bims-novged 2022-05-01 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2022–05–01
thirteen papers selected by
the Merkel lab, Ludwig-Maximilians University

In vivo delivery of plasmid DNA by lipid nanoparticles: the influence of ionizable cationic lipids on organ-selective gene expression.
Multiscale modeling of the cellular uptake of C6 peptide-siRNA complexes.
Synthesis and Characterization of Poly (β-amino Ester) and Applied PEGylated and Non-PEGylated Poly (β-amino ester)/Plasmid DNA Nanoparticles for Efficient Gene Delivery.
Small Molecule Modifications Significantly Increase the Transfection Efficiency of Low-Molecular Polymer.
Small Interfering RNA (siRNA) Transfection in Epiblast Stem Cells.
Nanodelivery of nucleic acids.
Delivery of Genetic Materials for the Management of Biological Disorders: Advancement and Roles of Polysaccharides and their Derivatives.
Gene delivery in adherent and suspension cells using the combined physical methods.
Leucine improves the aerosol performance of dry powder inhaler formulations of siRNA-loaded nanoparticles.
Optimization of large-scale manufacturing of biopolymeric and lipid nanoparticles using microfluidic swirl mixers.
The programmed site-specific delivery of LY3200882 and PD-L1 siRNA boosts immunotherapy for triple-negative breast cancer by remodeling tumor microenvironment.
PD-L1 siRNA-hyaluronic acid conjugate for dual-targeted cancer immunotherapy.
Design of siRNA molecules for silencing of membrane glycoprotein, nucleocapsid phosphoprotein, and surface glycoprotein genes of SARS-CoV2.

Biomater Sci. 2022 Apr 27.

In vivo delivery of plasmid DNA by lipid nanoparticles: the influence of ionizable cationic lipids on organ-selective gene expression.

Azizah Algarni, Emily H Pilkington, Estelle J A Suys, Hareth Al-Wassiti, Colin W Pouton, Nghia P Truong.

Ionizable cationic lipids play a critical role in developing new gene therapies for various biomedical applications, including COVID-19 vaccines. However, it remains unclear whether the formulation of lipid nanoparticles (LNPs) using DLin-MC3-DMA, an optimized ionizable lipid clinically used for small interfering RNA (siRNA) therapy, also facilitates high liver-selective transfection of other gene therapies such as plasmid DNA (pDNA). Here we report the first investigation into pDNA transfection efficiency in different mouse organs after intramuscular and intravenous administration of lipid nanoparticles (LNPs) where DLin-MC3-DMA, DLin-KC2-DMA or DODAP are used as the ionizable cationic lipid component of the LNP. We discovered that these three benchmark lipids previously developed for siRNA delivery followed an unexpected characteristic rank order in gene expression efficiency when utilized for pDNA. In particular, DLin-KC2-DMA facilitated higher in vivo pDNA transfection than DLin-MC3-DMA and DODAP, possibly due to its head group pKa and lipid tail structure. Interestingly, LNPs formulated with either DLin-KC2-DMA or DLin-MC3-DMA exhibited significantly higher in vivo protein production in the spleen than in the liver. This work sheds light on the importance of the choice of ionizable cationic lipid and nucleic acid cargo for organ-selective gene expression. The study also provides a new design principle towards the formulation of more effective LNPs for biomedical applications of pDNA, such as gene editing, vaccines and immunotherapies.

DOI: https://doi.org/10.1039/d2bm00168c
Comput Biol Chem. 2022 Apr 06. pii: S1476-9271(22)00059-7. [Epub ahead of print]98 107679

Multiscale modeling of the cellular uptake of C6 peptide-siRNA complexes.

P W C M Purijjala, P V G M Rathnayake, B T Kumara, B C M Gunathunge, R A A P Ranasinghe, D N Karunaratne, R J K U Ranatunga.

