bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2021‒08‒15
seventeen papers selected by
Benjamin Winkeljann
Ludwig-Maximilians University

  1. Mol Pharm. 2021 Aug 10.
      Small interfering RNA (siRNA)-based drugs have shown tremendous potential to date in cancer gene therapy. Despite the considerable efforts in siRNA design and manufacturing, unsatisfactory delivery systems persist as a limitation for the application of siRNA-based drugs. In this work, the cholesterol, cell-penetrating peptide conjugate cRGD (R8-cRGD), and polyethylene glycol (PEG) were introduced into low-molecular-weight polyethyleneimine (LMW PEI) to form cRGD-R9-cholesterol-PEI-PEG (RRCPP) nanoparticles with specific targeting and highly penetrating abilities. The enhanced siRNA uptake efficiency of the RRCPP delivery system benefited from R8-cRGD modification. Wee1 is an oncogenic nuclear kinase that can regulate the cell cycle as a crucial G2/M checkpoint. Overexpression of Wee1 in melanoma may lead to a poor prognosis. In the present study, RRCPP nanoparticles were designed for Wee1 siRNA delivery to form an RRCPP/siWee1 complex, which significantly silenced the expression of the WEE1 gene (>60% inhibition) and induced B16 tumor cell apoptosis by abrogating the G2M checkpoint and DNA damage in vitro. Furthermore, the RRCPP/siWee1 complex suppressed B16 tumor growth in a subcutaneous xenograft model (nearly 85% inhibition rate) and lung metastasis (nearly 66% inhibition rate) with ideal in vivo safety. Briefly, our results support the validity of RRCPP as a potential Wee1 siRNA carrier for melanoma gene therapy.
    Keywords:  RNA interference; WEE1 gene; low-molecular weight polyethyleneimine; melanoma gene therapy
  2. Mol Pharm. 2021 Aug 13.
      Gene therapy aims to treat patients by altering or controlling gene expression. The field of gene therapy has had increasing success in recent years primarily using viral-based approaches; however, there is still significant interest toward the use of polymeric materials due to their potential as flexible, low-cost scaffolds for gene delivery that do not suffer the mutagenesis and immunogenicity concerns of viral vectors. To address the challenges of efficiency and biocompatibility, a series of zwitterion-like polyethylenimine derivatives (zPEIs) were produced via the succinylation of 2-11.5% of polyethylenimine (PEI) amines. With increasing modification, zPEI polyplexes exhibited decreased serum-protein aggregation and dissociated more easily in the presence of a competitor polyanion when compared to unmodified PEI. Surprisingly, the gene delivery mediated in the presence of serum showed that succinylation of as few as 2% of PEI amines resulted in transgene expression 260- to 480-fold higher than that of unmodified PEI and 50- to 65-fold higher than that of commercial PEI-PEG2k in HEK293 and HeLa cells, respectively. Remarkably, the same zPEIs also produced 16-fold greater efficiency of CRISPR/Cas9 gene knock-in compared to unmodified PEI in the presence of serum. In addition, we show that 2% succinylation does not significantly decrease polymer/DNA binding ability or serum protein interaction to a significant extent, yet this small modification is still sufficient to provide a remarkable increase in transgene expression and gene knock-in in the presence of serum.
    Keywords:  gene delivery; in-serum stability; nonviral vectors; polyampholytes; polymer chemistry; transfection efficiency
  3. Commun Biol. 2021 08 11. 4(1): 956
      Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. Herein, we report a method that can predict the LNP pKa from the structure of the ionizable lipid. We used theoretical, NMR, fluorescent-dye binding, and electrophoretic mobility methods to comprehensively measure protonation of both the ionizable lipid and the formulated LNP. The pKa of the ionizable lipid was 2-3 units higher than the pKa of the LNP primarily due to proton solvation energy differences between the LNP and aqueous medium. We exploited these results to explain a wide range of delivery efficiencies in vitro and in vivo for intramuscular (IM) and intravascular (IV) administration of different ionizable lipids at escalating ionizable lipid-to-mRNA ratios in the LNP. In addition, we determined that more negatively charged LNPs exhibit higher off-target systemic expression of mRNA in the liver following IM administration. This undesirable systemic off-target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP.
