bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2021‒11‒28
eleven papers selected by
Benjamin Winkeljann
Ludwig-Maximilians University
  1. Supramolecular Surface Functionalization of Iron Oxide Nanoparticles with α-Cyclodextrin-Based Cationic Star Polymer for Magnetically-Enhanced Gene Delivery.
  2. Cyclic Peptide-Gadolinium Nanocomplexes as siRNA Delivery Tools.
  3. Fate of Tableted Freeze-Dried siRNA Lipoplexes in Gastrointestinal Environment.
  4. A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems.
  5. New insights on the mechanism of polyethylenimine transfection and their implications on gene therapy and DNA vaccines.
  6. Stimulus-responsive drug/gene delivery system based on polyethylenimine cyclodextrin nanoparticles for potential cancer therapy.
  7. Emerging landscape of cell-penetrating peptide-mediated nucleic acid delivery and their utility in imaging, gene-editing, and RNA-sequencing.
  8. Lipid nanoparticle chemistry determines how nucleoside base modifications alter mRNA delivery.
  9. Increasing Transfection Efficiency of Lipoplexes by Modulating Complexation Solution for Transient Gene Expression.
  10. Exosome-mediated delivery of inflammation-responsive Il-10 mRNA for controlled atherosclerosis treatment.
  11. Human umbilical cord mesenchymal stem cell-derived exosomes carrying hsa-miRNA-128-3p suppress pancreatic ductal cell carcinoma by inhibiting Galectin-3.
  1. Pharmaceutics. 2021 Nov 06. pii: 1884. [Epub ahead of print]13(11):
    Supramolecular Surface Functionalization of Iron Oxide Nanoparticles with α-Cyclodextrin-Based Cationic Star Polymer for Magnetically-Enhanced Gene Delivery.
    Hanyi Li, Erwin Peng, Feng Zhao, Jun Li, Junmin Xue.
      Supramolecular polymers formed through host-guest complexation have inspired many interesting developments of functional materials for biological and biomedical applications. Here, we report a novel design of a non-viral gene delivery system composed of a cationic star polymer forming supramolecular complexes with the surface oleyl groups of superparamagnetic iron oxide nanoparticles (SPIONs), for magnetically enhanced delivery of DNA into mammalian cells. The cationic star polymer was synthesized by grafting multiple oligoethylenimine (OEI) chains onto an α-cyclodextrin (α-CD) core. The SPIONs were synthesized from iron(III) acetylacetonate and stabilized by hydrophobic oleic acid and oleylamine in hexane, which were characterized in terms of their size, structure, morphology, and magnetic properties. The synthesized magnetic particles were found to be superparamagnetic, making them a suitable ferrofluid for biological applications. In order to change the hydrophobic surface of the SPIONs to a hydrophilic surface with functionalities for plasmid DNA (pDNA) binding and gene delivery, a non-traditional but simple supramolecular surface modification process was used. The α-CD-OEI cationic star polymer was dissolved in water and then mixed with the SPIONs stabilized in hexane. The SPIONs were "pulled" into the water phase through the formation of supramolecular host-guest inclusion complexes between the α-CD unit and the oleyl surface of the SPIONs, while the surface of the SPIONs was changed to OEI cationic polymers. The α-CD-OEI-SPION complex could effectively bind and condense pDNA to form α-CD-OEI-SPION/pDNA polyplex nanoparticles at the size of ca. 200 nm suitable for delivery of genes into cells through endocytosis. The cytotoxicity of the α-CD-OEI-SPION complex was also found to be lower than high-molecular-weight polyethylenimine, which was widely studied previously as a standard non-viral gene vector. When gene transfection was carried out in the presence of an external magnetic field, the α-CD-OEI-SPION/pDNA polyplex nanoparticles greatly increased the gene transfection efficiency by nearly tenfold. Therefore, the study has demonstrated a facile two-in-one method to make the SPIONs water-soluble as well as functionalized for enhanced magnetofection.
    Keywords:  cyclodextrin; gene transfection; iron oxide; magnetic nanoparticles; oligoethylenimine; supramolecular complexation
    DOI:  https://doi.org/10.3390/pharmaceutics13111884
  2. Pharmaceuticals (Basel). 2021 Oct 20. pii: 1064. [Epub ahead of print]14(11):
    Cyclic Peptide-Gadolinium Nanocomplexes as siRNA Delivery Tools.
