bims-novged 2022-11-20 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2022–11–20
ten papers selected by
the Merkel lab, Ludwig-Maximilians University

A novel peptide ligand-coated nano-siRNA-lipoplex technology for kidney targeted gene therapy.
The engineering challenges and opportunities when designing potent ionizable materials for the delivery of ribonucleic acids.
A zwitterionic polymer-inspired material mediated efficient CRISPR-Cas9 gene editing.
Lipid nanoparticles (LNP) induce activation and maturation of antigen presenting cells in young and aged individuals.
Mannosylated Polycations Target CD206+ Antigen-Presenting Cells and Mediate T-Cell-Specific Activation in Cancer Vaccination.
A simple self-assembly nanomicelle based on brain tumor-targeting peptide-mediated siRNA delivery for glioma immunotherapy via intranasal administration.
CNS Delivery of Nucleic Acid Therapeutics: Beyond the Blood-Brain Barrier and Towards Specific Cellular Targeting.
Preparation and characterization of cationic hyperbranched maltodextrins as potential carrier for siRNA encapsulation.
Characterisation of niosome nanoparticles prepared by microfluidic mixing for drug delivery.
Cell surface biotinylation to identify the receptors involved in nanoparticle uptake into endothelial cells.

Am J Transl Res. 2022 ;14(10): 7362-7377

A novel peptide ligand-coated nano-siRNA-lipoplex technology for kidney targeted gene therapy.

Xia Liu, Jiandong Zhang, Anna Tang, Liang Xu, Yufeng Huang.

OBJECTIVES: Small interfering RNA (siRNA) that silences specific disease-related genes holds the promise for the treatment of renal disease. However, delivery to the intended site of action remains a major obstacle. The goal of this study was to develop glomerulus-specific siRNA particles for targeted gene therapy of kidney diseases.
METHODS: We used a novel nanoparticle-based system comprised of siRNA in cationic liposomes (Lip) coated with non-inhibitory plasminogen activator inhibitor 1R (PAI-1R) that selectively targets glomerular cells and tested it with transforming growth factor-beta 1 (TGF-β1)-siRNA in nephritic rat model.
RESULTS: At the optimized ratio of components, three of PAI-1R, Lip and siRNA formed the compact nanostructured particles with close to neutral surface charge (+5.63 ± 1.45 mV) and relatively uniform size (68.9 ± 4.73 nm). When the fluorescence-conjugated siRNA was used, the labeled siRNA nanoparticles appeared specifically in glomeruli. Targeted delivery of siRNA specific to the TGFβ1 gene reduced elevated TGFβ1 mRNA expression and protein production in glomeruli, but had no effect on TGFβ1 mRNA levels in lung, spleen, artery or renal medulla, and in nephritic rats induced by injection of OX-7, for up to 5 days. PAI-1R-Lip-TGF-β1 siRNA administration significantly reduced increases in glomerular matrix accumulation and expression of PAI-1 and fibronectin.
CONCLUSIONS: We conclude that a single dose of PAI-1R-Lip-TGF-β1 siRNA inhibited glomerular TGF-β1 gene expression thereby ameliorating glomerulosclerosis specifically and efficiently in nephritic rats without affecting most of other organs. The target silencing of genes critical for glomerular diseases may represent a promising treatment strategy for kidney disease.

Keywords: Nanoparticles; gene therapy; glomerulosclerosis; kidney
Expert Opin Drug Deliv. 2022 Nov 15.

The engineering challenges and opportunities when designing potent ionizable materials for the delivery of ribonucleic acids.

Yan Ming Anson Lau, Janice Pang, Grayson Tilstra, Julien Couture-Senécal, Omar F Khan.

