nuclease degradation. In addition, the Fe3 O4 @PDA@PEI NPs has higher safety than PEI. Further, the Fe3 O4 @PDA@PEI/DNA polyplexes could be effectively absorbed by cells and successfully transfected, and exhibit higher cellular uptake and gene transfection efficiency than PEI/DNA polyplexes. The findings indicate that the Fe3 O4 @PDA@PEI NPs has the potential to be developed into a novel gene vector. This article is protected by copyright. All rights reserved.

Keywords:  Dopamine; Fe3O4 Nanoparticle; Gene delivery; Polyethyleneimine; Self-polymerization

DOI:  https://doi.org/10.1002/bab.2402
Macromol Biosci. 2022 Sep 10. e2200232

Construction of Degradable and Amphiphilic Triblock Polymer Carriers for Effective Delivery of siRNA.

Yunfeng Yan, Fangtao Zhu, Huahui Su, Xiaomin Liu, Qidi Ren, Fangqian Huang, Wenbo Ye, Mengdan Zhao, Yunchun Zhao, Junpeng Zhao, Qi Shuai.

  The development of effective and safe delivery carriers is one of the prerequisites for the clinical translation of siRNA-based therapeutics. In this study, we constructed a library of 144 functional triblock polymers using ring-opening polymerization (ROP) and thiol-ene click reaction. These triblock polymers are composed of hydrophilic poly (ethylene oxide) (PEO), hydrophobic poly (ε-caprolactone) (PCL), and cationic amine blocks. Three effective carriers were discovered by high-throughput screening of these polymers for siRNA delivery to HeLa-Luc cells. In vitro evaluation showed that siLuc-loaded nanoparticles (NPs) fabricated with leading polymer carriers exhibited sufficient knockdown of luciferase genes and relatively low cytotoxicity. The chemical structure of polymers significantly affected the physicochemical properties of the resulting siRNA-loaded NPs, which led to different cellular uptake of NPs and endosomal escape of loaded siRNA and thus the overall in vitro siRNA delivery efficacy. After systemic administration to mice with xenograft tumors, siRNA NPs based on P2-4.5A8 were substantially accumulated at tumor sites, suggesting that PEO and PCL blocks were beneficial for improving blood circulation and biodistribution of siRNA NPs. This functional triblock polymer platform may have great potential in the development of siRNA-based therapies for the treatment of cancers. This article is protected by copyright. All rights reserved.

Keywords:  gene therapy; polymeric nanoparticles; siRNA delivery; triblock polymer

DOI:  https://doi.org/10.1002/mabi.202200232
Int J Mol Sci. 2022 Aug 29. pii: 9791. [Epub ahead of print]23(17):

Co-Formulation of Amphiphilic Cationic and Anionic Cyclodextrins Forming Nanoparticles for siRNA Delivery in the Treatment of Acute Myeloid Leukaemia.

Ayse Kont, Monique C P Mendonça, Michael F Cronin, Mary R Cahill, Caitriona M O'Driscoll.

  Non-viral delivery of therapeutic nucleic acids (NA), including siRNA, has potential in the treatment of diseases with high unmet clinical needs such as acute myeloid leukaemia (AML). While cationic biomaterials are frequently used to complex the nucleic acids into nanoparticles, attenuation of charge density is desirable to decrease in vivo toxicity. Here, an anionic amphiphilic CD was synthesised and the structure was confirmed by Fourier-transform infrared spectroscopy (FT-IR), Nuclear Magnetic Resonance (NMR), and high-resolution mass spectrometry (HRMS). A cationic amphiphilic cyclodextrin (CD) was initially used to complex the siRNA and then co-formulated with the anionic amphiphilic CD. Characterisation of the co-formulated NPs indicated a significant reduction in charge from 34 ± 7 mV to 24 ± 6 mV (p < 0.05) and polydispersity index 0.46 ± 0.1 to 0.16 ± 0.04 (p < 0.05), compared to the cationic CD NPs. Size was similar, 161-164 nm, for both formulations. FACS and confocal microscopy, using AML cells (HL-60), indicated a similar level of cellular uptake (60% after 6 h) followed by endosomal escape. The nano co-formulation significantly reduced the charge while maintaining gene silencing (21%). Results indicate that blending of anionic and cationic amphiphilic CDs can produce bespoke NPs with optimised physicochemical properties and potential for enhanced in vivo performance in cancer treatment.