    Amir Nasrolahi Shirazi, Muhammad Imran Sajid, Dindyal Mandal, David Stickley, Stephanie Nagasawa, Joshua Long, Sandeep Lohan, Keykavous Parang, Rakesh Kumar Tiwari.
      We have recently reported that a cyclic peptide containing five tryptophan, five arginine, and one cysteine amino acids [(WR)5C], was able to produce peptide-capped gadolinium nanoparticles, [(WR)5C]-GdNPs, in the range of 240 to 260 nm upon mixing with an aqueous solution of GdCl3. Herein, we report [(WR)5C]-GdNPs as an efficient siRNA delivery system. The peptide-based gadolinium nanoparticles (50 µM) did not exhibit significant cytotoxicity (~93% cell viability at 50 µM) in human leukemia T lymphoblast cells (CCRF-CEM) and triple-negative breast cancer cells (MDA-MB-231) after 48 h. Fluorescence-activated cell sorting (FACS) analysis indicated that the cellular uptakes of Alexa-488-labeled siRNA were found to be enhanced by more than 10 folds in the presence of [(WR)5C]-GdNPs compared with siRNA alone in CCRF-CEM and MDA-MB-231 cells after 6 h of incubation at 37 °C. The gene silencing efficacy of the nanoparticles was determined via the western blot technique using an over-expressed gene, STAT-3 protein, in MDA-MB-231 cells. The results showed ~62% reduction of STAT-3 was observed in MDA-MB-231 with [(WR)5C]-GdNPs at N/P 40. The integrity of the cellular membrane of CCRF-CEM cells was found to be intact when incubated with [(WR)5C]-Gd nanoparticles (50 µM) for 2 h. Confocal microscopy reveals higher internalization of siRNA in MDA-MB-231 cells using [(WR)5C]-GdNPs at N/P 40. These results provided insight about the use of the [(WR)5C]-GdNPs complex as a potent intracellular siRNA transporter that could be a nontoxic choice to be used as a transfection agent for nucleic-acid-based therapeutics.
    Keywords:  cyclic peptides; gadolinium nanoparticles; intracellular transportation; nanocomplexes; siRNA delivery systems
    DOI:  https://doi.org/10.3390/ph14111064
  3. Pharmaceutics. 2021 Oct 28. pii: 1807. [Epub ahead of print]13(11):
    Fate of Tableted Freeze-Dried siRNA Lipoplexes in Gastrointestinal Environment.
    Asad Ur Rehman, Virginie Busignies, Marcela Coelho Silva Ribeiro, Nayara Almeida Lage, Pierre Tchoreloff, Virginie Escriou, Christine Charrueau.
      The incorporation of siRNA into nanocarriers is mandatory to facilitate its intracellular delivery, as siRNA itself cannot enter cells. However, the incorporation of these nanocarriers into oral, solid dosage forms and their fate in the gastrointestinal environment is yet to be explored. In the present work, the fate of, (i) naked siRNA, (ii) freshly prepared siRNA lipoplexes, and (iii) tableted siRNA lipoplexes, in simulated gastric and intestinal fluids was studied. The siRNA, either released from or protected within the lipoplexes, was quantified by gel electrophoresis and siRNA efficacy was assessed in cell transfection. The freshly prepared lipoplexes kept their siRNA load and transfection efficiency totally preserved during 1 h of incubation in simulated gastric fluid at 37 °C. However, in simulated intestinal fluid, despite no release of siRNA from lipoplexes after 6 h of incubation, gene silencing efficacy was dramatically decreased even after 1 h of exposure. The lipoplexes obtained from tablets efficiently protected siRNA in simulated gastric fluid, thus preserving the gene silencing efficacy, whereas their incubation in simulated intestinal fluid resulted in a marked siRNA release and decreased gene silencing efficacy. These results provided a detailed explanation for understanding the fate of siRNA in gastrointestinal conditions, when simply loaded in lipoplexes or formulated in the form of tablets.
    Keywords:  RNA interference; gel electrophoresis; nanocarriers; oral delivery; solid dosage form
    DOI:  https://doi.org/10.3390/pharmaceutics13111807
  4. Nat Commun. 2021 Nov 22. 12(1): 6777
    A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems.
    Meredith Packer, Dipendra Gyawali, Ravikiran Yerabolu, Joseph Schariter, Phil White.
      Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein, reversed-phase ion pair high performance liquid chromatography (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity analytical techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, electrophilic impurities derived from the ionizable cationic lipid component are shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidation and subsequent hydrolysis of the tertiary amine. It thus remains critical to ensure robust analytical methods and stringent manufacturing control to ensure mRNA stability and high activity in LNP delivery systems.
