bims-novged 2021-10-10 papers

on Non-viral vectors for gene delivery

Issue of 2021–10–10
ten papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Serum-Independent Nonviral Gene Delivery to Innate and Adaptive Immune Cells Using Immunoplexes.
Polyethylenimine-modified graphitic carbon nitride nanosheets: a label-free Raman traceable siRNA delivery system.
Sphingolipid extracts enhances gene delivery of cationic lipid vesicles into retina and brain.
DNA Origami Frameworks Enabled Self-Protective siRNA Delivery for Dual Enhancement of Chemo-Photothermal Combination Therapy.
Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs.
Intracranially injectable multi-siRNA nanomedicine for the inhibition of glioma stem cells.
Polydopamine nanoparticles with different sizes for NIR-promoted gene delivery and synergistic photothermal therapy.
Self-catalytic Small Interfering RNA Nanocarriers for Synergistic Treatment of Neurodegenerative Diseases.
Black phosphorus mediated photoporation: a broad absorption nanoplatform for intracellular delivery of macromolecules.
Evaluating the influence of Human Umbilical Cord Mesenchymal Stem Cells-derived exosomes loaded with miR-3182 on metastatic performance of Triple Negative Breast Cancer cells.

ACS Appl Bio Mater. 2020 Sep 21. 3(9): 6263-6272

Serum-Independent Nonviral Gene Delivery to Innate and Adaptive Immune Cells Using Immunoplexes.

Atanu Chakraborty, Jackline Joy Martín Lasola, Nhu Truong, Ryan M Pearson.

  Genetic engineering of innate and adaptive immune cells represents a potential solution to treat numerous immune-mediated pathologies. Current immune engineering methods to introduce nucleic acids into cells with high efficiency rely on physical mechanisms such as electroporation, viral vectors, or other chemical methods. Gene delivery using non-viral nanoparticles offers significant flexibility in biomaterial design to tune critical parameters such as nano-bio interactions, transfection efficiency, and toxicity profiles. However, their clinical utility has been limited due to complex synthetic procedures, high toxicity at increased polymer (nitrogen, N) to DNA ratios (phosphate, P) (N/P ratios), poor transfection efficiency and nanoparticle stability in the presence of serum, and short-term gene expression. Here, we describe the development of a simple, polymer-based non-viral gene delivery platform based on simple modifications of polyethylenimine (PEI) that displays potent and serum-independent transfection of innate and adaptive immune cells. Cationic acetylated PEI (Ac-PEI) was synthesized and complexed with plasmid DNA (pDNA) followed by enveloping with an anionic polyelectrolyte layer of poly(ethylene-alt-maleic acid) (PEMA) to form immunoplexes (IPs). Cellular interactions and gene expression could be precisely controlled in murine RAW 264.7 macrophages, murine DC2.4 dendritic cells, and human Jurkat T cells by altering the levels of PEMA envelopment, thus providing a strategy to engineer specific cell targeting into the IP platform. Optimally formulated IPs for immune cell transfection in the presence of serum utilized high N/P ratios to enable high stability, displayed reduced toxicity, high gene expression, and a lengthened duration of gene expression (>3 days) compared to non-enveloped controls. These results demonstrate the potential of engineered IPs to serve as simple, modular, targetable, and efficient non-viral gene delivery platform to efficiently alter gene expression within cells of the immune system.

Keywords:  Gene delivery; Immune cells; Nanoparticle; Polyethylenimine; Serum independent

DOI:  https://doi.org/10.1021/acsabm.0c00761
J Mater Chem B. 2021 Sep 14. 9(34): 6895-6901

Polyethylenimine-modified graphitic carbon nitride nanosheets: a label-free Raman traceable siRNA delivery system.

Lei Liu, Xin Du.

