bims-novged 2022-04-17 papers

on Non-viral vectors for gene delivery

Issue of 2022–04–17
thirteen papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Microfluidic-Based Cationic Cholesterol Lipid siRNA Delivery Nanosystem: Highly Efficient In Vitro Gene Silencing and the Intracellular Behavior.
A cationic cyclodextrin derivative-lipid hybrid nanoparticles for gene delivery effectively promotes stability and transfection efficiency.
Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device.
Rational design of engineered H-ferritin nanoparticles with improved siRNA delivery efficacy across an in vitro model of the mouse BBB.
Co-delivery of doxorubicin and CRISPR/Cas9 or RNAi-expressing plasmid by chitosan-based nanoparticle for cancer therapy.
pH-Responsive hyaluronic acid-cloaked polycation/gold nanohybrids for tumor-targeted synergistic photothermal/gene therapy.
Hydrophilic Random Cationic Copolymers as Polyplex-Formation Vectors for DNA.
A Robust Post-Insertion Method for the Preparation of Targeted siRNA LNPs.
Enhanced Gene Delivery Triggered by Dual pH/Redox Responsive Host-Guest Dimerization of Cyclooligosaccharide Star Polycations.
Modulation of Chitosan-TPP Nanoparticle Properties for Plasmid DNA Vaccines Delivery.
Albumin-Binding Aptamer Chimeras for Improved siRNA Bioavailability.
Current trends in delivery of non-viral nucleic acid-based therapeutics for improved efficacy.
Smartly responsive DNA-miRNA hybrids packaged in exosomes for synergistic enhancement of cancer cell apoptosis.

Int J Mol Sci. 2022 Apr 03. pii: 3999. [Epub ahead of print]23(7):

Microfluidic-Based Cationic Cholesterol Lipid siRNA Delivery Nanosystem: Highly Efficient In Vitro Gene Silencing and the Intracellular Behavior.

Zhaoyuan Zhu, Li Zhang, Ruilong Sheng, Jian Chen.

  Safe and efficient delivery of small interfering RNA (siRNA) is essential to gene therapy towards intervention of genetic diseases. Herein, we developed a novel cationic cholesterol lipid derivative (CEL) in which cholesterol hydrophobic skeleton was connected to L-lysine cationic headgroup via a hexanediol linker as the non-viral siRNA delivery carrier. Well-organized CEL/siRNA nanocomplexes (100-200 nm) were prepared by microfluidic-assisted assembly of CEL and siRNA at various N/P ratios. The CEL and CEL/siRNA nanocomplexes have lower cytotoxicity compared with bPEI25k. Delightfully, we disclosed that, in Hela-Luc and H1299-Luc cell lines, the micro-fluidic-based CEL/siRNA nanocomplexes exhibited high siRNA transfection efficiency under both serum-free condition (74-98%) and low-serum circumstances (80-87%), higher than that of lipofectamine 2000. These nanocomplexes also showed high cellular uptake through the caveolae/lipid-raft mediated endocytosis pathway, which may greatly contribute to transfection efficiency. Moreover, the time-dependent (0-12 h) dynamic intracellular imaging demonstrated the efficient delivery to cytoplasm after lysosomal co-localization. The results indicated that the microfluidic-based CEL/siRNA nanosystems possessed good stability, low cytotoxicity, high siRNA delivery efficiency, rapid cellular uptake and caveolae/lipid raft-dependent internalization. Additionally, this study provides a simple approach for preparing and applying a "helper lipid-free" cationic lipid siRNA delivery system as potential nanotherapeutics towards gene silencing treatment of (tumor) diseases.

Keywords:  cationic cholesterol lipid; endocytosis pathway; gene silencing; microfluidics; siRNA delivery

DOI:  https://doi.org/10.3390/ijms23073999
Drug Dev Ind Pharm. 2022 Apr 12. 1-15

A cationic cyclodextrin derivative-lipid hybrid nanoparticles for gene delivery effectively promotes stability and transfection efficiency.