    DOI:  https://doi.org/10.1038/s41467-021-26926-0
  5. Colloids Surf B Biointerfaces. 2021 Nov 17. pii: S0927-7765(21)00665-2. [Epub ahead of print] 112219
    New insights on the mechanism of polyethylenimine transfection and their implications on gene therapy and DNA vaccines.
    Juan Sabin, Manuel Alatorre-Meda, Jose Miñones, Vicente Domínguez-Arca, Gerardo Prieto.
      Polyethylenimine (PEI) has been demonstrated as an efficient DNA delivery vehicle both in vitro and in vivo. There is a consensus that PEI-DNA complexes enter the cells by endocytosis and escape from endosomes by the so-called "proton sponge" effect. However, little is known on how and where the polyplexes are de-complexed for DNA transcription and replication to occur inside the cell nucleus. To better understand this issue, we (i) tracked the cell internalization of PEI upon transfection to human epithelial cells and (ii) studied the interaction of PEI with phospholipidic layers mimicking nuclear membranes. Both the biological and physicochemical experiments provided evidence of a strong binding affinity between PEI and the lipidic bilayer. Firstly, confocal microscopy revealed that PEI alone could not penetrate the cell nucleus; instead, it arranged throughout the cytoplasm and formed a sort of aureole surrounding the nuclei periphery. Secondly, surface tension measurements, fluorescence dye leakage assays, and differential scanning calorimetry demonstrated that a combination of hydrophobic and electrostatic interactions between PEI and the phospholipidic monolayers/bilayers led to the formation of stable defects along the model membranes, allowing the intercalation of PEI through the monolayer/bilayer structure. Results are also supported by molecular dynamics simulation of the pore formation in PEI-lipidic bilayers. As discussed throughout the text, these results might shed light on a the mechanism in which the interaction between PEI and the nucleus membrane might play an active role on the DNA release: on the one hand, the PEI-membrane interaction is anticipated to facilitate the DNA disassembly from the polyplex by establishing a competition with DNA for the PEI binding and on the other hand, the forming defects are expected to serve as channels for the entrance of de-complexed DNA into the cell nucleus. A better understanding of the mechanism of transfection of cationic polymers opens paths to development of more efficiency vectors to improve gene therapy treatment and the new generation of DNA vaccines.
    Keywords:  DNA vectors; Mimicking nuclear membranes; Molecular dynamics simulations; Polyethylenimine; Pore formation
    DOI:  https://doi.org/10.1016/j.colsurfb.2021.112219
  6. Carbohydr Polym. 2022 Jan 15. pii: S0144-8617(21)01134-6. [Epub ahead of print]276 118747
    Stimulus-responsive drug/gene delivery system based on polyethylenimine cyclodextrin nanoparticles for potential cancer therapy.
    Hanieh Mousazadeh, Esat Bonabi, Nosratollah Zarghami.
      Combination therapy through simultaneous delivery of anti-cancer drugs and genes with nano-assembled structure has been proved to be a simple and effective approach for treating breast cancer. In this study, redox-sensitive folate-appended-polyethylenimine-β-cyclodextrin (roFPC) host-guest supramolecular nanoparticles (HGSNPs) were developed as a targeted co-delivery system of doxorubicin (Dox) and Human telomerase reverse transcriptase-small interfering RNA) hTERT siRNA) for potential cancer therapy. The nanotherapeutic system was prepared by loading adamantane-conjugated doxorubicin (Ad-Dox) into roFPC through the supramolecular assembly, followed by electrostatically-driven self-assembly between hTERT siRNA and roFPC/Ad-Dox. The roFPC' host-guest structures allow pH-dependent intracellular drug release in a sustained manner, as well as simultaneous and effective gene transfection. This co-delivery vector displayed combined anti-tumor properties of the Dox-enhanced gene transfection, good water-solubility, and biocompatibility, possesses considerably enhanced hemocompatibility, and especially targets folate receptor-positive cells only at low N/P levels to prompt effective cell apoptosis for cancer treatment.
    Keywords:  Cancer; Cyclodextrin; Host-guest; Nanoparticle; Supramolecular; hTERT siRNA
    DOI:  https://doi.org/10.1016/j.carbpol.2021.118747
  7. J Control Release. 2021 Nov 22. pii: S0168-3659(21)00627-1. [Epub ahead of print]
    Emerging landscape of cell-penetrating peptide-mediated nucleic acid delivery and their utility in imaging, gene-editing, and RNA-sequencing.