Keywords:  acute myeloid leukaemia (AML); modified cyclodextrins; nanomaterials; non-viral gene delivery; nucleic acids

DOI:  https://doi.org/10.3390/ijms23179791
Int J Pharm. 2022 Sep 04. pii: S0378-5173(22)00725-6. [Epub ahead of print] 122171

Simultaneous quantification of multiple RNA cargoes co-loaded into nanoparticle-based delivery systems.

Abhijeet Lokras, Akash Chakravarty, Thomas Rades, Dennis Christensen, Henrik Franzyk, Aneesh Thakur, Camilla Foged.

  Robust, sensitive, and versatile analytical methods are essential for quantification of RNA drug cargoes loaded into nanoparticle-based delivery systems. However, simultaneous quantification of multiple RNA cargoes co-loaded into nanoparticles remains a challenge. Here, we developed and validated the use of ion-pair reversed-phase high-performance liquid chromatography combined with UV detection (IP-RP-HPLC-UV) for simultaneous quantification of single- and double-stranded RNA cargoes. Complete extraction of RNA cargo from the nanoparticle carrier was achieved using a phenol:chloroform:isoamyl alcohol mixture. Separations were performed using either a C18 or a PLRP-S column, eluted with 0.1 M triethylammonium acetate solution (TEAA) as ion-pairing reagent (eluent A), and 0.1 M TEAA containing 25% (v/v) CH3CN as eluent B. These methods were applied to quantify mRNA and polyinosinic:polycytidylic acid co-loaded into lipid-polymer hybrid nanoparticles, and single-stranded oligodeoxynucleotide donors and Alt-R CRISPR single guide RNAs co-loaded into lipid nanoparticles. The developed methods were sensitive (limit of RNA quantitation < 60 ng), linear (R2 > 0.997), and accurate (≈ 100% recovery of RNA spiked in nanoparticles). Hence, the present study may facilitate convenient quantification of multiple RNA cargoes co-loaded into nanoparticle-based delivery systems.

Keywords:  IP-RP-HPLC; mRNA; nanomedicine; quantification; siRNA

DOI:  https://doi.org/10.1016/j.ijpharm.2022.122171
Sci Rep. 2022 Sep 03. 12(1): 15045

Delivery and assessment of a CRISPR/nCas9-based genome editing system on in vitro models of mucopolysaccharidoses IVA assisted by magnetite-based nanoparticles.

Andrés Felipe Leal, Javier Cifuentes, Carlos Emilio Torres, Diego Suárez, Valentina Quezada, Saúl Camilo Gómez, Juan C Cruz, Luis H Reyes, Angela Johana Espejo-Mojica, Carlos Javier Alméciga-Díaz.

Mucopolysaccharidosis IV A (MPS IVA) is a lysosomal disorder caused by mutations in the GALNS gene. Consequently, the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate accumulate in the lysosomal lumen. Although enzyme replacement therapy has shown essential advantages for the patients, several challenges remain to overcome, such as the limited impact on the bone lesion and recovery of oxidative profile. Recently, we validated a CRISPR/nCas9-based gene therapy with promising results in an in vitro MPS IVA model. In this study, we have expanded the use of this CRISPR/nCas9 system to several MPS IVA fibroblasts carrying different GALNS mutations. Considering the latent need to develop more safety vectors for gene therapy, we co-delivered the CRISPR/nCas9 system with a novel non-viral vector based on magnetoliposomes (MLPs). We found that the CRISPR/nCas9 treatment led to an increase in enzyme activity between 5 and 88% of wild-type levels, as well as a reduction in GAGs accumulation, lysosomal mass, and mitochondrial-dependent oxidative stress, in a mutation-dependent manner. Noteworthy, MLPs allowed to obtain similar results to those observed with the conventional transfection agent lipofectamine. Overall, these results confirmed the potential of CRISPR/nCas9 as a genome editing tool for treating MPS IVA. We also demonstrated the potential use of MLPs as a novel delivery system for CRISPR/nCas9-based therapies.