  In gene therapy utilising short interfering RNA (siRNA), delivery of the siRNA therapeutics to the target site is a major obstacle, due to low cellular uptake. Efficient delivery systems such as cell penetrating peptides (CPPs) are in the forefront for the development of efficient, safe, non-viral gene delivery. The C6 peptide series are a class of synthetic CPPs, developed specifically for the delivery of siRNA. This series of peptides are derivatives of the original C6 peptide, modified to increase cellular uptake and efficiency. In this study, multiscaled computational simulations of these peptides were performed in aqueous media, interrogating the relationship between the structure and behaviour. All atom molecular dynamic (MD) simulation results show that all CPPs show stable α-helical amphipathic secondary structures. Furthermore, docking calculations indicate that the C6 peptides can fit into the major groove of the siRNA double-helix, and once filled, could bind randomly along the minor grooves and to other, previously bound peptides. Coarse grained MD simulations were also used to generate free energy profiles for the dimerization of peptides, and binding of the peptide to siRNA. Simulation results confirm that all peptides favour binding to siRNA, they however, also favour dimerization. This affinity for aggregation may trigger the formation of larger complexes with siRNA and enhance the cellular uptake. These results indicate the capacity of C6 peptides as efficient delivery vehicles. As expected the amino acid sequence plays a crucial role in the helicity, peptide self-assembly, interaction of peptide with cell membrane and formation of stable siRNA-CPP complex.

Keywords:  Cell penetrating peptides; Free energy profiles; Molecular dynamics simulations; SiRNA

DOI:  https://doi.org/10.1016/j.compbiolchem.2022.107679
Front Pharmacol. 2022 ;13 854859

Synthesis and Characterization of Poly (β-amino Ester) and Applied PEGylated and Non-PEGylated Poly (β-amino ester)/Plasmid DNA Nanoparticles for Efficient Gene Delivery.

Sajid Iqbal, Alessandro F Martins, Muhammad Sohail, Jingjing Zhao, Qi Deng, Muhan Li, Zhongxi Zhao.

  Polymer-based nanocarriers require extensive knowledge of their chemistries to learn functionalization strategies and understand the nature of interactions that they establish with biological entities. In this research, the poly (β-amino ester) (PβAE-447) was synthesized and characterized, aimed to identify the influence of some key parameters in the formulation process. Initially; PβAE-447 was characterized for aqueous solubility, swelling capacity, proton buffering ability, and cytotoxicity study before nanoparticles formulation. Interestingly, the polymer-supported higher cell viability than the Polyethylenimine (PEI) at 100 μg/ml. PβAE-447 complexed with GFP encoded plasmid DNA (pGFP) generated nanocarriers of 184 nm hydrodynamic radius (+7.42 mV Zeta potential) for cell transfection. Transfection assays performed with PEGylated and lyophilized PβAE-447/pDNA complexes on HEK-293, BEAS-2B, and A549 cell lines showed better transfection than PEI. The outcomes toward A549 cells (above 66%) showed the highest transfection efficiency compared to the other cell lines. Altogether, these results suggested that characterizing physicochemical properties pave the way to design a new generation of PβAE-447 for gene delivery.

Keywords:  biodegradable polymer; gene delivery; poly (β-amino ester); stable polyplexes; transfection

DOI:  https://doi.org/10.3389/fphar.2022.854859
J Biomed Nanotechnol. 2022 Feb 01. 18(2): 435-445

Small Molecule Modifications Significantly Increase the Transfection Efficiency of Low-Molecular Polymer.

Pengfei Cui, Jianhe Ma, Huihui Zhang, Lin Qiu, Shuwen Zhou, Cheng Wang, Xinye Ni, Pengju Jiang, Jianhao Wang.

Gene vectors with high biocompatibility and transfection efficiency are critical for successful gene therapy. PEI 25K (Polyethyleneimine 25K) is a common polymeric gene vector that has been employed as a positive control material in gene transfection studies due to its good performance in endosome escape. PEI 25K's indegradability and abundance of positive charges, on the other hand, cause toxicity in cells, limiting its use. In this study, we developed the PEI-ER non-viral vector by adding an endoplasmic reticulum (ER) targeting ligand to low molecular weight PEI 1.8K. These small molecule modifications dramatically improved PEI transfection efficiency while barely interfering with compatibility. PEI-ER/DNA complexes were discovered to enter the cell via caveolin-mediated endocytosis, avoiding destruction in the endosome. We believe that this little chemical alteration is a simple solution to enhance the efficacy of cationic polymer vectors in gene transport, and it has a lot of medicinal applications.

