bims-novged 2022-01-02 papers

on Non-viral vectors for gene delivery

Issue of 2022–01–02
eight papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Novel Fluorinated Spermine and Small Molecule PEI to Deliver Anti-PD-L1 and Anti-VEGF siRNA for Highly Efficient Tumor Therapy.
Cross-linkable Polyion Complex Micelles from Polypept(o)ide-based ABC-triblock Copolymers for siRNA Delivery.
Mucus-producing epithelial models for investigating the activity of gene delivery systems in the lung.
Systemic delivery of siRNA to the colon using peptide modified PEG-PCL polymer micelles for the treatment of ulcerative colitis.
Ultrasound-sensitive siRNA-loaded nanobubbles fabrication and antagonism in drug resistance for NSCLC.
Added to pre-existing inflammation, mRNA-lipid nanoparticles induce inflammation exacerbation (IE).
Suppression of fibrin(ogen)-driven pathologies through controlled knockdown by lipid nanoparticle delivery of siRNA.
Physical-Chemical Characterization of Bilayer Membranes Derived from (±) α-Tocopherol-Based Gemini Lipids and Their Interaction with Phosphatidylcholine Bilayers and Lipoplex Formation with Plasmid DNA.

Pharmaceutics. 2021 Dec 02. pii: 2058. [Epub ahead of print]13(12):

Novel Fluorinated Spermine and Small Molecule PEI to Deliver Anti-PD-L1 and Anti-VEGF siRNA for Highly Efficient Tumor Therapy.

Yihui Zhang, Zihan Yuan, Yi Jin, Wenkai Zhang, Wei-En Yuan.

  Small interfering RNA (siRNA) can specifically silence disease gene expression. This project investigated the overexpression of programmed death receptor ligand 1 (PD-L1) and vascular endothelial growth factor (VEGF) on the surface of tumor cells. However, the main obstacle to the development of gene therapy drugs is the lack of an efficient delivery vector, which should be able to overcome multiple delivery barriers and protect siRNA to enter the target cells. Therefore, a novel fluorine-modified endogenous molecular carrier TFSPEI was constructed by linking fluorinated groups with hydrophobic and hydrophilic characteristics on the surface of PEI and spermine. The results showed that lower toxicity, higher endocytosis, and silencing efficiency were achieved. We found that the inhibition of VEGF targets can indirectly activate the immune response to promote the tumor-killing and invasion effects of T cells. The combined delivery of anti-VEGF siRNA and anti-PD-L1 siRNA could inhibit the expression of corresponding proteins, restore the anti-tumor function of T cells and inhibit the growth of neovascularization, and obtained significant anti-tumor effects. Therefore, this safe and efficient fluorinated spermine and small molecule PEI-based anti-PD-L1 and anti-VEGF siRNA delivery system is expected to provide a new strategy for gene therapy of tumors.

Keywords:  PD-L1; VEGF; anti-tumor; fluorine modified carrier; gene silencing

DOI:  https://doi.org/10.3390/pharmaceutics13122058
Macromol Rapid Commun. 2021 Dec 30. e2100698

Cross-linkable Polyion Complex Micelles from Polypept(o)ide-based ABC-triblock Copolymers for siRNA Delivery.

Leon Capelôa, Mina Yazdi, Heyang Zhang, Xiaobing Chen, Yu Nie, Ernst Wagner, Ulrich Lächelt, Matthias Barz.

  ABC-type triblock copolymers are a rising platform especially for oligonucleotide delivery as they offer an additional functionality beside the anyhow needed functions of shielding and complexation. We present a polypept(o)ide-based triblock copolymer synthesized by amine-initiated ring-opening polymerization (ROP) of N-carboxyanhydrides (NCAs), comprising a shielding block A of polysarcosine (pSar), a poly(S-ethylsulfonyl-l-cystein) (pCys(SO2 Et)) block B for bioreversible and chemo-selective cross-linking and a poly(l-lysine) (pLys) block C for complexation to construct polyion complex (PIC) micelles as vehicle for small interfering RNA (siRNA) delivery. We investigated the self-assembly behavior of ABC-type triblocks to derive correlations between block lengths of the polymer and PIC micelle structure, showing an enormous effect of the β-sheet forming pCys(SO2 Et) block. Moreover, the block enables the introduction of disulfide cross-links by reaction with multifunctional thiols to increase stability against dilution. The right content of the additional block leads to well-defined cross-linked 50-60 nm PIC micelles purified from production impurities and determinable siRNA loading. These PIC micelles can deliver functional siRNA into Neuro2A and KB cells evaluated by cellular uptake and specific gene knockdown assays. This article is protected by copyright. All rights reserved.