Since the nanotoxicity of gene delivery carriers has raised world-wide concerns, it is important to trace their intracellular performance, for example via uptake visualization. Here, we develop a novel ultrathin graphitic carbon nitride (g-C3N4) composite nanosystem for label-free Raman-traceable small interfering RNA (siRNA) delivery. Through low molecular weight polyethylenimine (PEI) modifications, these nanosystems can obtain siRNA loading capabilities. The lateral size of the PEI-g-C3N4 composite is around 100-150 nm with a thickness of nearly 0.6 nm. The designed label-free delivery system could avoid possible obstacles associated with artificial labels and it shows cytotoxicity toward cancer cells and good biocompatibility in normal human cells. The label-free PEI-g-C3N4 gene nanocarrier can be directly traced via Raman microscopy, which makes it suitable for intracellular visualization. Intracellular uptake of the self-fluorescent g-C3N4 nanosheets can also be traced via fluorescence imaging. The PEI modified g-C3N4 ultrathin nanosheets possess gene delivery capacity together with unique dual-traceable Raman and fluorescence features. Raman traces not only have higher specificity than fluorescence ones but they can also avoid background noises. Thus, they may replace widely implemented fluorescence tracing. This work could provide a label-free traceable platform for investigating the intracellular performances of gene delivery nanosystems.

DOI: https://doi.org/10.1039/d1tb00984b
Eur J Pharm Biopharm. 2021 Oct 01. pii: S0939-6411(21)00255-1. [Epub ahead of print]

Sphingolipid extracts enhances gene delivery of cationic lipid vesicles into retina and brain.

Nuseibah Al Qtaish, Idoia Gallego, Ilia Villate-Beitia, Myriam Sainz-Ramos, Gema Martínez-Navarrete, Cristina Soto-Sánchez, Eduardo Fernández, Patricia Gálvez Martín, Tania B Lopez-Mendez, Gustavo Puras, José Luis Pedraz.

  The aim was to evaluate relevant biophysic processes related to the physicochemical features and gene transfection mechanism when sphingolipids are incorporated into a cationic niosome formulation for non-viral gene delivery to central nervous system. For that, two formulations named niosphingosomes and niosomes devoid of sphingolipid extracts, as control, were developed by the oil-in water emulsion technique. Both formulations and the corresponding complexes, obtained upon the addition of the reporter EGFP plasmid, were physicochemically and biologically characterized and evaluated. Compared to niosomes, niosphingosomes, and the corresponding complexes decreased particle size and increased superficial charge. Although there were not significant differences in the cellular uptake, cell viability and transfection efficiency increased when human retinal pigment epithelial (ARPE-19) cells were exposed to niosphingoplexes. Endocytosis via caveolae decreased in the case of niosphingoplexes, which showed higher co-localization with lysosomal compartment, and endosomal escape properties. Moreover, niosphingoplexes transfected not only primary central nervous system cells, but also different cells in mouse retina, depending on the administration route, and brain cortex. These preliminary results suggest that niosphingosomes represent a promising non-viral vector formulation purposed for the treatment of both retinal and brain diseases by gene therapy approach.

Keywords:  Gene therapy; brain; niosomes; niosphingosomes; retina; sphingolipids

DOI:  https://doi.org/10.1016/j.ejpb.2021.09.011
Small. 2021 Oct 05. e2101780

DNA Origami Frameworks Enabled Self-Protective siRNA Delivery for Dual Enhancement of Chemo-Photothermal Combination Therapy.

Tingting Xu, Sha Yu, Yao Sun, Shaojun Wu, Di Gao, Mingyang Wang, Zhenzhen Wang, Ye Tian, Qianhao Min, Jun-Jie Zhu.

  Although chemotherapy and photothermal therapy are widely used to combat cancer, their efficacy is often limited by multidrug resistance. Small interfering RNAs (siRNAs) have ability to suppress the expression of target genes, which has been extensively employed for combating the multidrug resistance to chemodrugs and hyperthermia in cancer therapy. However, efficient delivery of siRNAs along with chemo-photothermal agents in vivo is still an enormous challenge. Herein, octahedral DNA origami frameworks (OctDOFs) are constructed as a nanovehicle for precise organization and orchestrated delivery of siRNAs, chemodrugs (doxorubicin, Dox), and photothermal agents (gold nanorods, AuNRs) in combinatorial treatment of cancer. The inner cavity of the rigid OctDOFs structure is able to shield the encapsulated siRNAs during transportation by sterically hindering RNase degradation and protein binding, thus achieving effective downregulation of connective tissue growth factor (CTGF) and heat shock protein 72 (HSP72) for dual sensitization of cancer cells to chemodrugs and hyperthermia. By amplifying chemo-photothermal therapeutic potency with siRNAs, the proposed OctDOFs exhibited superior cytotoxicity and tumor inhibition efficacy in vitro and in vivo. This nanovehicle creates a promising siRNA delivery platform for precise medication and combination therapy.