   INTRODUCTION: Ionizable lipids are critical components in lipid nanoparticles. These molecules sequester nucleic acids for delivery to cells. However, to build more efficacious delivery molecules, the field must continue to broaden structure-function studies for greater insight. While nucleic acid binding efficiency, degradability and nanoparticle stability are vitally important, this review offers perspective on additional factors that must be addressed to improve delivery efficiency.
AREAS COVERED: We discuss how administration route, cellular heterogeneity, uptake pathway, endosomal escape timing, age, sex and threshold effects can change depending on the type of LNP ionizable lipid.
EXPERT OPINION: Ionizable lipid structure-function studies often focus on the efficiency of RNA utilization and biodistribution. While these focus areas are critical, they remain high-level observations. As our tools for observation and system interrogation improve, we believe that the field should begin collecting additional data. On the cellular level, this data should include age (dividing or senescent cells), sex and phenotype, cell entry pathway, and endosome type. Additionally, administration route and dose are essential to track. This additional data will allow us to identify and understand heterogeneity in LNP efficacy across patient populations, which will help us provide better ionizable lipid options for different groups.

Keywords:  Endosomal escape; Functional delivery; Ionizable lipid; LNP; Lipid nanoparticle; Patient stratification; RNA; Ribonucleic acid; Structure-function; Uptake mechanism

DOI:  https://doi.org/10.1080/17425247.2022.2144827
Asian J Pharm Sci. 2022 Aug;17(5): 666-678

A zwitterionic polymer-inspired material mediated efficient CRISPR-Cas9 gene editing.

Lingmin Zhang, Langyu Yang, Jionghua Huang, Sheng Chen, Chuangjia Huang, Yinshan Lin, Ao Shen, ZhouYikang Zheng, Wenfu Zheng, Shunqing Tang.

  The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR/Cas9) adaptive immune system is a cutting-edge genome-editing toolbox. However, its applications are still limited by its inefficient transduction. Herein, we present a novel gene vector, the zwitterionic polymer-inspired material with branched structure (ZEBRA) for efficient CRISPR/Cas9 delivery. Polo-like kinase 1 (PLK1) acts as a master regulator of mitosis and overexpresses in multiple tumor cells. The Cas9 and single guide sgRNA (sgRNA)-encoded plasmid was transduced to knockout Plk1 gene, which was expected to inhibit the expression of PLK1. Our studies demonstrated that ZEBRA enabled to transduce the CRISPR/Cas9 system with large size into the cells efficiently. The transduction with ZEBRA was cell line dependent, which showed ∼10-fold higher in CD44-positive cancer cell lines compared with CD44-negative ones. Furthermore, ZEBRA induced high-level expression of Cas9 proteins by the delivery of CRISPR/Cas9 and efficient gene editing of Plk1 gene, and inhibited the tumor cell growth significantly. This zwitterionic polymer-inspired material is an effective and targeted gene delivery vector and further studies are required to explore its potential in gene delivery applications.

Keywords:  CD44; CRISPR/Cas9; Gene editing; PLK1; Zwitterionic polymers

DOI:  https://doi.org/10.1016/j.ajps.2022.08.001
Res Sq. 2022 Nov 07. pii: rs.3.rs-2199652. [Epub ahead of print]

Lipid nanoparticles (LNP) induce activation and maturation of antigen presenting cells in young and aged individuals.

Jennifer Connors, David Joyner, Nathan Mege, Gina Cusimano, Matthew Bell, Jennifer Marcy, Bhavani Taramangalam, Paulo Lin, Ying Tam, Paulo Lin, Drew Weissman, Michele Kutzler, Mohamad-Gabriel Alameh, Elias Haddad.

Despite the overwhelming success of mRNA-based vaccine in protecting against SARS-CoV-2 infection and reducing disease severity and hospitalization, little is known about the role lipid nanoparticles (LNP) play in initiating immune response. In this report we studied the adjuvantive impact of empty LNP with no mRNA cargo (eLNP) on anti-viral pathways and immune function of cells from young and aged individuals. We found that eLNP induced maturation of monocyte derived dendritic cells by measuring the expression of CD40, CD80, HLA-DR and production of cytokines including IFN-α,IL-6, IFN-γ, IL-12, and IL-21. Flow cytometry analysis of specific dendritic cell subsets showed that eLNP can induce CD40 expression and cytokine production in cDC1, cDC2 and monocytes. Empty LNP (eLNP) effects on dendritic cells and monocytes coincided with induction pIRF7 and pTBK1, which are both important in mitigating innate immune signaling. Interestingly our data show that in response to eLNP stimulus at 6 and 24 hrs, aged individuals have decreased CD40 expression and reduced IFN- γ output compared to young adults. Furthermore, we show that cDC1, cDC2, and CD14 dim CD16 + monocytes from healthy aged individuals have dysregulated anti-viral signaling response to eLNP stimulation as measured by the defect in type I IFN production, phosphorylation of IRF7, TBK-1, and immune function like phagocytosis. These data showed a novel function of eLNP in eliciting DC maturation and innate immune signaling pathways and that some of these functions are impaired in older individuals providing some suggestion of why older individuals (> 65 yrs of age) respond display lower immune responses and adverse events to SARS-CoV-2 mRNA-based vaccines.