    Jingping Geng, Xuan Xia, Lin Teng, Lidan Wang, Linlin Chen, Xiangli Guo, Bonn Belingon, Jason Li, Xuemei Feng, Xianghui Li, Wendou Shang, Yingying Wan, Hu Wang.
      The safety issues like immunogenicity and unacceptable cancer risk of viral vectors for DNA/mRNA vaccine delivery necessitate the development of non-viral vectors with no toxicity. Among the non-viral strategies, cell-penetrating peptides (CPPs) have been a topic of interest recently because of their ability to cross plasma membranes and facilitate nucleic acids delivery both in vivo and in vitro. In addition to the application in the field of gene vaccine and gene therapy, CPPs based nucleic acids delivery have been proved by its potential application like gene editing, RNA-sequencing, and imaging. Here, we focus on summarizing the recent applications and progress of CPPs-mediated nucleic acids delivery and discuss the current problems and solutions in this field.
    Keywords:  CPP; Delivery; Gene editing; Gene therapy; Imaging; RNA-sequencing
    DOI:  https://doi.org/10.1016/j.jconrel.2021.11.032
  8. J Control Release. 2021 Nov 18. pii: S0168-3659(21)00617-9. [Epub ahead of print]
    Lipid nanoparticle chemistry determines how nucleoside base modifications alter mRNA delivery.
    Jilian R Melamed, Khalid A Hajj, Namit Chaudhary, Daria Strelkova, Mariah L Arral, Norbert Pardi, Mohamad-Gabriel Alameh, Jason B Miller, Lukas Farbiak, Daniel J Siegwart, Drew Weissman, Kathryn A Whitehead.
      Therapeutic mRNA has the potential to revolutionize the treatment of myriad diseases and, in 2020, facilitated the most rapid vaccine development in history. Among the substantial advances in mRNA technology made in recent years, the incorporation of base modifications into therapeutic mRNA sequences can reduce immunogenicity and increase translation. However, experiments from our lab and others have shown that the incorporation of base modifications does not always yield superior protein expression. We hypothesized that the variable benefit of base modifications may relate to lipid nanoparticle chemistry, formulation, and accumulation within specific organs. To test this theory, we compared IV-injected lipid nanoparticles formulated with reporter mRNA incorporating five base modifications (ψ, m1ψ, m5U, m5C/ψ, and m5C/s2U) and four ionizable lipids (C12-200, cKK-E12, ZA3-Ep10, and 200Oi10) with tropism for different organs. In general, the m1ψ base modification best enhanced translation, producing up to 15-fold improvements in total protein expression compared to unmodified mRNA. Expression improved most dramatically in the spleen (up to 50-fold) and was attributed to enhanced protein expression in monocytic lineage splenocytes. The extent to which these effects were observed varied with delivery vehicle and correlated with differences in innate immunogenicity. Through comparison of firefly luciferase and erythropoietin mRNA constructs, we also found that mRNA modification-induced enhancements in protein expression are limited outside of the spleen, irrespective of delivery vehicle. These results highlight the complexity of mRNA-loaded lipid nanoparticle drug design and show that the effectiveness of mRNA base modifications depend on the delivery vehicle, the target cells, and the site of endogenous protein expression.
    Keywords:  Lipid nanoparticles; Lipidoid; Non-viral gene delivery; Pseudouridine; mRNA
    DOI:  https://doi.org/10.1016/j.jconrel.2021.11.022
  9. Int J Mol Sci. 2021 Nov 16. pii: 12344. [Epub ahead of print]22(22):
    Increasing Transfection Efficiency of Lipoplexes by Modulating Complexation Solution for Transient Gene Expression.
    Jaemun Kim, Ji Yul Kim, Hyeonkyeong Kim, Eunsil Kim, Soonyong Park, Kyoung-Hwa Ryu, Eun Gyo Lee.
      Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.
    Keywords:  CHO-S; DC-Chol/DOPE; complexation solution; transfection; transient gene expression
    DOI:  https://doi.org/10.3390/ijms222212344
  10. Theranostics. 2021 ;11(20): 9988-10000
    Exosome-mediated delivery of inflammation-responsive Il-10 mRNA for controlled atherosclerosis treatment.