Keywords:  3D nanoscaffolds; Combination therapy; DNA origami; dual sensitization; siRNAs delivery

DOI:  https://doi.org/10.1002/smll.202101780
Nat Biomed Eng. 2021 Sep;5(9): 1059-1068

Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs.

Melissa P Lokugamage, Daryll Vanover, Jared Beyersdorf, Marine Z C Hatit, Laura Rotolo, Elisa Schrader Echeverri, Hannah E Peck, Huanzhen Ni, Jeong-Kee Yoon, YongTae Kim, Philip J Santangelo, James E Dahlman.

Lipid nanoparticles (LNPs) for the efficient delivery of drugs need to be designed for the particular administration route and type of drug. Here we report the design of LNPs for the efficient delivery of therapeutic RNAs to the lung via nebulization. We optimized the composition, molar ratios and structure of LNPs made of lipids, neutral or cationic helper lipids and poly(ethylene glycol) (PEG) by evaluating the performance of LNPs belonging to six clusters occupying extremes in chemical space, and then pooling the lead clusters and expanding their diversity. We found that a low (high) molar ratio of PEG improves the performance of LNPs with neutral (cationic) helper lipids, an identified and optimal LNP for low-dose messenger RNA delivery. Nebulized delivery of an mRNA encoding a broadly neutralizing antibody targeting haemagglutinin via the optimized LNP protected mice from a lethal challenge of the H1N1 subtype of influenza A virus, and delivered mRNA more efficiently than LNPs previously optimized for systemic delivery. A cluster approach to LNP design may facilitate the optimization of LNPs for other administration routes and therapeutics.

DOI: https://doi.org/10.1038/s41551-021-00786-x
Neurooncol Adv. 2021 Jan-Dec;3(1):3(1): vdab104

Intracranially injectable multi-siRNA nanomedicine for the inhibition of glioma stem cells.

Cheripelil Abraham Manju, Kottarapat Jeena, Ranjith Ramachandran, Maneesh Manohar, Anna Mathew Ambily, Koythatta Meethalveedu Sajesh, Genekehal Siddaramana Gowd, Krishnakumar Menon, Keechilat Pavithran, Ashok Pillai, Shantikumar V Nair, Manzoor Koyakutty.

   Background: Nanoparticle siRNA-conjugates are promising clinical therapeutics as indicated by recent US-FDA approval. In glioma stem cells (GSC), multiple stemness associated genes were found aberrant. We report intracranially injectable, multi-gene-targeted siRNA nanoparticle gel (NPG) for the combinatorial silencing of 3 aberrant genes, thus inhibiting the tumorogenic potential of GSCs.
Methods: NPG loaded with siRNAs targeted against FAK, NOTCH-1, and SOX-2 were prepared by the self-assembly of siRNAs with protamine-hyaluronic acid combination. Electron microscopy, DLS, and agarose gel electrophoresis were used for the physicochemical characterization. Cell transfection and gene-silencing efficiency were studied using human mesenchymal stem cells and rat C6 glioma-derived GSCs. Neurosphere inhibition was tested in vitro using GSCs derived from C6 cell line and glioma patient samples. Patient-derived xenograft model and orthotopic rat glioma model were used to test the effect of NPG on in vivo tumorigenicity.
Results: The siRNA nanoparticles with an average size ~ 250 nm and ~ 95% loading efficiency showed cellular uptake in ~95.5% GSCs. Simultaneous gene silencing of FAK, NOTCH-1, and SOX-2 led to the inhibition of neurosphere formation by GSCs, whereas normal stem cells remained unaffected and retained neuronal differentiation capability. GBM PDX models manifested significant impairment in the tumorigenic potential of NPG treated GSCs. Intracranial injection of NPG inhibited tumor growth in orthotopic rat brain tumor model.
Conclusion: Intracranially injectable n-siRNA NPG targeted to multiple stem-cell signaling impairs glioma initiation capabilities of GSCs and inhibited tumor growth in vivo.