DOI: https://doi.org/10.21203/rs.3.rs-2199652/v1
Biomacromolecules. 2022 Nov 17.

Mannosylated Polycations Target CD206+ Antigen-Presenting Cells and Mediate T-Cell-Specific Activation in Cancer Vaccination.

Federica Bellato, Sara Feola, Gloria Dalla Verde, Greta Bellio, Marco Pirazzini, Stefano Salmaso, Paolo Caliceti, Vincenzo Cerullo, Francesca Mastrotto.

Immunotherapy is deemed one of the most powerful therapeutic approaches to treat cancer. However, limited response and tumor specificity are still major challenges to address. Herein, mannosylated polycations targeting mannose receptor- are developed as vectors for plasmid DNA (pDNA)-based vaccines to improve selective delivery of genetic material to antigen-presenting cells and enhance immune cell activation. Three diblock glycopolycations (M15A12, M29A25, and M58A45) and two triblock copolymers (M29A29B9 and M62A52B32) are generated by using mannose (M), agmatine (A), and butyl (B) derivatives to target CD206, complex nucleic acids, and favor the endosomal escape, respectively. All glycopolycations efficiently complex pDNA at N/P ratios <5, protecting the pDNA from degradation in a physiological milieu. M58A45 and M62A52B32 complexed with plasmid encoding for antigenic ovalbumin (pOVA) trigger the immune activation of cultured dendritic cells, which present the SIINFEKL antigenic peptide via specific major histocompatibility complex-I. Importantly, administration of M58A45/pOVA elicits SIINFEKL-specific T-cell response in C56BL/6 mice bearing the melanoma tumor model B16-OVA, well in line with a reduction in tumor growth. These results qualify mannosylation as an efficient strategy to target immune cells in cancer vaccination and emphasize the potential of these glycopolycations as effective delivery vehicles for nucleic acids.

DOI: https://doi.org/10.1021/acs.biomac.2c00993
Acta Biomater. 2022 Nov 13. pii: S1742-7061(22)00740-1. [Epub ahead of print]

A simple self-assembly nanomicelle based on brain tumor-targeting peptide-mediated siRNA delivery for glioma immunotherapy via intranasal administration.

Lin Tang, Rui Zhang, Yusi Wang, Xiaoyu Zhang, Yuling Yang, Binyan Zhao, Li Yang.

  The blood-brain barrier (BBB) has a key role in preventing drugs from entering the brain. Non-invasive intranasal drug delivery routes that bypass the BBB are increasing in popularity because of their ability to shorten the journey and reduce the loss of genetic drugs such as siRNA in transit. However, the complex synthesis and quality control process of most nose-to-brain delivery carriers and the limited mass production are the main obstacles to their clinical application. Here, we constructed a siRNA delivery system with simple synthesis and quality control methods using cholesterol-modified T7 (T7-C), in which T7 can bind to the transferrin receptor (TfR) expressed on glioma cells to target gliomas. In our results, T7-C had dual functions as a glioma-targeting carrier and immune adjuvant. As a targeted delivery carrier, T7-C intranasally delivered siRNA into the mouse brain through the olfactory bulb pathway and was taken up by glioma cells by the caveolin- and transferrin-dependent pathway. As an immune adjuvant, T7-C could promote DC maturation and combined with slit2 siRNA could promote polarization of M2 subtype macrophages to M1 subtype macrophages and then increase the proportion of effector T cells to remodel the tumor environment. In conclusion, T7-C with glioma targeting as a delivery system of slit2 siRNA showed a good therapeutic effect in the treatment of glioma after intranasal administration and had potential application prospects. STATEMENT OF SIGNIFICANCE: In contrast to the existing literature that uses complex materials to deliver drugs across the blood-brain barrier (BBB) in an invasive manner for glioma treatment, we developed a simple, self-assembling siRNA delivery system (T7-C) based on brain tumor-targeted T7 peptide to treat glioma by intranasal administration. T7-C/siRNA could reach the tumor site through the olfactory bulb route and adjust the "cold" tumor microenvironment to the "hot" tumor microenvironment and non-invasive intranasal delivery route could shorten the journey and reduce the loss of genetic drugs. Therefore, our design has good application prospects and is expected to serve as a general strategy for intranasal drug delivery in the treatment of brain tumors.