    Te Bu, Zhelong Li, Ying Hou, Wenqi Sun, Rongxin Zhang, Lianbi Zhao, Mengying Wei, Guodong Yang, Lijun Yuan.
      Rationale: Tailored inflammation control is badly needed for the treatment of kinds of inflammatory diseases, such as atherosclerosis. IL-10 is a potent anti-inflammatory cytokine, while systemic and repeated delivery could cause detrimental side-effects due to immune repression. In this study, we have developed a nano-system to deliver inflammation-responsive Il-10 mRNA preferentially into macrophages for tailored inflammation control. Methods: Il-10 was engineered to harbor a modified HCV-IRES (hepatitis C virus internal ribosome entry site), in which the two miR-122 recognition sites were replaced by two miR-155 recognition sites. The translational responsiveness of the engineered mRNA to miR-155 was tested by Western blot or ELISA. Moreover, the engineered Il-10 mRNA was passively encapsulated into exosomes by forced expression in donor cells. Therapeutic effects on atherosclerosis and the systemic leaky expression effects in vivo of the functionalized exosomes were analyzed in ApoE-/- (Apolipoprotein E-deficient) mice. Results: The engineered IRES-Il-10 mRNA could be translationally activated in cells when miR-155 was forced expressed or in M1 polarized macrophages with endogenous miR-155 induced. In addition, the engineered IRES-Il-10 mRNA, when encapsulated into the exosomes, could be efficiently delivered into macrophages and some other cell types in the plaque in ApoE-/- mice. In the recipient cells of the plaque, the encapsulated Il-10 mRNA was functionally translated into protein, with relatively low leaky in other tissues/organs without obvious inflammation. Consistent with the robust Il-10 induction in the plaque, exosome-based delivery of the engineered Il-10 could alleviate the atherosclerosis in ApoE-/- mice. Conclusion: Our study established a potent platform for controlled inflammation control via exosome-based systemic and repeated delivery of engineered Il-10 mRNA, which could be a promising strategy for atherosclerosis treatment.
    Keywords:  Atherosclerosis; exosomes; inflammation-responsive; interleukin-10; internal ribosome entry site
    DOI:  https://doi.org/10.7150/thno.64229
  11. Clin Transl Oncol. 2021 Nov 23.
    Human umbilical cord mesenchymal stem cell-derived exosomes carrying hsa-miRNA-128-3p suppress pancreatic ductal cell carcinoma by inhibiting Galectin-3.
    X Xie, J Ji, X Chen, W Xu, H Chen, S Zhu, J Wu, Y Wu, Y Sun, W Sai, Z Liu, M Xiao, B Bao.
      BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal malignant tumors of the digestive system. Many patients are diagnosed at an advanced stage and lose eligibility for surgery. Moreover, there are few effective methods for treating pancreatic ductal cell carcinoma. Increasing attention has been given to microRNAs (miRNAs) and their regulatory roles in tumor progression. In this study, we investigated the effects of exosomes extracted from human umbilical cord mesenchymal stem cells (HUCMSCs) carrying hsa-miRNA-128-3p on pancreatic cancer cells.METHODS: Based on existing experimental and database information, we selected Galectin-3, which is associated with pancreatic cancer, and the corresponding upstream hsa-miRNA-128-3p. We extracted HUCMSCs from a fresh umbilical cord, hsa-miRNA-128-3p was transfected into HUCMSCs, and exosomes containing hsa-miRNA-128-3p were extracted and collected. The effect of exosomes rich in hsa-miRNA-128-3p on pancreatic cancer cells was analyzed.
    RESULTS: The expression of Galectin-3 in normal pancreatic duct epithelial cells was significantly lower than that in PDAC cell lines. We successfully extracted HUCMSCs from the umbilical cord and transfected hsa-miRNA-128-3p into HUCMSCs. Then we demonstrated that HUCMSC-derived exosomes with hsa-miRNA-128-3p could suppress the proliferation, invasion, and migration of PANC-1 cells in vitro by targeting Galectin-3.
    CONCLUSION: Hsa-miRNA-128-3p could be considered as a potential therapy for pancreatic cancer. We provided a new idea for targeted therapy of PDAC.
    Keywords:  Exosome; Galectin-3; Hsa-miRNA-128-3p; Human umbilical cord mesenchymal stem cells; Pancreatic ductal cell carcinoma
    DOI:  https://doi.org/10.1007/s12094-021-02705-7
This page is being maintained by Thomas Krichel. It was last updated on 2021‒11‒28 at 14:20:56.