DOI: https://doi.org/10.1166/jbn.2022.3252
Methods Mol Biol. 2022 ;2490 47-55

Small Interfering RNA (siRNA) Transfection in Epiblast Stem Cells.

Georgia R Kafer.

  Lipid-based transfection of siRNA is a technique routinely used to investigate gene function in experiments using mammalian cells cultured in vitro. Due to innate differences in cellular characteristics, the efficiency of lipid-based transfection is variable across cell types. Pluripotent cells which exist in a "primed" state such as human embryonic stem cells (hESCs) and mouse epiblast stem cells (mEpiSCs) are notorious for being refractory to lipid-based transfection systems. Herein we describe a forward transfection protocol which we routinely use to achieve upwards of 70% transfection efficiency rates in mEpiSCs. Our protocol also includes a suggested transfection timeline and details pertaining to the techniques we use to validate transfection success.

Keywords:  Epiblast; Lipid; Lipofectamine; Mouse; Small interfering RNA; Transfection; mEpiSC; siRNA

DOI:  https://doi.org/10.1007/978-1-0716-2281-0_5
Nat Rev Methods Primers. 2022 ;pii: 24. [Epub ahead of print]2

Nanodelivery of nucleic acids.

Bárbara B Mendes, João Conniot, Aviram Avital, Dongbao Yao, Xingya Jiang, Xiang Zhou, Noga Sharf-Pauker, Yuling Xiao, Omer Adir, Haojun Liang, Jinjun Shi, Avi Schroeder, João Conde.

There is growing need for a safe, efficient, specific and non-pathogenic means for delivery of gene therapy materials. Nanomaterials for nucleic acid delivery offer an unprecedented opportunity to overcome these drawbacks; owing to their tunability with diverse physico-chemical properties, they can readily be functionalized with any type of biomolecules/moieties for selective targeting. Nucleic acid therapeutics such as antisense DNA, mRNA, small interfering RNA (siRNA) or microRNA (miRNA) have been widely explored to modulate DNA or RNA expression Strikingly, gene therapies combined with nanoscale delivery systems have broadened the therapeutic and biomedical applications of these molecules, such as bioanalysis, gene silencing, protein replacement and vaccines. Here, we overview how to design smart nucleic acid delivery methods, which provide functionality and efficacy in the layout of molecular diagnostics and therapeutic systems. It is crucial to outline some of the general design considerations of nucleic acid delivery nanoparticles, their extraordinary properties and the structure-function relationships of these nanomaterials with biological systems and diseased cells and tissues.

DOI: https://doi.org/10.1038/s43586-022-00104-y
Curr Drug Deliv. 2022 Apr 22.

Delivery of Genetic Materials for the Management of Biological Disorders: Advancement and Roles of Polysaccharides and their Derivatives.

Nandan Gupta, Rishabha Malviya.

  Advancement in nanotechnology leads to the development of polysaccharides which are very effective carriers in carrying therapeutic ingredients like drugs, proteins, and genes. This review describes the role of polysaccharides and their derivatives in the cellular targeting of genetic materials for the treatment of various biological disorders. Applications, challenges, advantages, and disadvantages of polysaccharides used in gene delivery are discussed in the manuscript. Cationic and natural polysaccharides are generally used for RNA and DNA delivery and exhibit better performance in gene transfection. After a substantial literature survey, it can be concluded that different polysaccharides and their derivatives are effectively used in the delivery of genetic material. Natural polysaccharides are widely used due to their advantageous properties like biocompatibility, biodegradability, and low toxicity in the biological environment.

Keywords:  Gene delivery; Genetic material; Nanoparticles; Polysaccharide; Targeted drug delivery

DOI:  https://doi.org/10.2174/1567201819666220422154504
Cytotechnology. 2022 Apr;74(2): 245-257

Gene delivery in adherent and suspension cells using the combined physical methods.

Kimia Kardani, Alireza Milani, Azam Bolhassani.