Zhongjuan Wang, Shaobin Xu, Hongying Xia, Yanqiu Liu, Bin Li, Yueqin Liang, Zhongkun Li.

  Genetic medicines hold great promise for treatment of a number of diseases, however, the development of effective gene delivery carrier is still a challenge. The commonly used gene carrier liposomes and cationic polymers have limited their clinical application due to their respective disadvantages. Lipid-polymer hybrid nanoparticles (LHNPs) are novel drug delivery system which exhibit complementary characteristics of both polymeric nanoparticles and liposomes. In this account, we developed the α-cyclodextrin conjugated generation-2 polyamidoamine dendrimers-lipids hybrid nanoparticles (CDG2-LHNPs) for gene delivery. The pDNA/CDG2-LHNPs was stable during 15 days storage period both at 4 °C, 25 °C and 37 °C, whereas the particle size of pDNA/CDG2 and pDNA/liposomes dramatically increased after storage at 4 °C for 8 h. CDG2-LHNPs showed significantly superior transfection efficiencies compared to either CDG2 or liposomes. The mechanism of high transfection efficiency of pDNA/CDG2-LHNPs was further explored using pharmacological inhibitors chlorpromazine, filipin and cytochalasion D. The result demonstrated that cell uptake of pDNA/CDG2-LHNPs was mediated by clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME) and macropinocytosis together. pDNA/CDG2-LHNPs were more likely be taken up by cells through CVME, which avoided lysosomal degradation to a large extent. Moreover, the liposome component of pDNA/CDG2-LHNPs increased its cell uptake efficiency, and the CDG2 polymer component increased its proton buffer capacity, so the hybrid nanoparticles taken up by CME could also successfully escape from the lysosome. This CDG2-LHNPs with stability and high transfection efficiency overcome the shortcomings of liposomes and polymers applied separately, and has great potential for gene drug delivery.

Keywords:  cyclodextrin; gene delivery; high transfection efficiency; hybrid nanoparticles; stability

DOI:  https://doi.org/10.1080/03639045.2022.2059499
J Vis Exp. 2022 Mar 22.

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device.

Masatoshi Maeki, Yuto Okada, Shuya Uno, Ayuka Niwa, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi.

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

DOI: https://doi.org/10.3791/62999
Nanoscale. 2022 Apr 13.

Rational design of engineered H-ferritin nanoparticles with improved siRNA delivery efficacy across an in vitro model of the mouse BBB.

Ziwei Yuan, Bin Wang, Yilong Teng, William Ho, Bin Hu, Kofi Oti Boakye-Yiadom, Xiaoyang Xu, Xue-Qing Zhang.

Gene therapy holds tremendous potential for the treatment of incurable brain diseases including Alzheimer's disease (AD), stroke, glioma, and Parkinson's disease. The main challenge is the lack of effective gene delivery systems traversing the blood-brain barrier (BBB), due to the complex microvessels present in the brain which restrict substances from the circulating blood passing through. Recently, increasing efforts have been made to develop promising gene carriers for brain-related disease therapies. One such development is the self-assembled heavy chain ferritin (HFn) nanoparticles (NPs). HFn NPs have a unique hollow spherical structure that can encapsulate nucleic acid drugs (NADs) and specifically bind to cancer cells and BBB endothelial cells (BBB ECs) via interactions with the transferrin receptor 1 (TfR1) overexpressed on their surfaces, which increases uptake through the BBB. However, the gene-loading capacity of HFn is restricted by its limited interior volume and negatively charged inner surface; therefore, these drawbacks have prompted the demand for strategies to remould the structure of HFn. In this work, we analyzed the three-dimensional (3D) structure of HFn using Chimera software (v 1.14) and developed a class of internally cationic HFn variants (HFn+ NPs) through arginine mutation on the lumenal surface of HFn. These HFn+ NPs presented powerful electrostatic forces in their cavities, and exhibited higher gene encapsulation efficacy than naive HFn. The top-performing candidate, HFn2, effectively delivered siRNA to glioma cells after traversing the BBB and achieved the highest silencing efficacy among HFn+ NPs. Overall, our findings demonstrate that HFn+ NPs obtained by this genetic engineering method provide critical insights into the future development of nucleic acid delivery carriers with BBB-crossing ability.