  4. J Nanobiotechnology. 2021 Aug 11. 19(1): 236
      Recently, we designed novel amphipathic cell-penetrating peptides, called WRAP, able to transfer efficiently siRNA molecules into cells. In order to gain more information about the relationship between amino acid composition, nanoparticle formation and cellular internalization of these peptides composed of only three amino acids (leucine, arginine and tryptophan), we performed a structure-activity relationship (SAR) study. First, we compared our WRAP1 and WRAP5 peptides with the C6M1 peptide also composed of the same three amino acids and showing similar behaviors in siRNA transfection. Afterwards, to further define the main determinants in the WRAP activity, we synthesized 13 new WRAP analogues harboring different modifications like the number and location of leucine and arginine residues, the relative location of tryptophan residues, as well as the role of the α-helix formation upon proline insertions within the native WRAP sequence. After having compared the ability of these peptides to form peptide-based nanoparticles (PBNs) using different biophysical methods and to induce a targeted gene silencing in cells, we established the main sequential requirements of the amino acid composition of the WRAP peptide. In addition, upon measuring the WRAP-based siRNA transfection ability into cells compared to several non-peptide transfection agents available on the markets, we confirmed that WRAP peptides induced an equivalent level of targeted gene silencing but in most of the cases with lower cell toxicity as clearly shown in clonogenic assays.
    Keywords:  Cell-penetrating peptides; Nanoparticle; Structure–activity relationship; siRNA delivery
  5. Adv Healthc Mater. 2021 Aug 11. e2101202
      The therapeutic use of RNA interference is limited by the inability of siRNA molecules to reach their site of action, the cytosol of target cells. Lipid nanoparticles, including liposomes, are commonly employed as siRNA carrier systems to overcome this hurdle, although their widespread use remains limited due to a lack of delivery efficiency. More recently, nature's own carriers of RNA, extracellular vesicles (EVs), are increasingly being considered as alternative siRNA delivery vehicles due to their intrinsic properties. However, they are difficult to load with exogenous cargo. Here, EV-liposome hybrid nanoparticles (hybrids) are prepared and evaluated as an alternative delivery system combining properties of both liposomes and EVs. It is shown that hybrids are spherical particles encapsulating siRNA, contain EV-surface makers, and functionally deliver siRNA to different cell types. The functional behavior of hybrids, in terms of cellular uptake, toxicity, and gene-silencing efficacy, is altered as compared to liposomes and varies among recipient cell types. Moreover, hybrids produced with cardiac progenitor cell (CPC) derived-EVs retain functional properties attributed to CPC-EVs such as activation of endothelial signaling and migration. To conclude, hybrids combine benefits of both synthetic and biological drug delivery systems and might serve as future therapeutic carriers of siRNA.
    Keywords:  drug delivery; exosomes; extracellular vesicles; liposomes; nucleic acids; siRNA
  6. Pharmaceutics. 2021 Jul 01. pii: 1003. [Epub ahead of print]13(7):
      In brain-targeted delivery, the transport of drugs or genes across the blood-brain barrier (BBB) is a major obstacle. Recent reports found that focused ultrasound (FUS) with microbubbles enables transient BBB opening and improvement of drug or gene delivery. We previously developed nano-sized bubbles (NBs), which were prepared based on polyethylene glycol (PEG)-modified liposomes containing echo-contrast gas, and showed that our NBs with FUS could also induce BBB opening. The aim of this study was to enhance the efficiency of delivery of pDNA into neuronal cells following transportation across the BBB using neuron-binding peptides. This study used the RVG-R9 peptide, which is a chimeric peptide synthesized by peptides derived from rabies virus glycoprotein and nonamer arginine residues. The RVG peptide is known to interact specifically with the nicotinic acetylcholine receptor in neuronal cells. To enhance the stability of the RVG-R9/pDNA complex in vivo, PEGylated polyethyleneimine (PEG-PEI) was also used. The ternary complexes composed of RVG-R9, PEG-PEI, and pDNA could interact with mouse neuroblastoma cells and deliver pDNA into the cells. Furthermore, for the in vivo experiments using NBs and FUS, gene expression was observed in the FUS-exposed brain hemispheres. These results suggest that this systemic gene delivery system could be useful for gene delivery across the BBB.