  Physical methods are widely utilized to deliver nucleic acids into cells such as electro-transfection or heat shock. An efficient gene electro-transfection requires the best conditions including voltage, the pulse length or number, buffer, incubation time and DNA form. In this study, the delivery of pEGFP-N1 vector into two adherent cell lines (HEK-293 T and COS-7) with the same origin (epithelial cells), and also mouse bone marrow-derived dendritic cells (DCs) was evaluated using electroporation under different conditions alone and along with heat treatment. Our data showed that the highest green fluorescent protein (GFP) expression in HEK-293 T and COS-7 cells was observed in serum-free RPMI cell culture medium as electroporation buffer, voltage (200 V), the pulse number (2), the pulse length (15 ms), the circular form of DNA, and 48 h after electro-transfection. In addition, the highest GFP expression in DCs was detected in serum-free RPMI, voltage (300 V), the pulse number (1), the pulse length (5 ms), and 48 h after electro-transfection. The use of sucrose as electroporation buffer, the pulse number (2), and the pulse length (25 ms) led to further cytotoxicity and lower transfection in HEK293T and COS-7 cells than other conditions. Moreover, the high voltage (700 V) increased the cell cytotoxicity, and decreased electro-transfection efficiency in DCs. On the other hand, the best conditions of electroporation along with heat treatment could significantly augment the transfection efficiency in all the cells. These data will be useful for gene delivery in other cells with the same properties using physical methods.
Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00524-4.

Keywords:  Cytotoxic effect; Electroporation; Heat; Non-viral delivery system; Physical delivery; Transfection

DOI:  https://doi.org/10.1007/s10616-022-00524-4
Int J Pharm. 2022 Apr 25. pii: S0378-5173(22)00313-1. [Epub ahead of print] 121758

Leucine improves the aerosol performance of dry powder inhaler formulations of siRNA-loaded nanoparticles.

You Xu, Laure Harinck, Abhijeet G Lokras, Per Gerde, Ewa Selg, Carl-Olof Sjöberg, Henrik Franzyk, Aneesh Thakur, Camilla Foged.

  Thermostable dry powder inhaler (DPI) formulations with high aerosol performance are attractive inhalable solid dosage forms for local treatment of inflammatory lung diseases. We recently demonstrated that lipidoid-polymer hybrid nanoparticles (LPNs) loaded with small interfering RNA (siRNA) directed against tumor necrosis factor alpha (TNF-α) mediate efficient intracellular siRNA delivery and reduce inflammation in vivo. Here, we show that mixtures of the stabilizing excipients trehalose (Tre) and dextran (Dex), in combination with the shell-forming dispersion enhancer leucine (Leu), stabilize TNF-α siRNA-loaded LPNs during spray drying into nanocomposite microparticles (DPI formulations), and result in DPI formulations with high aerosol performance. At low Leu content (0 to 10%, w/w), the DPI formulations were amorphous, and exhibited poor aerosol performance. When the Leu content was increased from 20 to 60% (w/w), the surface content of Leu increased from 39.2 to 68.1 mol%, and the flowability was significantly improved. Microscopy analyses suggest that the improved powder dispersibility is the result of a wrinkled surface morphology, which reduces the surface area available for interparticle interactions. Increasing the Leu content further (above 10%, w/w) did not influence the aerosol performance, and the aerosol yield was maximal at 30-40% Leu (w/w). Formulations containing 40% Leu and a Tre:Dex ratio of 10:90 (w/w) displayed a high fine particle fraction and aerosol properties suitable for inhalation. The chemical integrity of TNF-α siRNA was preserved in the solid state, and biodistribution studies in mice showed that pulmonary administration of DPI formulations with high aerosol performance resulted in homogenous deep lung deposition. Our results demonstrate that at optimal ratios, ternary excipient mixtures of Leu, Tre and Dex protect TNF-α siRNA-loaded LPNs during spray drying. Hence, this study shows that microparticles with an amorphous Tre/Dex matrix and a crystalline Leu shell are required for stabilizing the nanocomposite LPNs in the solid state, and for ensuring aerosol properties suitable for inhalation.