DOI: https://doi.org/10.1039/d1nr07880a
Carbohydr Polym. 2022 Jul 01. pii: S0144-8617(22)00219-3. [Epub ahead of print]287 119315

Co-delivery of doxorubicin and CRISPR/Cas9 or RNAi-expressing plasmid by chitosan-based nanoparticle for cancer therapy.

Qiuping Li, Xinrong Lv, Cui Tang, Chunhua Yin.

  Folic acid (FA) and 2-(Diisopropylamino) ethyl methacrylate (DPA) double grafted trimethyl chitosan (TMC) nanoparticles (FTD NPs) were synthesized for the co-delivery of doxorubicin (DOX) and Survivin CRISPR/Cas9-expressing plasmid (sgSurvivin pDNA) or Survivin shRNA-expressing plasmid (iSur pDNA). FA modification enhanced the uptake of DOX and pDNA loaded into FTD NPs in tumor cells. A rapid release of DOX was triggered under acidic conditions due to pH-sensitiveness of FTD NPs arising from DPA conjugation. Negligible differences between FTD/sgSurvivin pDNA NPs and FTD/iSur pDNA NPs demonstrated that RNA interference (RNAi) and CRISPR/Cas9 technologies possessed comparable antitumor efficiency. Notably, the in vitro and in vivo antitumor efficacies of FTD/DOX/sgSurvivin pDNA NPs were superior to those of single delivery of DOX or sgSurvivin pDNA, while were comparable to those of FTD/DOX/iSur pDNA NPs. These results suggested that the combination of chemotherapeutics and CRISPR/Cas9 systems would provide a potential modality for cancer therapy.

Keywords:  CRISPR/Cas9; Chitosan-based nanoparticles; FA modification; RNAi; Synergistic antitumor effects; pH-responsiveness

DOI:  https://doi.org/10.1016/j.carbpol.2022.119315
Biomater Sci. 2022 Apr 12.

pH-Responsive hyaluronic acid-cloaked polycation/gold nanohybrids for tumor-targeted synergistic photothermal/gene therapy.

Yanjun Liu, Xiaoguang Dai, Bingran Yu, Meiwan Chen, Nana Zhao, Fu-Jian Xu.

The combination of photothermal therapy (PTT) and gene therapy (GT) has attracted intense interest in cancer treatment. However, the lack of long circulation and active tumor targeting reduces the therapeutic efficacy of complementary PTT/GT. In this work, hyaluronic acid (HA)-cloaked gold nanorods-PGED (prepared by ring-opening of polyglycidyl methacrylate (PGMA) with ethylenediamine (ED))/pDNA (AP/pDNA-HA) complexes were prepared to achieve long circulation and tumor targeting for photoacoustic imaging (PAI)-guided synergistic PTT/GT. Gold nanorods endow the complexes with photothermal effect and PAI function. Benefiting from the HA cloak, the AP/pDNA-HA complexes exhibit excellent stability, biocompatibility, long circulation behavior and active targeting. In addition, the pH-responsive characteristic of the Schiff base bonds helps the AP/pDNA-HA complexes to effectively escape from the endosome/lysosome. The antioncogene p53 was employed to investigate the gene transfection efficiency of the delivery system both in vitro and in vivo. The superiority of synergistic PTT/GT is established in a mouse 4T1 breast tumor model. The current study provides a facile strategy for constructing multifunctional gene delivery systems with long circulation and tumor targeting features, which can achieve effective imaging-guided synergistic tumor treatment.

