bims-novged 2021-06-27 papers

on Non-viral vectors for gene delivery

Issue of 2021–06–27
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Spherical Nucleic Acids for Near-Infrared Light-Responsive Self-Delivery of Small-Interfering RNA and Antisense Oligonucleotide.
Effects of sterol derivatives in cationic liposomes on biodistribution and gene-knockdown in the lungs of mice systemically injected with siRNA lipoplexes.
Systemic delivery of mRNA and DNA to the lung using polymer-lipid nanoparticles.
Therapeutic delivery of siRNA with polymeric carriers to down-regulate STAT5A expression in high-risk B-cell acute lymphoblastic leukemia (B-ALL).
Tailoring the Architecture of Cationic Polymer Brush-Modified Carbon Nanotubes for Efficient siRNA Delivery in Cancer Immunotherapy.
Multi-component bioresponsive nanoparticles for synchronous delivery of docetaxel and TUBB3 siRNA to lung cancer cells.
Scalable Purification of Plasmid DNA Nanoparticles by Tangential Flow Filtration for Systemic Delivery.
Down-regulation of TNF-α via macrophage-targeted RNAi system for the treatment of acute inflammatory sepsis.
Surmounting tumor resistance to metallodrugs by co-loading a metal complex and siRNA in nanoparticles.
Controlled Delivery of MicroRNAs into Primary Cells Using Nanostraw Technology.
Coordination-based self-assembled capsules (SACs) for protein, CRISPR-Cas9, DNA and RNA delivery.

ACS Nano. 2021 Jun 25.

Spherical Nucleic Acids for Near-Infrared Light-Responsive Self-Delivery of Small-Interfering RNA and Antisense Oligonucleotide.

Lei Chen, Gaigai Li, Xingxing Wang, Jinbo Li, Yan Zhang.

  Herein, we developed a photolabile spherical nucleic acid (PSNA) for carrier-free and near-infrared (NIR) photocontrolled self-delivery of small-interfering RNA (siRNA) and antisense oligonucleotide (ASO). PSNA comprised a hydrophilic siRNA shell with a hydrophobic core containing a peptide nucleic acid-based ASO (pASO) and NIR photosensitizer (PS). The incorporation of a singlet oxygen (1O2)-cleavable linker between the siRNA and pASO allowed on-demand disassembly of PSNA in tumor cells once 1O2 was produced by the inner PS upon NIR light irradiation. The generated 1O2 could also concurrently promote lysosomal escape of the released siRNA and pASO to reach cytosolic targets. Both in vitro and in vivo results demonstrated that, under NIR light irradiation, PSNA could suppress hypoxia inducible factor-1α (HIF-1α) and B-cell lymphoma 2 (Bcl-2) for gene therapy (GT), which further combined photodynamic therapy (PDT) favored by the released PS to inhibit tumor cell growth. Given its carrier-free, NIR-sensitive, designable, and biocompatible merits, PSNA represents a promising self-delivery nanoplatform for cancer therapy.

Keywords:  ASO; gene therapy; near-infrared photoregulation; photodynamic therapy; self-delivery; siRNA; spherical nucleic acid

DOI:  https://doi.org/10.1021/acsnano.1c03072
Mol Med Rep. 2021 Aug;pii: 598. [Epub ahead of print]24(2):

Effects of sterol derivatives in cationic liposomes on biodistribution and gene-knockdown in the lungs of mice systemically injected with siRNA lipoplexes.

Yoshiyuki Hattori, Hiromu Saito, Teruaki Oku, Kei-Ichi Ozaki.

  Cationic liposomes can be intravenously injected to deliver short interfering (si)RNAs into the lungs. The present study investigated the effects of sterol derivatives in systemically injected siRNA/cationic liposome complexes (siRNA lipoplexes) on gene‑knockdown in the lungs of mice. Cationic liposomes composed of 1,2‑dioleoyl‑3‑trimethylammonium‑propane or dimethyldioctadecylammonium bromide (DDAB) were prepared as a cationic lipid, with sterol derivatives such as cholesterol (Chol), β‑sitosterol, ergosterol (Ergo) or stigmasterol as a neutral helper lipid. Transfected liposomal formulations composed of DDAB/Chol or DDAB/Ergo did not suppress the expression of the luciferase gene in LLC‑Luc and Colon 26‑Luc cells in vitro, whereas other formulations induced moderate gene‑silencing. The systemic injection of siRNA lipoplexes formulated with Chol or Ergo into mice resulted in abundant siRNA accumulation in the lungs. In comparison, systemically injected DDAB/Chol or DDAB/Ergo lipoplexes of Tie2 siRNA effectively increased the suppression of the Tie2 mRNA expression in the lungs of mice. These findings indicated that DDAB/Chol and DDAB/Ergo liposomes could function as vectors for siRNA delivery to the lungs.