Keywords:  Glioma; Intranasal administration; T7-C; Tumor microenvironment; siRNA

DOI:  https://doi.org/10.1016/j.actbio.2022.11.013
Pharm Res. 2022 Nov 15.

CNS Delivery of Nucleic Acid Therapeutics: Beyond the Blood-Brain Barrier and Towards Specific Cellular Targeting.

Anisha D'Souza, Saeideh Nozohouri, Benjamin S Bleier, Mansoor M Amiji.

  Nucleic acid-based therapeutic molecules including small interfering RNA (siRNA), microRNA(miRNA), antisense oligonucleotides (ASOs), messenger RNA (mRNA), and DNA-based gene therapy have tremendous potential for treating diseases in the central nervous system (CNS). However, achieving clinically meaningful delivery to the brain and particularly to target cells and sub-cellular compartments is typically very challenging. Mediating cell-specific delivery in the CNS would be a crucial advance that mitigates off-target effects and toxicities. In this review, we describe these challenges and provide contemporary evidence of advances in cellular and sub-cellular delivery using a variety of delivery mechanisms and alternative routes of administration, including the nose-to-brain approach. Strategies to achieve subcellular localization, endosomal escape, cytosolic bioavailability, and nuclear transfer are also discussed. Ultimately, there are still many challenges to translating these experimental strategies into effective and clinically viable approaches for treating patients.

Keywords:  CNS delivery; blood–brain barrier; cell-specificity; nucleic acid therapeutics

DOI:  https://doi.org/10.1007/s11095-022-03433-5
Int J Biol Macromol. 2022 Nov 15. pii: S0141-8130(22)02701-5. [Epub ahead of print]

Preparation and characterization of cationic hyperbranched maltodextrins as potential carrier for siRNA encapsulation.

Yue Sun, Li Cheng, Yan Hong, Zhaofeng Li, Caiming Li, Xiaofeng Ban, Zhengbiao Gu.

  The present study sought to investigate the physicochemical properties of cationic branched maltodextrins with similar degrees of substitution but different degrees of branching and their siRNA delivery capacity. The results showed that the ratio of α-1,6 glycosidic bonds was significantly increased in the sample treated with dual enzymes. The structural characterization results showed that abundant short chains reassembled by 1,4-α-glucan branching enzyme (GBEs) hydrolysis formed hyperbranched short clustered structure. The absorption peaks that appeared in the FT-IR spectrum confirmed the occurrence of quaternization. The complexes formed by self-assembly of cationic maltodextrins and siRNA were verified by the gel retardation assay and atomic force microscopy, demonstrating a uniform spherical structure with a size close to 300-350 nm. Meanwhile, cationic branched maltodextrins could effectively reduce the change of secondary structure of siRNA. Overall, the results suggested that branched maltodextrins with a cationic surface had significant potential as siRNA carriers.

Keywords:  Cationic modification; Highly branched maltodextrins; Nanoparticles; Self-assembly complexes; siRNA deliver

DOI:  https://doi.org/10.1016/j.ijbiomac.2022.11.142
Int J Pharm X. 2022 Dec;4 100137

Characterisation of niosome nanoparticles prepared by microfluidic mixing for drug delivery.