Keywords:  TNF-α siRNA; aerosol performance; leucine; lipidoid-polymer hybrid nanoparticles; pulmonary administration; spray drying

DOI:  https://doi.org/10.1016/j.ijpharm.2022.121758
Int J Pharm. 2022 Apr 23. pii: S0378-5173(22)00317-9. [Epub ahead of print] 121762

Optimization of large-scale manufacturing of biopolymeric and lipid nanoparticles using microfluidic swirl mixers.

Mhd Anas Tomeh, Muhamad Hawari Mansor, Roja Hadianamrei, Weizhen Sun, Xiubo Zhao.

  Nanoparticles (NPs) have great potential as efficient drug delivery systems (DDSs) that have been widely used in cancer therapy and vaccines especially in the past decade. The rise in demand from the pharmaceutical industry drives the growth of the global NPs market. However, complex production processes have hindered the market growth. Therefore, the development of advanced preparation techniques such microfluidics is required to improve productivity and controllability. In this study, we present a novel microfluidic design (swirl mixer) that helps accelerating the translation of many DDSs from laboratory to clinical application. The new swirl mixer provides high production rate, reproducibility, and precise control of particle size with low polydispersity index (PDI). To assess the performance of the swirl mixer, two different types of nanoformulations were used: silk nanoparticles (SNPs) and lipid nanoparticles (LNPs). The microfluidic device produced NPs efficiently with high productivity and allow for tuning the mean size and size distribution by changing multiple processing parameters.

Keywords:  Doxorubicin; Drug delivery; Lipid nanoparticles; Microfluidic; Nanoparticle manufacturing; Silk fibroin; Swirl mixer

DOI:  https://doi.org/10.1016/j.ijpharm.2022.121762
Biomaterials. 2022 Apr 12. pii: S0142-9612(22)00157-0. [Epub ahead of print]284 121518

The programmed site-specific delivery of LY3200882 and PD-L1 siRNA boosts immunotherapy for triple-negative breast cancer by remodeling tumor microenvironment.

Pan Zhang, Chao Qin, Nan Liu, Xinyuan Zhou, Xuxin Chu, Fangnan Lv, Yongwei Gu, Lifang Yin, Jiyong Liu, Jianping Zhou, Meirong Huo.

  Despite the remarkable success of immunotherapies over the past decade, their effectiveness against triple-negative breast cancer (TNBC) is limited to a small subset of patients, mainly due to the low immunogenicity and unfavorable tumor microenvironment. In this study, we successfully constructed a programmed site-specific delivery nanosystem for the combined delivery of transforming growth factor beta (TGF-β) receptor inhibitor LY3200882 (LY) and PD-L1 siRNA (siPD-L1) to boost anti-tumor immunotherapy. As expected, LY in the outer layer of the nanosystem was released by stimulation of MMP2, and dramatically down-regulated the expression of extracellular matrix (ECM) in the tumor-associated fibroblasts (TAFs), and thus promoted the infiltration of effector T cells and penetration of nanomedicines. Simultaneously, the blockade of TGF-β by LY also triggered immunogenic cell death (ICD) of tumor cells and induced the maturation of dendritic cells. Moreover, the programmed design provided the siPD-L1/protamine cationic inner core with easier access to tumor cells and TAFs after MMP2-stimulated breakup of the outer layer, down-regulating the expression of PD-L1 in both types of cells. Notably, the synergistic effect of LY and siPD-L1 remarkably enhanced the tumor antigen presentation and immunosuppressive microenvironment remodeling, thus efficiently inhibiting the TNBC growth, metastasis, and recurrence. Therefore, the programmed site-specific delivery nanosystem is a promising drug delivery platform for boosting anti-tumor immunotherapy efficacy for TNBC.

Keywords:  Immunogenic cell death; Immunosuppressive tumor microenvironment; Immunotherapy; PD-L1; TGF-β; Tumor-associated fibroblasts

DOI:  https://doi.org/10.1016/j.biomaterials.2022.121518
J Control Release. 2022 Apr 25. pii: S0168-3659(22)00219-X. [Epub ahead of print]346 226-239

PD-L1 siRNA-hyaluronic acid conjugate for dual-targeted cancer immunotherapy.