Keywords:  cationic liposome; gene-knockdown; lung; short interfering RNA delivery; sterol derivative

DOI:  https://doi.org/10.3892/mmr.2021.12237
Biomaterials. 2021 Jun 10. pii: S0142-9612(21)00322-7. [Epub ahead of print]275 120966

Systemic delivery of mRNA and DNA to the lung using polymer-lipid nanoparticles.

James C Kaczmarek, Asha Kumari Patel, Luke H Rhym, Umberto Capasso Palmiero, Balkrishen Bhat, Michael W Heartlein, Frank DeRosa, Daniel G Anderson.

  Non-viral vectors offer the potential to deliver nucleic acids including mRNA and DNA into cells in vivo. However, designing materials that effectively deliver to target organs and then to desired compartments within the cell remains a challenge. Here we develop polymeric materials that can be optimized for either DNA transcription in the nucleus or mRNA translation in the cytosol. We synthesized poly(beta amino ester) terpolymers (PBAEs) with modular changes to monomer chemistry to investigate influence on nucleic acid delivery. We identified two PBAEs with a single monomer change as being effective for either DNA (D-90-C12-103) or mRNA (DD-90-C12-103) delivery to lung endothelium following intravenous injection in mice. Physical properties such as particle size or charge did not account for the difference in transfection efficacy. However, endosome co-localization studies revealed that D-90-C12-103 nanoparticles resided in late endosomes to a greater extent than DD-90-C12-103. We compared luciferase expression in vivo and observed that, even with nucleic acid optimized vectors, peak luminescence using mRNA was two orders of magnitude greater than pDNA in the lungs of mice following systemic delivery. This study indicates that different nucleic acids require tailored delivery vectors, and further support the potential of PBAEs as intracellular delivery materials.

Keywords:  DNA; Gene delivery; Lung; Nanoparticles; Non-viral vectors; mRNA

DOI:  https://doi.org/10.1016/j.biomaterials.2021.120966
PLoS One. 2021 ;16(6): e0251719

Therapeutic delivery of siRNA with polymeric carriers to down-regulate STAT5A expression in high-risk B-cell acute lymphoblastic leukemia (B-ALL).

Mahsa Mohseni, Cezary Kucharski, Remant Bahadur K C, Mohammad Nasrullah, Xiaoyan Jiang, Hasan Uludağ, Joseph Brandwein.

Overexpression and persistent activation of STAT5 play an important role in the development and progression of acute lymphoblastic leukemia (ALL), the most common pediatric cancer. Small interfering RNA (siRNA)-mediated downregulation of STAT5 represents a promising therapeutic approach for ALL to overcome the limitations of current treatment modalities such as high relapse rates and poor prognosis. However, to effectively transport siRNA molecules to target cells, development of potent carriers is of utmost importance to surpass hurdles of delivery. In this study, we investigated the use of lipopolymers as non-viral delivery systems derived from low molecular weight polyethylenimines (PEI) substituted with lauric acid (Lau), linoleic acid (LA) and stearic acid (StA) to deliver siRNA molecules to ALL cell lines and primary samples. Among the lipid-substituted polymers explored, Lau- and LA-substituted PEI displayed excellent siRNA delivery to SUP-B15 and RS4;11 cells. STAT5A gene expression was downregulated (36-92%) in SUP-B15 and (32%) in RS4;11 cells using the polymeric delivery systems, which consequently reduced cell growth and inhibited the formation of colonies in ALL cells. With regard to ALL primary cells, siRNA-mediated STAT5A gene silencing was observed in four of eight patient cells using our leading polymeric delivery system, 1.2PEI-Lau8, accompanied by the significant reduction in colony formation in three of eight patients. In both BCR-ABL positive and negative groups, three of five patients demonstrated marked cell growth inhibition in both MTT and trypan blue exclusion assays using 1.2PEI-Lau8/siRNA complexes in comparison with their control siRNA groups. Three patient samples did not show any positive results with our delivery systems. Differential therapeutic responses to siRNA therapy observed in different patients could result from variable genetic profiles and patient-to-patient variability in delivery. This study supports the potential of siRNA therapy and the designed lipopolymers as a delivery system in ALL therapy.