DOI: https://doi.org/10.1038/s41598-022-19407-x
Eur J Pharm Biopharm. 2022 Sep 02. pii: S0939-6411(22)00187-4. [Epub ahead of print]

Pulmonary delivery of liposomes co-loaded with SN38 prodrug and curcumin for the treatment of lung cancer.

Chengzhi Gao, Lanfang Zhang, Minhao Xu, Yi Luo, Ben Wang, Meiyan Kuang, Xingyou Liu, Meng Sun, Yue Guo, Lesheng Teng, Chenhui Wang, Yan Zhang, Jing Xie.

  A co-delivery system of SN38 (7-ethyl-10-hydroxyl camptothecin) prodrug and CUR (curcumin) was designed for the treatment of lung cancer by pulmonary delivery. SN38 was linked to cell-penetrating peptide (CPP) TAT via a polyethylene glycol (PEG) linker to form the SN38 prodrug (TAT-PEG-SN38). Liposomes co-loaded with amphiphilic TAT-PEG-SN38 and curcumin (Lip-TAT-PEG-SN38/CUR) were successfully prepared by a microfluidic method for the treatment of lung cancer via pulmonary delivery. Lip-TAT-PEG-SN38/CUR showed nanometer-sized sphericity and a particle size of 171.21 nm. Besides, Lip-TAT-PEG-SN38/CUR exhibited enhanced antiproliferative effect, increased cell apoptosis induction and improved cell cycle arrest compared to the single agents in vitro. The combination induced significant tumor inhibition in a BALB/c mouse lung cancer model. These results indicated that our SN38 prodrug and curcumin co-delivery system was a promising candidate for lung cancer treatment.

Keywords:  Curcumin; Pulmonary delivery; SN38; lung cancer

DOI:  https://doi.org/10.1016/j.ejpb.2022.08.021
Annu Int Conf IEEE Eng Med Biol Soc. 2022 Jul;2022 3207-3210

A Coarse-to-Fine Pathology Patch Selection for Improving Gene Mutation Prediction in Acute Myeloid Leukemia.

Chun-Chia Chiu, Jeng-Lin Li, Yu-Fen Wang, Bor-Sheng Ko, Chi-Chun Lee.

Identifying gene mutation is essential to prognosis and therapeutic decisions for acute myeloid leukemia (AML) but the current gene analysis is inefficient and non-scalable. Pathological images are readily accessible and can be effectively modeled using deep learning. This work aims at predicting gene mutation directly by modeling bone marrow smear images. Traditionally, bone marrow smear slides are cropped into patches with manual segmentation for patch-level modeling. Slide-level modeling, such as multi-instance learning, could aggregate patches for holistic modeling, though suffer from excessive redundancy. In this study, we propose a discriminative multi-instance approach to select useful patches in a coarse-to-fine process. Specifically, we preprocess a slide into patches by using a trained pre-selector network. Then, we rule out low quality patches in the coarse selection with known prior knowledge, and refine the model using gene-discriminative patches in the fine selection. We evaluate the framework for CEBPA, FLT3, and NPM1 gene mutation prediction and obtain 71.67%, 56.26%, and 56.34% F1-score. Further analysis show the effect of different selection criteria on prediction gene mutations using pathological images.

DOI: https://doi.org/10.1109/EMBC48229.2022.9871814
Pest Manag Sci. 2022 Sep 10.

Influence of diverse storage conditions of double-stranded RNA in vitro on the RNA interference efficiency in vivo insect Tribolium castaneum.

Mei Bai, Zi-Ling Liu, Yu-Yu Zhou, Qiu-Xuan Xu, Tong-Xian Liu, Hong-Gang Tian.