Keywords:  cancer stem cells; gene silencing; nanoparticle; neurosphere; self-assembly

DOI:  https://doi.org/10.1093/noajnl/vdab104
Colloids Surf B Biointerfaces. 2021 Sep 22. pii: S0927-7765(21)00569-5. [Epub ahead of print]208 112125

Polydopamine nanoparticles with different sizes for NIR-promoted gene delivery and synergistic photothermal therapy.

Peng Zhang, Xinfang Li, Qinan Xu, Youxiang Wang, Jian Ji.

  The combination of photothermal therapy and gene therapy has received increasing attention in tumor treatment. However, how to improve synergistic efficacy has become a new challenge. NIR light has a great potential in tumor treatment because of its considerable penetration depth and spatiotemporal controllability. Polydopamine is a popular photothermal conversion agent, which has desirable photothermal conversion ability and good biocompatibility. In this research, polydopamine-polyethyleneimine nanoparticles with diameters of 13 nm (SPPNPs) and 236 nm (LPPNPs) were prepared as gene carriers. The size of polydopamine nanoparticles had great effect on the complexes formation, photothermal conversion ability and gene transfection efficiency. After loading gene, the SPPNPs/gene and LPPNPs/gene complexes were about 60-80 nm and 240 nm respectively, indicating different styles of complexes formation. Both SPPNPs/gene and LPPNPs/gene complexes without NIR irradiation could achieve similar gene transfection efficiency as commercial lipofectamine 2000, while with lower cytotoxicity. Due to better photothermal conversion ability, the transfection level of LPPNPs/pGL-3 complexes increased to 4.5 times after NIR irradiation (2.6 W/cm2, 15 min), which ascribed to the quick escape of gene complexes from the endosome. The produced heat under NIR irradiation could also ablate tumor cells. So LPPNPs were chosen to deliver tumor suppressor gene p53 DNA to investigate the synergistic efficacy of gene/photothermal therapy. The tumor in KB tumor-bearing mice was almost eliminated after intratumoral injection, and the tumor inhibition efficacy of gene/photothermal synergistic therapy achieved to 99%. By combining NIR-promoted gene transfection and gene/photothermal synergistic therapy, the LPPNPs hold great promise in practical tumor treatment.

Keywords:  Gene therapy; Photothermal therapy; Polydopamine; Size effect; Synergistic therapy

DOI:  https://doi.org/10.1016/j.colsurfb.2021.112125
Adv Mater. 2021 Oct 03. e2105711

Self-catalytic Small Interfering RNA Nanocarriers for Synergistic Treatment of Neurodegenerative Diseases.

Weihong Ji, Yan Li, Huan Peng, Ruichen Zhao, Jie Shen, Yanyue Wu, Jianze Wang, Qiulian Hao, Zhiguo Lu, Jun Yang, Xin Zhang.

  Gene therapy has shown great potential for neurodegenerative diseases with complex pathology. However, its therapeutic effect is limited due to the delivery barriers and its own single function. Herein, self-catalytic small interfering RNA (siRNA) nanocarriers (S/Ce-PABMS) are developed to catalyze delivery process and treatment process for synergistic treatment of neurodegenerative diseases. On the one hand, the rough surface of S/Ce-PABMS mediated by ceria (CeO2 ) nanozymes can catalyze cellular uptake in the delivery process, so that S/Ce-PABMS with acetylcholine analogs penetrate the blood-brain barrier and enter neurons more effectively. On the other hand, CeO2 nanozymes can catalyze the treatment process by scavenging excess reactive oxygen species, and cooperate with siRNA targeting SNCA to decrease the α-synuclein (α-syn) aggregation and alleviate the parkinsonian pathology. Moreover, the S/Ce-PABMS treatment reduces the number of activated microglia and regulates the release of inflammatory cytokine, thereby relieving neuroinflammation. After treatment with S/Ce-PABMS, dyskinesia in Parkinson's disease model mice has been significantly alleviated. The finding shows that the self-catalytic nanocarriers S/Ce-PABMS have great potential in the treatment of neurodegenerative diseases. This article is protected by copyright. All rights reserved.