DOI: https://doi.org/10.1371/journal.pone.0251719
ACS Appl Mater Interfaces. 2021 Jun 25.

Tailoring the Architecture of Cationic Polymer Brush-Modified Carbon Nanotubes for Efficient siRNA Delivery in Cancer Immunotherapy.

Danyang Li, Momina Ahmed, Anisah Khan, Lizhou Xu, Adam A Walters, Belén Ballesteros, Khuloud T Al-Jamal.

  The facile and controlled fabrication of homogeneously grafted cationic polymers on carbon nanotubes (CNTs) remains poorly investigated, which further hinders the understanding of interactions between functionalized CNTs with different nucleic acids and the rational design of appropriate gene delivery vehicles. Herein, we describe the controlled grafting of cationic poly(2-dimethylaminoethylmethacrylate) brushes on CNTs via surface-initiated atom transfer radical polymerization integrated with mussel-inspired polydopamine chemistry. The binding of nucleic acids with different brush-CNT hybrids discloses the highly architectural-dependent behavior with dense short brush-coated CNTs displaying the highest binding among all the other hybrids, namely, dense long, sparse long, and sparse short brush-coated CNTs. Additionally, different chemistries of the brush coatings were shown to influence the biocompatibility, cellular uptake, and silencing efficiency in vitro. This platform provides great flexibility for the design of polymer brush-CNT hybrids with precise control over their structure-activity relationship for the rational design of nucleic acid delivery systems.

Keywords:  atom transfer radical polymerization; carbon nanotubes; cationic polymer brush; nucleic acid interaction; polydopamine chemistry; siRNA delivery

DOI:  https://doi.org/10.1021/acsami.1c02627
Nanoscale. 2021 Jun 23.

Multi-component bioresponsive nanoparticles for synchronous delivery of docetaxel and TUBB3 siRNA to lung cancer cells.

Claudia Conte, Patrícia F Monteiro, Pratik Gurnani, Snow Stolnik, Francesca Ungaro, Fabiana Quaglia, Philip Clarke, Anna Grabowska, Maria Kavallaris, Cameron Alexander.

Bioresponsive nanoparticles (NPs) are of interest for anticancer nanomedicines, owing to the possibility to 'design in' selective modulation of drug release at target sites. Here we describe the double emulsion formulation of redox-responsive NPs based on modified polyethylene glycol (PEG)-co-poly(lactic-co-glycolic acid) (PLGA) block copolymers and oligo (β-aminoesters) (OBAE), both of which contained disulfide linkages, for the co-delivery of a cytotoxic small molecule drug and a nucleic acid. In particular, we focused our attention on docetaxel (DTX) and a siRNA against TUBB3, a gene that encodes for βIII-tubulin, in order to have a synergistic effect in the treatment of lung cancer. Spherical NPs of around 150 nm with negative zeta potential and high loading efficiencies of both drugs were obtained. Stability and release studies showed "on demand" drug release under reducing conditions. Unloaded NPs containing PEG-disulfide-PLGA and OBAE were well-tolerated by lung cancer cells, thus masking the intrinsic cytotoxicity of OBAE, while for intracellular siRNA delivery, redox responsive NPs demonstrated a higher cell internalization with a preferential cytoplasmic accumulation of siRNA, with a subsequent fast gene-silencing efficiency. The viability of cells treated with combined DTX/TUBB3-siRNA NPs significantly decreased as compared to NPs loaded only with DTX, thus showing an efficient combined anticancer effect, due to a substantial reduction of β-tubulin expression. Finally, in an in vivo feasibility study employing an orthotopic lung cancer model, NPs formulated with an anti-luciferase siRNA distributed throughout the lungs following oro-tracheal administration, and demonstrated effective gene knockdown and no apparent cytotoxicity. Taken together, these results show that the double emulsion formulated redox responsive PEG-PLGA and OBAE systems represent a promising new therapeutic approach for the local combined chemo- and gene-therapy of lung cancer.