   BACKGROUND: A significant variation of RNA interference (RNAi) efficiency hinders further functional gene studies and pest control application in many insects. The available double-stranded RNA (dsRNA) molecules introduced into the target cells are regarded as the crucial factor for efficient RNAi response. However, numerous studies have only focused on dsRNA stability in vivo; it is uncertain whether different dsRNA storage conditions in vitro play a role in variable RNAi efficiency among insects.
RESULTS: A marker gene cardinal, which leads to white eyes when knocked-down in the red flour beetle Tribolium castaneum, was used to evaluate the effects of RNAi efficiency under different dsRNA storage conditions. We demonstrated that the dsRNA molecule is very stable under typical cryopreservation temperatures (-80 °C and -20 °C) within 180 days, and RNAi efficiency shows no significant differences under either low temperature. Unexpectedly, while dsRNA molecules were treated with multiple freeze-thaw cycles up to 50 times between -80/-20 °C and room temperature, we discovered that dsRNA integrity and RNAi efficiency were comparable to fresh dsRNA. Finally, when the stability of dsRNA was further measured under refrigerated storage conditions (4 °C), we surprisingly found that dsRNA is still stable within 180 days and can induce an efficient RNAi response as initial dsRNA.
CONCLUSION: Our results indicated that dsRNA is extraordinarily stable under various temperature storage conditions, which did not significantly impact RNAi efficiency in vivo insects. This article is protected by copyright. All rights reserved.

Keywords:  RNA interference; RNA storage; RNAi efficiency; Tribolium castaneum; dsRNA stability

DOI:  https://doi.org/10.1002/ps.7171
ACS Macro Lett. 2022 Sep 09. 1167-1173

Coarse-Grained Simulation of Bottlebrush: From Single-Chain Properties to Self-Assembly.

Juhae Park, Vikram Thapar, Yeojin Choe, Luis Adrian Padilla Salas, Abelardo Ramírez-Hernández, Juan J de Pablo, Su-Mi Hur.

Bottlebrush polymers consist of a linear backbone with densely grafted side chains. They are known to have a range of properties of interest, such as enhanced mechanical strength and rapid self-assembly into large domains, and have attracted attention as promising candidates for applications in photonics, lithography, energy storage, organic optoelectronics, and drug delivery. Here, we present a coarse-grained model of bottlebrush polymers that is able to reproduce their experimentally observed persistence lengths and chain conformations in the melt. The model is then used to investigate the morphologies of this class of materials for various chain architectures and grafting densities.

DOI: https://doi.org/10.1021/acsmacrolett.2c00310
Biopreserv Biobank. 2022 Sep 02.

The Application of Light-Assisted Drying to the Thermal Stabilization of Nucleic Acid Nanoparticles.

Phuong Anh Lam, Daniel P Furr, Allison Tran, Riley Q McKeough, Damian Beasock, Morgan Chandler, Kirill A Afonin, Susan R Trammell.

  Background: Cold-chain storage can be challenging and expensive for the transportation and storage of biologics, especially in low-resource settings. Nucleic acid nanoparticles (NANPs) are an example of new biological products that require refrigerated storage. Light-assisted drying (LAD) is a new processing technique to prepare biologics for anhydrous storage in a trehalose amorphous solid matrix at ambient temperatures. In this study, LAD was used to thermally stabilize four types of NANPs with differing structures and melting temperatures. Methods: Small volume samples (10 μL) containing NANPs were irradiated with a 1064 nm laser to speed the evaporation of water and create an amorphous trehalose preservation matrix. Samples were then stored for 1 month at 4°C or 20°C. A FLIR C655 mid-IR camera was used to record the temperature of samples during processing. The trehalose matrix was characterized using polarized light imaging (PLI) to determine if crystallization occurred during processing or storage. Damage to LAD-processed NANPs was assessed after processing and storage using gel electrophoresis. Results: Based on the end moisture content (EMC) as a function time and the thermal histories of samples, a LAD processing time of 30 min is sufficient to achieve low EMCs for the 10 μL samples used in this study. PLI demonstrates that the trehalose matrix was resistant to crystallization during processing and after storage at 4°C and at room temperature. The native-polyacrylamide gel electrophoresis results for DNA cubes, RNA cubes, and RNA rings indicate that the main structures of these NANPs were not damaged significantly after LAD processing and being stored at 4°C or at room temperature for 1 month. Conclusions: These preliminary studies indicate that LAD processing can stabilize NANPs for dry-state storage at room temperature, providing an alternative to refrigerated storage for these nanomedicine products.

Keywords:  anhydrous preservation; nucleic acid nanoparticles; thermal stabilization

DOI:  https://doi.org/10.1089/bio.2022.0035