DOI: https://doi.org/10.1039/d2bm00296e
Materials (Basel). 2022 Apr 04. pii: 2650. [Epub ahead of print]15(7):

Hydrophilic Random Cationic Copolymers as Polyplex-Formation Vectors for DNA.

Varvara Chrysostomou, Hector Katifelis, Maria Gazouli, Konstantinos Dimas, Costas Demetzos, Stergios Pispas.

  Research on the improvement and fabrication of polymeric systems as non-viral gene delivery carriers is required for their implementation in gene therapy. Random copolymers have not been extensively utilized for these purposes. In this regard, double hydrophilic poly[(2-(dimethylamino) ethyl methacrylate)-co-(oligo(ethylene glycol) methyl ether methacrylate] [P(DMAEMA-co-OEGMA)] random copolymers were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The copolymers were further modified by quaternization of DMAEMA tertiary amine, producing the cationic P(QDMAEMA-co-OEGMA) derivatives. Fluorescence and ultraviolet-visible (UV-vis) spectroscopy revealed the efficient interaction of copolymers aggregates with linear DNAs of different lengths, forming polyplexes, with the quaternized copolymer aggregates exhibiting stronger binding affinity. Light scattering techniques evidenced the formation of polyplexes whose size, molar mass, and surface charge strongly depend on the N/P ratio (nitrogen (N) of the amine group of DMAEMA/QDMAEMA over phosphate (P) groups of DNA), DNA length, and length of the OEGMA chain. Polyplexes presented colloidal stability under physiological ionic strength as shown by dynamic light scattering. In vitro cytotoxicity of the empty nanocarriers was evaluated on HEK293 as a control cell line. P(DMAEMA-co-OEGMA) copolymer aggregates were further assessed for their biocompatibility on 4T1, MDA-MB-231, MCF-7, and T47D breast cancer cell lines presenting high cell viability rates.

Keywords:  DNA; RAFT polymerization; cationic polymers; gene delivery; in vitro cytotoxicity; non-viral vectors; nucleic acids; polyplexes; random copolymers

DOI:  https://doi.org/10.3390/ma15072650
Int J Pharm. 2022 Apr 11. pii: S0378-5173(22)00296-4. [Epub ahead of print] 121741

A Robust Post-Insertion Method for the Preparation of Targeted siRNA LNPs.

L E Swart, C A Koekman, C W Seinen, H Issa, M Rasouli, R M Schiffelers, O Heidenreich.

  Targeted delivery of nucleic acids is gaining momentum due to improved efficacy, selectivity, increased circulation time and enhanced tissue retention in target cells. Using nucleic acid-based therapies previously undruggable targets have proven now to be amenable for treatment. Currently, several methods for preparing targeted or labelled delivery vehicles for nucleic acids are based on liposomal formulations. Lipid nanoparticles (LNPs) are structurally different from liposomes and these methods should therefore be evaluated before being translated to siRNA LNPs preparation protocols. Here, we describe a robust and facile method for the preparation of targeted or fluorescently labelled siRNA LNPs. Using a copper free strain-promoted azide-alkyne cycloaddition (SPAAC) we demonstrate that post-insertion of ligand-lipid conjugates into preformed LNPs is superior to direct-surface modification because it preserves the physicochemical parameters of the LNPs. We found that the time point of solvent removal by dialysis is critical and affects the hydrodynamic diameter of the LNPs; post-insertion after dialysis shows the smallest increase in hydrodynamic diameter and polydispersity index (PDI). The post-insertion of ligand-lipid conjugates also proceeded with rapid kinetics and high efficacy over a wide temperature range. Using this optimised protocol, we generated siRNA LNPs containing both targeting and fluorescent tracking ligands allowing us to monitor siRNA LNP uptake kinetics in dependence of the targeting ligand. In aggregate, we describe a robust approach for the generation of targeted and labelled siRNA LNPs that allows their controlled and facile decoration with ligand combinations.