DOI: https://doi.org/10.1039/d1nr02179f
ACS Appl Mater Interfaces. 2021 Jun 23.

Scalable Purification of Plasmid DNA Nanoparticles by Tangential Flow Filtration for Systemic Delivery.

Heng-Wen Liu, Yizong Hu, Yong Ren, Hwanhee Nam, Jose Luis Santos, Shirley Ng, Like Gong, Mary Brummet, Christine A Carrington, Christopher G Ullman, Martin G Pomper, Il Minn, Hai-Quan Mao.

  Plasmid DNA (pDNA) nanoparticles synthesized by complexation with linear polyethylenimine (lPEI) are one of the most effective non-viral gene delivery vehicles. However, the lack of scalable and reproducible production methods and the high toxicity have hindered their clinical translation. Previously, we have developed a scalable flash nanocomplexation (FNC) technique to formulate pDNA/lPEI nanoparticles using a continuous flow process. Here, we report a tangential flow filtration (TFF)-based scalable purification method to reduce the uncomplexed lPEI concentration in the nanoparticle formulation and improve its biocompatibility. The optimized procedures achieved a 60% reduction of the uncomplexed lPEI with preservation of the nanoparticle size and morphology. Both in vitro and in vivo studies showed that the purified nanoparticles significantly reduced toxicity while maintaining transfection efficiency. TFF also allows for gradual exchange of solvents to isotonic solutions and further concentrating the nanoparticles for injection. Combining FNC production and TFF purification, we validated the purified pDNA/lPEI nanoparticles for future clinical translation of this gene nanomedicine.

Keywords:  DNA nanoparticles; biocompatibility; gene delivery; linear PEI; tangential flow filtration

DOI:  https://doi.org/10.1021/acsami.1c05750
J Control Release. 2021 Jun 17. pii: S0168-3659(21)00309-6. [Epub ahead of print]

Down-regulation of TNF-α via macrophage-targeted RNAi system for the treatment of acute inflammatory sepsis.

Jieun Lee, Wooic Son, Juhyeong Hong, Yoonsung Song, Chul-Su Yang, Yong-Hee Kim.

  Sepsis is a systemic inflammatory response syndrome caused by bacterial infection. The sepsis therapy has involved prescription of adequate antibiotics, requiring several days to determine the proper type without reducing the inflammatory response. Thus, it is necessary to rapidly decrease fundamental inflammation, which can induce serious organ damage. In the inflammatory mechanism, tumor necrosis factor-alpha (TNF-α) produced by macrophages has an important role in infiltration of macrophages into infected sites and as a trigger for secretion of pro-inflammatory cytokines. However, commercialized TNF-α antibody medicines have limits such as fibrosis, cytokine storms, and high production costs. There is a growing need for anti-inflammatory sepsis treatment free from side effects. For this reason, TNF-α converting enzyme (TACE) could be an innovative target to break the positive feedback loop of inflammatory mediators (TNF-α) since it converts the inactive TNF-α membrane bound form to the activated soluble form in macrophages. A non-viral gene delivery system was developed in this study to deliver siRNA into inflammation-mediated macrophages without toxicity. The peptide-based gene carrier created by conjugating positively-charged nine arginine (9R) and the TKPR (Thr-Lys-Pro-Arg) sequence from the Fc region of Immunoglobulin G (IgG) specifically binds to the neuropilin-1 (NRP-1) receptor on the macrophage surface. Our results demonstrated that siTACE/TKPR-9R complexes were internalized in macrophages and successfully down-regulated TACE mRNA level. Finally, RNA interference with cell-targeted peptide carriers indicates a fundamental therapy for acute inflammatory sepsis free of off-target effects.

Keywords:  Anti-inflammatory gene therapy; Combination therapy; RNA interference; Targeted gene carrier

DOI:  https://doi.org/10.1016/j.jconrel.2021.06.022
Chem Sci. 2021 Feb 09. 12(12): 4547-4556

Surmounting tumor resistance to metallodrugs by co-loading a metal complex and siRNA in nanoparticles.