Keywords:  cross-linking; gene silencing; polyion complex micelles; polypept(o)ides; self-assembly; siRNA delivery

DOI:  https://doi.org/10.1002/marc.202100698
Int J Pharm. 2021 Dec 24. pii: S0378-5173(21)01229-1. [Epub ahead of print] 121423

Mucus-producing epithelial models for investigating the activity of gene delivery systems in the lung.

Thomas Sonntag, Mickael Rapp, Pascal Didier, Luc Lebeau, Françoise Pons, Anne Casset.

  Inhaled transfection particles have to penetrate the mucus layer lining the airways to successfully deliver their therapeutic nucleic acid payload to target cells in the underlying epithelium. However, the in vitro models used for evaluating gene carrier efficiency often disregard this viscous defensive barrier. In this study, the two mucus-secreting cell lines NCI-H292 and Calu-3 were selected to develop a series of epithelial models displaying gradual mucus production. In NCI-H292 models, a gradual increase in the MUC5AC mucin was obtained after cell exposure to inducers. In Calu-3 models, MUC5AC production increased as a function of culture duration (3, 7, 14 days) at the air-liquid interface (ALI). Six DOPC-derived cationic lipids were designed and their pDNA delivery activity was evaluated to validate these cellular models. The strongest impairment of the lipid delivery activity was observed in the Calu-3 14-d ALI model. The MUC5AC production in this model was the greatest and the mucus layer was 20 µm thick. The mucus exhibited a solid viscoelastic behaviour, and represented a major hindrance to lipoplex diffusion. The Calu-3 14-d ALI model will be highly useful for accurate evaluation of gene carriers intended for airway administration and characterization of their interactions with the mucus.

Keywords:  airway mucus; cell models; gene delivery; lipoplexes; multiple particle tracking; rheology

DOI:  https://doi.org/10.1016/j.ijpharm.2021.121423
Eur J Pharm Biopharm. 2021 Dec 25. pii: S0939-6411(21)00358-1. [Epub ahead of print]

Systemic delivery of siRNA to the colon using peptide modified PEG-PCL polymer micelles for the treatment of ulcerative colitis.

Hisako Ibaraki, Naruhiro Hatakeyama, Naoki Arima, Akihiro Takeda, Yasuo Seta, Takanori Kanazawa.

  Ulcerative colitis (UC) is a refractory inflammatory bowel disease that causes inflammation and ulcers in the digestive tract, and significantly reduces the patient's quality of life. While existing UC treatments have many challenges, nanotechnology, and small interfering RNA (siRNA) based formulations are novel and promising for UC treatment. We previously reported that intravenous administration of MPEG-PCL-CH2R4H2C nanomicelles had high inflammatory site accumulation and remarkable therapeutic effects on rheumatoid arthritis by a phenomenon similar to enhanced permeability and retention effect. In this study, we investigated the effects of siRNA delivered using MPEG-PCL-CH2R4H2C nanomicelles through intravenous administration to the inflammation site of dextran sulfate sodium-induced colitis mice. The MPEG-PCL-CH2R4H2C micelles had optimum physical properties and high siRNA compaction ability. Moreover, model-siRNA delivered through MPEG-PCL-CH2R4H2C showed higher accumulation in the inflammatory site than that of the naked siRNA. Furthermore, intravenous administration of MPEG-PCL-CH2R4H2C/siRelA micelles, targeting siRelA, a subunit of NF-κB, significantly decreased the shortening of large intestine, clinical score, and production of inflammatory cytokines compared the 5-ASA and naked siRelA. These results suggest that MPEG-PCL-CH2R4H2C is a useful carrier for the systemic delivery and accumulation of siRNA, thus improving its therapeutic effect.