Mohammad A Obeid, Ibrahim Khadra, Alaa A A Aljabali, Haneen Amawi, Valerie A Ferro.

  Lipid nanoparticles have gained much attention due to their potential as drug delivery systems. They are safe, effective, and be targeted to particular tissues to deliver their payload. Niosomes are one type of lipid nanoparticles that comprise non-ionic surfactants which have proven to be effective due to their stability and biocompatibility. Different manufacturing processes have been reported for niosome preparation, but many of them are not scalable or reproducible for pharmaceutical use. In this study, microfluidic mixing was used to prepare niosomes with different lipid compositions by changing the type of non-ionic surfactant. Niosomes were evaluated for their physicochemical characteristics, morphology, encapsulation efficacy, release profiles of atenolol as a model hydrophilic compound, and cytotoxic activities. Microfluidic mixing allows for particle self-assembly and drug loading in a single step, without the need for post-preparation size reduction. Depending on the lipid composition, the empty particles were <90 nm in size with a uniform distribution. A slight but not significant increase in these values was observed when loading atenolol in most of the prepared formulations. All formulations were spherical and achieved variable levels of atenolol encapsulation. Atenolol release was slow and followed the Korsmeyer-Peppas model regardless of the surfactant type or the percentage of cholesterol used.

Keywords:  Atenolol; Drug delivery; Drug release; Microfluidic mixing; Niosomes

DOI:  https://doi.org/10.1016/j.ijpx.2022.100137
Acta Biomater. 2022 Nov 09. pii: S1742-7061(22)00738-3. [Epub ahead of print]

Cell surface biotinylation to identify the receptors involved in nanoparticle uptake into endothelial cells.

Aldy Aliyandi, Catharina Reker Smit, Inge S Zuhorn, Anna Salvati.

  Targeted drug delivery requires -among others- specific interaction of nanocarriers with cell surface receptors enabling efficient internalization into the targeted cells. Thus, identification of receptors allowing efficient nanocarrier uptake is essential to improve the design of targeted nanomedicines. Here we used methods based on cell surface biotinylation to identify cell surface receptors mediating nanoparticle uptake by cells. We used human brain and liver endothelial cells, as representative examples of cells typically showing very low and very high nanoparticle uptake, respectively. Amino-modified and carboxylated silica were used as model nanoparticles that typically show high and low uptake into cells, respectively, and that carry different coronas after exposure in full human plasma. Using cell surface biotinylation of live cells and receptor pull-down assays, we compared the receptors internalized in control untreated cells and those internalized upon exposure to nanoparticles. In this way, we identified receptors associated with (high) nanoparticle uptake. The candidate receptors were further validated by decorating the nanoparticles with an artificial corona consisting of the respective receptor ligands. We found that a vitronectin corona can be used to target integrin receptors and strongly enhances nanoparticle uptake in brain and liver endothelial cells. The increased uptake was maintained in the presence of serum, suggesting that the vitronectin-corona could resist interaction and competition with serum. Furthermore, plasminogen-coated nanoparticles promoted uptake in endothelial cells of the liver, but not of the brain. The presented approach using reversible biotinylation of cell surface receptors in live cells allows for the identification of receptors that are instrumental in nanoparticle uptake, which can be exploited for targeted drug delivery. STATEMENT OF SIGNIFICANCE: In order to deliver drugs to their site of action, drug-loaded nanocarriers can be targeted to cell receptors enabling efficient uptake into target cells. Thus, methods to identify nanocarrier receptors are invaluable. Here we used reversible biotinylation of live cells and receptor pull down approaches for receptor identification. By comparative analysis of the individual receptors internalized in untreated cells and cells exposed to nanoparticles, we identified receptors enabling high nanoparticle uptake into liver and brain endothelial cells. Their role was confirmed by decorating nanoparticles with an artificial corona composed of the receptor ligands. In conclusion, live cell reversible biotinylation of cell receptors is a powerful tool for the identification of potential receptors for receptor-based targeting of nanocarriers.

Keywords:  Cellular receptors; Endothelial cells; Protein corona; Reversible biotinylation; Targeted drug delivery

DOI:  https://doi.org/10.1016/j.actbio.2022.11.010