    Keywords:  brain; gene delivery; nanobubble; ultrasound
  7. Adv Funct Mater. 2021 Jan 27. pii: 2007096. [Epub ahead of print]31(5):
      Cancer is a genetic disease originating from the accumulation of gene mutations in a cellular subpopulation. Although many therapeutic approaches have been developed to treat cancer, recent studies have revealed an irrefutable challenge that tumors evolve defenses against some therapies. Gene therapy may prove to be the ultimate panacea for cancer by correcting the fundamental genetic errors in tumors. The engineering of nanoscale inorganic carriers of cancer therapeutics has shown promising results in the efficacious and safe delivery of nucleic acids to treat oncological diseases in small-animal models. When these nanocarriers are used for co-delivery of gene therapeutics along with auxiliary treatments, the synergistic combination of therapies often leads to an amplified health benefit. In this review, an overview of the inorganic nanomaterials developed for combinatorial therapies of gene and other treatment modalities is presented. First, the main principles of using nucleic acids as therapeutics, inorganic nanocarriers for medical applications and delivery of gene/drug payloads are introduced. Next, the utility of recently developed inorganic nanomaterials in different combinations of gene therapy with each of chemo, immune, hyperthermal, and radio therapy is examined. Finally, current challenges in the clinical translation of inorganic nanomaterial-mediated therapies are presented and outlooks for the field are provided.
    Keywords:  combinatorial therapy; gene delivery; inorganic nanomaterials; nanomedicine; tumors
  8. Pharmaceutics. 2021 Jul 13. pii: 1075. [Epub ahead of print]13(7):
      Despite the apparent advantages for long-term treatment and local therapies against intestinal diseases, the oral delivery of nucleic acids has been challenging due to unfavorable physiological conditions for their stability. In this study, a novel nanodelivery system of PEG-PCL nanoparticles with encapsulated nucleic acids-mannosylated PEI (Man-PEI) complexes was developed for intestinal delivery. We complexed model nucleic acids with Man-PEI at the optimal N/P ratio of 20:1 for in vitro and in vivo analyses. Cells were transfected in vitro and analyzed for gene expression, receptor-mediated uptake, and PEG-PCL nanoparticles' toxicity. We also evaluated the nucleic acid's stability in the nanocarrier during formulation, and under simulated gastrointestinal environments or the presence of nucleases. Finally, we assessed the biodistribution for the PEG-PCL nanoparticles with encapsulated complexes and their ability to transfect intestinal cells in vivo. Nucleic acids complexed with Man-PEI were protected from degradation against nucleases. In comparison to the parent compound PEI, Man-PEI transfected the cells with an overall higher potency. Competition assay indicated receptor-mediated endocytosis promoted by mannose receptors. The PEG-PCL nanoparticles with Man-PEI/plasmid complexes indicated minimal cytotoxicity. The nanocarrier successfully protected the complexes in a simulated gastric fluid environment and released them in a simulated intestinal fluid environment, promoted by the presence of lipases. The oral administration of the PEG-PCL nanoparticles with encapsulated Man-PEI/plasmid complexes transfected intestinal cells with the plasmid in vivo, while presenting a time-dependent progression through the intestines. Conclusively, our carrier system can deliver genetic material to the GI tract and actively target mannose receptor overexpressing cells.