Keywords:  Allergies; Autoimmune disease; Drug delivery system; MPEG-PCL-CH2R4H2C; NF- κB; Nanomicelle; Systemic delivery; Ulcerative colitis; siRNA

DOI:  https://doi.org/10.1016/j.ejpb.2021.12.009
Drug Deliv. 2022 Dec;29(1): 99-110

Ultrasound-sensitive siRNA-loaded nanobubbles fabrication and antagonism in drug resistance for NSCLC.

Chunhong Su, XiaoJun Ren, Fang Yang, Bin Li, Hao Wu, Hui Li, Fang Nie.

  Due to the lack of safe, effective, and gene-targeted delivery technology. In this study, we have prepared nanobubbles loaded PDLIM5 siRNA (PDLIM5siRNA-NBs) to investigate the transfection efficiency and their antagonism in drug resistance in combination with ultrasound irradiation for non-small-cell lung cancer (NSCLC). Research results show that the PDLIM5 siRNA are effectively bound to the shell of NBs with a mean diameter of 191.6 ± 0.50 nm and a Zeta potential of 11.8 ± 0.68 mV. And the ultrasonic imaging indicated that the PDLIM5 siRNA NBs maintain the same signals as the microbubbles (SonoVue). Under the optimized conditions of 0.5 W/m2 ultrasound intensity and 1 min irradiation duration, the highest transfection efficiency of PC9GR cells was 90.23 ± 1.45%, which resulted in the inhibition of PDLIM5 mRNA and protein expression. More importantly, the anti-tumor effect of fabricated PDLIM5siRNA-NBs with the help of ultrasound irradiation has been demonstrated to significantly inhibit tumor cell growth and promote apoptosis. Therefore, NBs carrying PDLIM5siRNA may have the potential to act as gene vectors combined with ultrasound irradiation to antagonize drug resistance for NSCLC.

Keywords:  Targeted delivery; drug resistance; nanobubbles; ultrasound image; ultrasound irradiation

DOI:  https://doi.org/10.1080/10717544.2021.2021321
J Control Release. 2021 Dec 22. pii: S0168-3659(21)00680-5. [Epub ahead of print]

Added to pre-existing inflammation, mRNA-lipid nanoparticles induce inflammation exacerbation (IE).

Hamideh Parhiz, Jacob S Brenner, Priyal Patel, Tyler E Papp, Hamna Shahnawaz, Qin Li, Ruiqi Shi, Marco Zamora, Amir Yadegari, Oscar A Marcos-Contreras, Ambika Natesan, Norbert Pardi, Vladimir V Shuvaev, Raisa Kiseleva, Jacob Myerson, Thomas Uhler, Rachel S Riley, Xuexiang Han, Michael J Mitchell, Kieu Lam, James Heyes, Drew Weissman, Vladimir Muzykantov.

  Current nucleoside-modified RNA lipid nanoparticle (modmRNA-LNP) technology has successfully paved the way for the highest clinical efficacy data from next-generation vaccinations against SARS-CoV-2 during the COVID-19 pandemic. However, such modmRNA-LNP technology has not been characterized in common pre-existing inflammatory or immune-challenged conditions, raising the risk of adverse clinical effects when administering modmRNA-LNPs to deliver therapeutic proteins or vaccinate against infectious diseases. Herein, we induce an acute-inflammation model in mice with lipopolysaccharide (LPS) intratracheally (IT), 1 mg kg-1, or intravenously (IV), 2 mg kg-1, and then IV administer modmRNA-LNP, 0.32 mg kg-1, after 4 h, and screen for inflammatory markers, such as pro-inflammatory cytokines. ModmRNA-LNP at this dose caused no significant elevation of cytokine levels in naive mice. In contrast, shortly after LPS immune stimulation, modmRNA-LNP enhanced inflammatory cytokine responses, Interleukin-6 (IL-6) in serum and Macrophage Inflammatory Protein 2 (MIP-2) in liver by 95-fold and 52-fold, respectively. Our report identifies this phenomenon as inflammation exacerbation (IE), which was proven to be specific to the LNP, acting independent of mRNA cargo, and was demonstrated to be time- and dose-dependent. Macrophage depletion and TLR3 -/- and TLR4-/- knockout mouse studies revealed macrophages were the immune cells involved or responsible for IE. Finally, we show that pretreatment with anti-inflammatory drugs, such as corticosteroids, can partially alleviate IE response in mice. Our findings characterize the importance of LNP-mediated IE phenomena in gram negative bacterial inflammation, the generalizability of modmRNA-LNP in other forms of chronic or acute inflammatory and immune contexts needs to be addressed.