Hongzhi Qiao, Lei Zhang, Dong Fang, Zhenzhu Zhu, Weijiang He, Lihong Hu, Liuqing Di, Zijian Guo, Xiaoyong Wang.

Copper complexes are promising anticancer agents widely studied to overcome tumor resistance to metal-based anticancer drugs. Nevertheless, copper complexes per se encounter drug resistance from time to time. Adenosine-5'-triphosphate (ATP)-responsive nanoparticles containing a copper complex CTND and B-cell lymphoma 2 (Bcl-2) small interfering RNA (siRNA) were constructed to cope with the resistance of cancer cells to the complex. CTND and siRNA can be released from the nanoparticles in cancer cells upon reacting with intracellular ATP. The resistance of B16F10 melanoma cells to CTND was terminated by silencing the cellular Bcl-2 gene via RNA interference, and the therapeutic efficacy was significantly enhanced. The nanoparticles triggered a cellular autophagy that amplified the apoptotic signals, thus revealing a novel mechanism for antagonizing the resistance of copper complexes. In view of the extensive association of Bcl-2 protein with cancer resistance to chemotherapeutics, this strategy may be universally applicable for overcoming the ubiquitous drug resistance to metallodrugs.

DOI: https://doi.org/10.1039/d0sc06680j
Adv Nanobiomed Res. 2021 Jun;1(6): 2000061

Controlled Delivery of MicroRNAs into Primary Cells Using Nanostraw Technology.

Mara A Pop, Benjamin D Almquist.

  MicroRNAs (miRNAs) are small noncoding RNAs that play key roles in post- transcriptional gene regulation. Being involved in regulating virtually all cellular processes, from proliferation and differentiation to migration and apoptosis, they have emerged as important epigenetic players. While most interest has gone into which miRNAs are involved in specific cellular processes or pathologies, the dosage-dependent effects of miRNAs remain vastly unexplored. Different doses of miRNAs can cause selective downregulation of target genes, in turn determining what signaling pathways and cellular responses are triggered. To explore this behavior, the effects of incremental miRNA dosage need to be studied; however, current delivery methods for miRNAs are unable to control how much miRNA enters a cell. Herein, an approach is presented based on a nanostrawelectroporation delivery platform that decouples the delivery from biological mechanisms (e.g., endocytosis) to enable precise control over the amount of miRNA delivered, along with demonstrating ratiometric intracellular delivery into primary dermal fibroblasts for miR-181a and miR-27a. In addition, it is shown that the nanostraw delivery platform allows efficient delivery of miRNAs into primary keratinocytes, opening new opportunities for successful miRNA delivery into this hard-to-transfect cell type.

Keywords:  controlled delivery; epigenetics; microRNA; nanobiotechnology; nanostraw

DOI:  https://doi.org/10.1002/anbr.202000061
Chem Sci. 2021 Feb 01. 12(7): 2329-2344

Coordination-based self-assembled capsules (SACs) for protein, CRISPR-Cas9, DNA and RNA delivery.

Lukman O Alimi, Mram Z Alyami, Santanu Chand, Walaa Baslyman, Niveen M Khashab.

Biologics, such as functional proteins and nucleic acids, have recently dominated the drug market and comprise seven out of the top 10 best-selling drugs. Biologics are usually polar, heat sensitive, membrane impermeable and subject to enzymatic degradation and thus require systemic routes of administration and delivery. Coordination-based delivery vehicles, which include nanosized extended metal-organic frameworks (nMOFs) and discrete coordination cages, have gained a lot of attention because of their remarkable biocompatibility, in vivo stability, on-demand biodegradability, high encapsulation efficiency, easy surface modification and moderate synthetic conditions. Consequently, these systems have been extensively utilized as carriers of biomacromolecules for biomedical applications. This review summarizes the recent applications of nMOFs and coordination cages for protein, CRISPR-Cas9, DNA and RNA delivery. We also highlight the progress and challenges of coordination-based platforms as a promising approach towards clinical biomacromolecule delivery and discuss integral future research directions and applications.

DOI: https://doi.org/10.1039/d0sc05975g