Suyeon Kim, Roun Heo, Seok Ho Song, Kwon-Ho Song, Jung Min Shin, Se Jin Oh, Hyo-Jung Lee, Jo Eun Chung, Jae Hyung Park, Tae Woo Kim.

  "Foreignization" of tumor cells via delivery of a non-self foreign antigen (Ag) into tumors is an appealing strategy to initiate anti-tumor immunity that can facilitate tumor rejection by pre-existing foreign-Ag-reactive T cells. However, the immune-suppressive factors in the tumor microenvironment (TME) limit the durable and potent immune response of these cells against tumor antigens, stressing the need for improved tumor-foreignization strategies. Here, we demonstrate that blockade of programmed cell death ligand 1 (PD-L1) on both tumor cells and dendritic cells (DCs) can markedly potentiate the induction of tumor-reactive T cells, thereby strengthening the anti-tumor immunity ignited by tumor-foreignization. Specifically, we developed a polymeric nanoconjugate (PEG-HA-OVA/PPLs), consisting of siPD-L1-based polyplexes, PEGylated hyaluronic acid as the CD44-targeting moiety, and ovalbumin (OVA) as a model foreign antigen. Notably, PEG-HA-OVA/PPLs were simultaneously delivered into CD44high tumor cells and CD44high DCs, leading to efficient cross-presentation of OVA and downregulation of PD-L1 in both cell types. Importantly, the nanoconjugate not only allowed OVA-specific T cells to vigorously reject the foreignized tumor cells but also reprogrammed the TME to elicit robust T-cell responses specific to the endogenous tumor Ags, eventually generating long-lasting protective immunity. Thus, our combination strategy represents an innovative approach for the induction of potent tumor immunity via a two-step consecutive immune boost against exogenous and endogenous tumor Ags.

Keywords:  CD44; Immune evasion; PD-L1; Tumor foreignization; Tumor microenvironment

DOI:  https://doi.org/10.1016/j.jconrel.2022.04.023
J Genet Eng Biotechnol. 2022 Apr 28. 20(1): 65

Design of siRNA molecules for silencing of membrane glycoprotein, nucleocapsid phosphoprotein, and surface glycoprotein genes of SARS-CoV2.

Vijaya Sai Ayyagari.

  The global COVID-19 pandemic caused by SARS-CoV2 infected millions of people and resulted in more than 4 million deaths worldwide. Apart from vaccines and drugs, RNA silencing is a novel approach for treating COVID-19. In the present study, siRNAs were designed for the conserved regions targeting three structural genes, M, N, and S, from forty whole-genome sequences of SARS-CoV2 using four different software, RNAxs, siDirect, i-Score Designer, and OligoWalk. Only siRNAs which were predicted in common by all the four servers were considered for further shortlisting. A multistep filtering approach has been adopted in the present study for the final selection of siRNAs by the usage of different online tools, viz., siRNA scales, MaxExpect, DuplexFold, and SMEpred. All these web-based tools consider several important parameters for designing functional siRNAs, e.g., target-site accessibility, duplex stability, position-specific nucleotide preference, inhibitory score, thermodynamic parameters, GC content, and efficacy in cleaving the target. In addition, a few parameters like GC content and dG value of the entire siRNA were also considered for shortlisting of the siRNAs. Antisense strands were subjected to check for any off-target similarities using BLAST. Molecular docking was carried out to study the interactions of guide strands with AGO2 protein. A total of six functional siRNAs (two for each gene) have been finally selected for targeting M, N, and S genes of SARS-CoV2. The siRNAs have not shown any off-target effects, interacted with the domain(s) of AGO2 protein, and were efficacious in cleaving the target mRNA. However, the siRNAs designed in the present study need to be tested in vitro and in vivo in the future.

Keywords:  COVID-19; Membrane glycoprotein; Nucleocapsid phosphoprotein; SARS-CoV2; Surface glycoprotein; siRNA; siRNA design tools

DOI:  https://doi.org/10.1186/s43141-022-00346-z