    Keywords:  mannosylated PEI; nanoparticles; nucleic acids; oral delivery; targeting
  9. Ther Deliv. 2021 Aug 10.
      Background: The current study sought to formulate a dry powder inhalant (DPI) for pulmonary delivery of lipopolymeric nanoparticles (LPNs) consisting of cisplatin and siRNA for multidrug-resistant lung cancer. siRNA against ABCC3 gene was used to silence drug efflux promoter. Results & discussion: The formulation was optimized through the quality by design system by nanoparticle size and cisplatin entrapment. The lipid concentration, polymer concentration and lipid molar ratio were selected as variables. The DPI was characterized by in vitro deposition study using the Anderson cascade impactor. DPI formulation showed improved pulmonary pharmacokinetic parameters of cisplatin with higher residence time in lungs. Conclusion: Local delivery of siRNA and cisplatin to the lung tissue resulted into an enhanced therapeutic effectiveness in combating drug resistance.
    Keywords:  Cryo-TEM; DPI; MTT assay; PEG-PLA; RT-PCR; chemosensitization; cisplatin; gene knockdown; lung cancer; siRNA
  10. Methods Mol Biol. 2021 ;2355 275-286
      Successful delivery of mRNA into the cytosol of professional antigen-presenting cells (APCs) poses one of the biggest challenges in developing effective mRNA vaccines to treat various cancers and viral infectious diseases. However, most polymeric mRNA delivery systems fail to transfect APCs. We have discovered that decoration of pH-sensitive endosome-disruptive GALA peptides on the surface of mRNA polyplexes leads to efficient targeting and transfection of APCs. GALA peptides not only enhance specific uptake in APCs through binding to sialic acid moieties, they also facilitate the endosomal escape of mRNA especially in dendritic cells (DCs). Here, we describe in detail the production of stabilized mRNA polyplexes post-conjugated with GALA peptides via copper-free click chemistry. Methods described here include the synthesis and purification of GALA peptides and its conjugation to mRNA polyplexes.
    Keywords:  Click chemistry; GALA peptide; Nanoparticle surface decoration; Peptide conjugation; Post-PEGylation; Post-modification; mRNA polyplexes
  11. J Control Release. 2021 Aug 05. pii: S0168-3659(21)00408-9. [Epub ahead of print]
      The development of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems has created a tremendous wave that is sweeping the world of genome editing. The ribonucleoprotein (RNP) method has evolved to be the most advantageous form for in vivo application. Modification of the CRISPR/Cas9 RNP method to adapt delivery through a variety of carriers can either directly improve the stability and specificity of the gene-editing tool in vivo or indirectly endow the system with high gene-editing efficiency that induces few off-target mutations through different delivery methods. The exploration of in vivo applications mediated by various delivery methods lays the foundation for genome research and variety improvements, which is especially promising for better in vivo research in the field of translational biomedicine. In this review, we illustrate the modifiable structures of the Cas9 nuclease and single guide RNA (sgRNA), summarize the latest research progress and discuss the feasibility and advantages of various methods. The highlighted results will enhance our knowledge, stimulate extensive research and application of Cas9 and provide alternatives for the development of rational delivery carriers in multiple fields.
    Keywords:  CRISPR/Cas9; Genome editing; In vivo delivery; Modification; Nonviral vector; Ribonucleoprotein; sgRNA
  12. J Extracell Vesicles. 2021 Aug;10(10): e12111
      Small extracellular vesicles (sEVs), through their natural ability to interact with biological membranes and exploit endogenous processing pathways to convey biological information, are quintessential for the delivery of therapeutically relevant compounds, such as microRNAs (miRNAs) and proteins. Here, we used a fluorescently-labelled miRNA to quantify the efficiency of different methods to modulate the cargo of sEVs. Our results showed that, compared with electroporation, heat shock, permeation by a detergent-based compound (saponin) or cholesterol-modification of the miRNA, Exo-Fect was the most efficient method with > 50% transfection efficiency. Furthermore, qRT-PCR data showed that, compared with native sEVs, Exo-Fect modulation led to a > 1000-fold upregulation of the miRNA of interest. Importantly, this upregulation was observed for sEVs isolated from multiple sources. The modulated sEVs were able to delivery miR-155-5p into a reporter cell line, confirming the successful delivery of the miRNA to the target cell and, more importantly, its functionality. Finally, we showed that the membrane of Exo-Fect-loaded sEVs was altered compared with native sEVs and that enhanced the internalization of Exo-Fect-loaded sEVs within the target cells and decreased the interaction of those modulated sEVs with lysosomes.