Keywords:  Adverse effect; Inflammation; Lipid nanoparticle; Nanoparticle; Toxicity; mRNA

DOI:  https://doi.org/10.1016/j.jconrel.2021.12.027
Blood. 2021 Dec 27. pii: blood.2021014559. [Epub ahead of print]

Suppression of fibrin(ogen)-driven pathologies through controlled knockdown by lipid nanoparticle delivery of siRNA.

Lih Jiin Juang, Woosuk Steve Hur, Lakmali Munasinghage Silva, Amy W Strilchuk, Brenton Francisco, Jerry Leung, Madelaine K Robertson, Dafna J Groeneveld, Bridget La Prairie, Elizabeth M Chun, Andrew P Cap, James P Luyendyk, Joseph S Palumbo, Pieter R Cullis, Thomas H Bugge, Matthew J Flick, Christian J Kastrup.

Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated siRNA targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga mRNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16% and 4% of normal within one-week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with a 0.5, 1, and 2 mg/kg dose, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumour cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provide the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models.

DOI: https://doi.org/10.1182/blood.2021014559
Langmuir. 2021 Dec 25.

Physical-Chemical Characterization of Bilayer Membranes Derived from (±) α-Tocopherol-Based Gemini Lipids and Their Interaction with Phosphatidylcholine Bilayers and Lipoplex Formation with Plasmid DNA.

Bappa Maiti, Krishan Kumar, Subhasis Datta, Santanu Bhattacharya.

Membrane formation and aggregation properties of two series of (±) α-tocopherol-based cationic gemini lipids without and with hydroxyl functionalities at the headgroup region (TnS n = 3, 4, 5, 6, 8, and 12; THnS n = 4, 5, 6, 8, and 12) with varying polymethylene spacer lengths were investigated extensively while comparing with the corresponding properties of the monomeric counterparts (TM and THM). Liposomal suspensions of each cationic lipid were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), zeta potential measurements, and small-angle X-ray diffraction studies. The length of the spacer and the presence of hydroxyl functionalities at the headgroup region strongly contribute to the aggregation behavior of these gemini lipids in water. The interaction of each tocopherol lipid with a model phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)-derived vesicles, was thoroughly examined by differential scanning calorimetry (DSC) and 1,6-diphenyl-1,3,5-hexatriene (DPH)-doped fluorescence anisotropy measurements. The binding efficiency of the cationic tocopherol liposomes with plasmid DNA (pDNA) was followed by an ethidium bromide (EB) exclusion assay and zeta potential measurements, whereas negatively charged micellar sodium dodecyl sulfate (SDS)-mediated release of the pDNA from various preformed pDNA-liposomal complexes (lipoplex) was studied by an ethidium bromide (EB) reintercalation assay. The structural transformation of pDNA upon complexation with liposome was characterized using circular dichroism (CD) spectroscopic measurements. Gemini lipid-pDNA interactions depend on both the presence of hydroxyl functionalities at the headgroups and the length of the spacer chain between the headgroups. Succinctly, we performed a detailed physical-chemical characterization of the membranes formed from cationic monomeric and gemini lipids bearing tocopherol as their hydrophobic backbone and describe the role of inserting the -OH group at the headgroup of such lipids.

DOI: https://doi.org/10.1021/acs.langmuir.1c01039