Keywords:  brain delivery; nanozymes; neurodegenerative diseases; self-catalytic; siRNA; synergistic treatment

DOI:  https://doi.org/10.1002/adma.202105711
Nanoscale. 2021 Oct 08.

Black phosphorus mediated photoporation: a broad absorption nanoplatform for intracellular delivery of macromolecules.

Jielin Wang, Aranit Harizaj, Yongbo Wu, Xiaofang Jiang, Toon Brans, Juan C Fraire, Julián Mejía Morales, Stefaan C De Smedt, Zhilie Tang, Ranhua Xiong, Kevin Braeckmans.

Nanoparticle-sensitized photoporation for intracellular delivery of external compounds usually relies on the use of spherical gold nanoparticles as sensitizing nanoparticles. As they need stimulation with visible laser light, they are less suited for transfection of cells in thick biological tissues. In this work, we have explored black phosphorus quantum dots (BPQDs) as alternative sensitizing nanoparticles for photoporation with a broad and uniform absorption spectrum from the visible to the near infra-red (NIR) range. We demonstrate that BPQD sensitized photoporation allows efficient intracellular delivery of both siRNA (>80%) and mRNA (>40%) in adherent cells as well as in suspension cells. Cell viability remained high (>80%) irrespective of whether irradiation was performed with visible (532 nm) or near infrared (800 nm) pulsed laser light. Finally, as a proof of concept, we used BPQD sensitized photoporation to deliver macromolecules in cells with thick phantom tissue in the optical path. NIR laser irradiation resulted in only 1.3× reduction in delivery efficiency as compared to photoporation without the phantom gel, while with visible laser light the delivery efficiency was reduced 2×.

DOI: https://doi.org/10.1039/d1nr05461a
Life Sci. 2021 Oct 03. pii: S0024-3205(21)01002-X. [Epub ahead of print] 120015

Evaluating the influence of Human Umbilical Cord Mesenchymal Stem Cells-derived exosomes loaded with miR-3182 on metastatic performance of Triple Negative Breast Cancer cells.

Yalda Khazaei-Poul, Samaneh Shojaei, Ameneh Koochaki, Hossein Ghanbarian, Samira Mohammadi-Yeganeh.

   AIMS: Deregulation of microRNA (miRNA) function has been linked to numerous human cancers, such as Triple Negative Breast Cancer (TNBC). Exosomes, a subgroup of extracellular vehicles (EVs), can efficiently deliver many different cargo types to the target cell and have an extensive role in delivering therapeutic cargo for treatment. The present study intended to interrogate the effects of exosomal delivery of miR-3182 on TNBC cellular processes.
MAIN METHODS: Human Umbilical Cord Mesenchymal Stem Cells (HUCMSCs) were cultured and exosomes were isolated and characterized using TEM, SEM, DLS, and Western blot. Exosomes were transfected with miR-3182 and added to the treatment groups. The expression level of miR-3182 and their target genes including mTOR and S6KB1 were evaluated using RT-qPCR. The effects of miR-3182 loaded HUCMSC-exosomes treatment on the cellular aspect of MDA-MB-231 cells including their viability, migration potency, cell cycle status and apoptosis were investigated.
KEY FINDINGS: According to the results, exosomal miR-3182 significantly abolished cell proliferation and migration (P < 0.05). miR-3182 loaded exosomes also induced apoptosis in TNBC cells by down-regulating mTOR and S6KB1 genes (P < 0.05).
SIGNIFICANCE: In nutshell, miR-3182-loaded HUCMSC-exosomes can suppress TNBC invasion, suggesting that exosomes containing miR-3182 could be a reliable therapeutic paradigm in TNBC therapy.

Keywords:  Exosome; Human Umbilical Cord Mesenchymal Stem Cells; S6KB1; Triple Negative Breast Cancer (TNBC); mTOR; miR-3182

DOI:  https://doi.org/10.1016/j.lfs.2021.120015