    Keywords:  extracellular vesicles; microRNA; modulation; post‐isolation
  13. Pharmaceutics. 2021 Jul 11. pii: 1063. [Epub ahead of print]13(7):
      Resistance to chemotherapy, enhanced proliferation, invasion, angiogenesis, and metastasis (RPIAM) represent major obstacles that limit the efficacy of cancer treatment especially in advanced stages of cancer. Overcoming or suppressing RPIAM can dramatically improve the treatment outcome. Non-small cell lung cancer (NSCLC) is frequently diagnosed in an advanced stage and often possesses intrinsic resistance to chemotherapy accompanied by the fast development of acquired resistance during the treatment. Oncogenic receptor tyrosine kinases (TKs), specifically epidermal growth factor (EGF) TKs, play an important role in the activation of MAPK/PI3K/Akt/STAT pathways, finally leading to the development of RPIAM. However, the suppression of EGF-TK by different drugs is limited by various defensive mechanisms and mutations. In order to effectively prevent the development of RPIAM in NSCLC, we formulated and tested a multicomponent and multifunctional cancer targeted delivery system containing Nanostructured Lipid Carriers (NLCs) as vehicles, luteinizing hormone release hormone (LHRH) as a cancer targeting moiety, EFG-TK inhibitor gefitinib and/or paclitaxel as anticancer drug(s), siRNA targeted to EGF receptor (EGFR) mRNA as a suppressor of EGF receptors, and an imaging agent (rhodamine) for the visualization of cancer cells. Experimental data obtained show that this complex delivery system possesses significantly enhanced anticancer activity that cannot be achieved by individual components applied separately.
    Keywords:  EGFR mutated and gefitinib-resistant NSCLC; LHRH; lipid nanoparticles; paclitaxel; siRNA
  14. J Control Release. 2021 Aug 10. pii: S0168-3659(21)00421-1. [Epub ahead of print]
      So far, most reported delivery of CRISPR/Cas9 is achieved by internalized or encapsulated multiple ribonucleoprotein units into only one carrier unit, with relatively large size. Here, we report a novel, small-sized, individual package of CRISPR/Cas9, via using tetralysine modified H-chian apoferritin (TL-HFn) as packaging material. In this paper, each CRISPR/Cas9 complex is proved to be successfully installed into one TL-HFn (~26 nm), and delivered into the targeting cell via TfR1-mediated endocytosis. We found that after 6 h of treatment, the CRISPR/Cas9 complex can be tracked within the nuclear of Hela cells for the purpose of gene editing of enhanced green fluorescent protein (EGFP). Moreover, TL-HFn individually packed CRISPR/Cas9 displayed higher genome editing activity compared with that of free CRISPR/Cas9 treated group both in vitro (up to 28.96%) and in vivo. Such satisfied genome editing efficiency could be attributed to the endosomal escape and pH-induced disassembly abilities given by TL-HFn after uptake into cytoplasm, which had been verified in our previous research. In all, those results prompted that TL-HFn possessed more potential for intracellular delivery of CRISPR/Cas9, with potential biocompatibility, stability and delivery efficiency.
    Keywords:  CRISPR/Cas9; Endosomal escape; Gene editing; H-chain apoferritin; Tetralysine
  15. Biomater Sci. 2021 Aug 11.
      The discovery of CRISPR/Cas9 genome-editing technology enables the precise manipulation of mammalian DNA sequences for treating genetic disorders. Despite its high efficiency for genome editing, the introduction of CRISPR/Cas9 machinery, which is composed of Cas9 nuclease protein and guide RNA, into cells challenges its clinical translation potential. Therefore, the intracellular delivery of genome-editing machinery determines the efficacy of gene manipulation via the CRISPR/Cas9 technology. Recently, metallosupramolecules including metal-organic frameworks (MOFs) and metal-organic cages (MOCs) have been designed to selfassemble with Cas9 nuclease and guide RNA for CRISPR/Cas9 delivery and genome editing. Herein, we review the most recent advances and strategies of constructing metallosupramolecules for CRISPR/Cas9 delivery. In particular, we discuss nanoscale MOFs and MOCs that could be assembled and regulated by the intracellular environment for the spatiotemporal delivery of genome editing machinery. We also provide a perspective view of the future development of metallosupramolecules for genome editing and gene therapy in vivo.