Keywords:  Targeted Delivery; click chemistry; copper free strain-promoted azide alkyne cycloaddition (SPAAC); ligand post-insertion; siRNA lipid nanoparticles (LNPs)

DOI:  https://doi.org/10.1016/j.ijpharm.2022.121741
Macromol Rapid Commun. 2022 Apr 14. e2200145

Enhanced Gene Delivery Triggered by Dual pH/Redox Responsive Host-Guest Dimerization of Cyclooligosaccharide Star Polycations.

Ana I Carbajo-Gordillo, José López-Fernández, Juan M Benito, José L Jiménez Blanco, María L Santana-Armas, Gema Marcelo, Christophe Di Giorgio, Cédric Przybylski, Carmen Ortiz Mellet, Conchita Tros de Ilarduya, Francisco Mendicuti, José M García Fernández.

  A robust strategy is reported to build perfectly monodiperse star polycations combining a trehalose-based cyclooligosaccharide (cyclotrehalan, CT) central core onto which oligoethyleneimine radial arms are installed. The architectural perfection of the compounds is demonstrated by a variety of physicochemical techniques, including NMR, MS, DLS, TEM, and GPC. Key to the strategy is the possibility of customizing the cavity size of the macrocyclic platform to enable/preventing inclusion of adamantane motifs. These properties could be taken in advantage to implement sequential levels of stimuli responsiveness by combining computational design, precision chemistry and programmed host-guest interactions. Specifically, it is shown that supramolecular dimers implying a trimeric CT-tetraethyleneimine star polycation and purposely designed bis-adamantane guests are preorganized to efficiently complex plasmid DNA (pDNA) into transfection-competent nanocomplexes. The stability of the dimer species is responsive to the protonation state of the cationic clusters, resulting in dissociation at acidic pH. This process facilitates endosomal escape, but reassembling can take place in the cytosol then handicapping pDNA nuclear import. By equipping the ditopic guest with a redox-sensitive disulfide group, recapturing phenomena are prevented, resulting in drastically improved transfection efficiencies both in vivo and in vitro. This article is protected by copyright. All rights reserved.

Keywords:  cyclooligosaccharides; host-guest systems; non-viral gene delivery; star-shaped polycations; stimuli-sensitive vectors

DOI:  https://doi.org/10.1002/marc.202200145
Polymers (Basel). 2022 Apr 01. pii: 1443. [Epub ahead of print]14(7):

Modulation of Chitosan-TPP Nanoparticle Properties for Plasmid DNA Vaccines Delivery.

Renato Nunes, Ana Sofia Serra, Aiva Simaite, Ângela Sousa.

  Nucleic acid vaccines have become a revolutionary technology to give a fast, safe, cost-effective and efficient response against viral infections, such as SARS-CoV-2 or Human papillomavirus (HPV). However, to ensure their effectiveness, the development of adequate methods to protect, carry, and deliver nucleic acids is fundamental. In this work, nanoparticles (NPs) of chitosan (CS)-tripolyphosphate (TPP)-plasmid DNA (pDNA) were thoroughly modulated and characterized, by measuring the charge and size through dynamic light scattering (DLS) and morphology by scanning electron microscopy (SEM). Stability, cytotoxicity and cellular uptake of NPs were also evaluated. Finally, the effect of polyplexes on the expression of HPV E7 antigen in human fibroblast and RAW cells was investigated through polymerase chain reaction (PCR) and real-time PCR. The results showed NPs with a spherical/oval shape, narrow size distribution <180 nm and positive zeta potentials (>20 mV) and good stability after one month of storage at 4 °C in formulation buffer or when incubated in culture medium and trypsin. In vitro studies of NPs cytotoxicity revealed that the elimination of formulation buffers led to an improvement in the rate of cell viability. The E7 antigen transcription was also increased for NPs obtained with high pDNA concentration (60 μg/mL). The analyzed CS-TPP-pDNA polyplexes can offer a promising vehicle for nucleic acid vaccines, not only in the prevention or treatment of viral infections, but also to fight emergent and future pathogens.