  16. Carbohydr Polym. 2021 Oct 15. pii: S0144-8617(21)00771-2. [Epub ahead of print]270 118384
      To achieve the co-delivery of chemotherapeutic drugs, genes, and immune agents in a single nanoparticulate system, p-mercaptobenzoic acid-grafted N, N, N-trimethyl chitosan nanoparticles (MT NPs) were successfully synthesized. Paclitaxel (PTX) was encapsulated into the hydrophobic core of the MT NPs, and meanwhile, survivin shRNA-expressing plasmid (iSur-pDNA) and recombinant human interleukin-2 (rhIL-2) were loaded onto the hydrophilic shell of the MT NPs. Owing to the redox-sensitiveness of MT NPs, a rapid release of PTX was triggered by the high concentration of glutathione. The synergistic effects of PTX (1.5 mg/kg), iSur-pDNA (1.875 mg/kg), and rhIL-2 (6 × 105 IU/kg) at a low dose endowed the MT/PTX/pDNA/rhIL-2 NPs with enhanced antitumor efficacies and improved tumor-induced immunosuppression. These results demonstrated that the co-delivery of PTX, iSur-pDNA, and rhIL-2 by the amphiphilic chitosan based NPs with redox-sensitiveness could be a promising strategy in the treatment of tumors.
    Keywords:  Chitosan; Paclitaxel; Recombinant human interleukin-2; Redox-sensitiveness nanoparticles; Survivin shRNA-expressing plasmid; Synergistic antitumor treatment
  17. Front Chem. 2021 ;9 716771
      Development of small organic chromophores as DNA condensing agents, which explore supramolecular interactions and absorbance or fluorescence-based tracking of condensation and gene delivery processes, is in the initial stages. Herein, we report the synthesis and electrostatic/groove binding interaction-directed synergistic self-assembly of the aggregates of two viologen-functionalized tetraphenylethylene (TPE-V) molecules with CT-DNA and subsequent concentration-dependent DNA condensation process. TPE-V molecules differ in their chemical structure according to the number of viologen units. Photophysical and morphological studies have revealed the interaction of the aggregates of TPE-V in Tris buffer with CT-DNA, which transforms the fibrous network structure of CT-DNA to partially condensed beads-on-a-string-like arrangement with TPE-V aggregates as beads via electrostatic and groove binding interactions. Upon further increasing the concentration of TPE-V, the "beads-on-a-string"-type assembly of TPE-V/CT-DNA complex changes to completely condensed compact structures with 40-50 nm in diameter through the effective charge neutralization process. Enhancement in the melting temperature of CT-DNA, quenching of the fluorescence emission of ethidium bromide/CT-DNA complex, and the formation of induced CD signal in the presence of TPE-V molecules support the observed morphological changes and thereby verify the DNA condensation abilities of TPE-V molecules. Decrease in the hydrodynamic size, increase in the zeta potential value with the addition of TPE-V molecules to CT-DNA, failure of TPE-V/cucurbit(8)uril complex to condense CT-DNA, and the enhanced DNA condensation ability of TPE-V2 with two viologen units compared to TPE-V1 with a single viologen unit confirm the importance of positively charged viologen units in the DNA condensation process. Initial cytotoxicity analysis on A549 cancer and WI-38 normal cells revealed that these DNA condensing agents are non-toxic in nature and hence could be utilized in further cellular delivery studies.
    Keywords:  DNA condensation; aggregates; electrostatic interaction; self-assembly; tetraphenylethylene; viologen