Keywords:  cervical cancer; chitosan-TPP nanoparticles; ionotropic gelation; plasmid DNA vaccine

DOI:  https://doi.org/10.3390/polym14071443
Cell Mol Bioeng. 2022 Apr;15(2): 161-173

Albumin-Binding Aptamer Chimeras for Improved siRNA Bioavailability.

Jonah C Rosch, Ella N Hoogenboezem, Alexander G Sorets, Craig L Duvall, Ethan S Lippmann.

   Introduction: Short interfering RNAs (siRNAs) are potent nucleic acid-based drugs designed to target disease driving genes that may otherwise be undruggable with small molecules. However, therapeutic potential of siRNA in vivo is limited by poor pharmacokinetic properties, including rapid renal clearance and nuclease degradation. Backpacking on natural carriers such as albumin, which is present at high concentration and has a long half-life in serum, is an effective way to modify pharmacokinetics of biologic drugs that otherwise have poor bioavailability. In this work, we sought to develop albumin-binding aptamer-siRNA chimeras to improve the bioavailability of siRNA.
Methods: A Systematic Evolution of Ligands through Exponential Enrichment (SELEX) approach was used to obtain modified RNA-binding aptamers, which were then fused directly to siRNA via in vitro transcription. Molecular and pharmacokinetic properties of the aptamer-siRNA chimeras were subsequently measured in vitro and in vivo.
Results: In vitro assays show that albumin-binding aptamers are stable in serum while maintaining potent gene knockdown capabilities in the chimera format. In vivo, the absolute circulation half-life of the best-performing aptamer-siRNA chimera (Clone 1) was 1.6-fold higher than a scrambled aptamer chimera control.
Conclusions: Aptamer-siRNA chimeras exhibit improved bioavailability without compromising biological activity. Hence, this albumin-binding aptamer-siRNA chimera approach may be a promising strategy for drug delivery applications.
Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00718-y.

Keywords:  Albumin; Aptamer; Drug delivery; siRNA

DOI:  https://doi.org/10.1007/s12195-022-00718-y
Adv Drug Deliv Rev. 2022 Apr 12. pii: S0169-409X(22)00187-9. [Epub ahead of print] 114297

Current trends in delivery of non-viral nucleic acid-based therapeutics for improved efficacy.

Meltem Avci-Adali, Hélder Santos.

DOI: https://doi.org/10.1016/j.addr.2022.114297
Nanoscale. 2022 Apr 14.

Smartly responsive DNA-miRNA hybrids packaged in exosomes for synergistic enhancement of cancer cell apoptosis.

Fan Zhang, Albertina N Isak, Shiqi Yang, Yuchen Song, Lingjie Ren, Chang Feng, Guifang Chen.

Endogenous and exogenous tumor-related microRNAs (miRNAs) are considered promising tumor biomarkers and tumor therapeutic agents. In this work, we propose a miRNA self-responsive drug delivery system (miR-SR DDS), which enables the association between endogenous and exogenous miRNAs, so as to achieve a smart responsive and synergistic drug delivery. The miR-SR DDS consists of DNA-miRNA hybrids of let-7a and the complementary DNA of miR-155, which was packaged in exosomes. In response to the overexpressed miR-155 in breast cancer cells, the hybrids disintegrate and release let-7a and the complementary DNA of miR-155 to inhibit the expression of HMGA1 and relieve the inhibition of SOX1, respectively. Under the dual-targeted gene regulation, results show that the growth, migration and invasion of breast cancer cells can be synergistically inhibited through the Wnt/β-catenin signaling pathway. The concept and successful practice of the miR-SR DDS can be used as a reference for the development of miRNA drugs.

DOI: https://doi.org/10.1039/d1nr08539e