bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2023‒08‒20
fourteen papers selected by
the Merkel lab, Ludwig-Maximilians University

  1. Mol Pharm. 2023 Aug 14.
      Polyethylenimine (PEI) is a highly efficient cationic polymer for nucleic acid delivery, and although it is commonly used in preclinical studies, its clinical application is limited because of concerns regarding its cytotoxicity. Poly(β-amino ester)s are a new group of biodegradable and biocompatible cationic polymers that can be used for siRNA delivery. In this study, we synthesized Boc-protected and deprotected poly(β-amino ester)s, P(BSpBAE) and P(SpBAE), respectively, based on spermine and 1,4-butanediol diacrylate to deliver siRNA. The polymers were synthesized by Michael addition in a step-growth polymerization and characterized via 1H NMR spectroscopy and size-exclusion chromatography (SEC). The polymers can encapsulate siRNA as determined by SYBR gold assays. Both polymers and polyplexes were biocompatible in vitro. Furthermore, the cellular uptake of P(BSpBAE) and P(SpBAE) polyplexes was more efficient than for branched PEI (25 kDa) polyplexes at the same N/P ratios. P(BSpBAE) polyplexes achieved 60% eGFP knockdown in vitro, which indicates that the Boc-protection can improve the siRNA delivery and gene silencing efficiency of PBAEs. P(BSpBAE) polyplexes and P(SpBAE) polyplexes showed different cellular uptake mechanisms, and P(BSpBAE) polyplexes demonstrated decreased endosomal entrapment, which could explain why P(BSpBAE) polyplexes more efficiently mediated gene silencing than P(SpBAE) polyplexes. Furthermore, transfection of an siRNA against mutated KRAS in KRAS-mutated lung cancer cells led to around 35% (P(BspBAE)) to 45% (P(SpBAE)) inhibition of KRAS expression and around 33% (P(SpBAE)) to 55% (P(BspBAE)) decreased motility in a migration assay. These results suggest that the newly developed spermine-based poly(β-amino ester)s are promising materials for therapeutic siRNA delivery.
    Keywords:  KRAS; PBAE; lung cancer; poly(β-amino ester)s; siRNA delivery; spermine
  2. Eur J Pharm Biopharm. 2023 Aug 12. pii: S0939-6411(23)00205-9. [Epub ahead of print]
      RNA therapeutics represents a powerful strategy for diseases where other approaches have failed, especially given the recent successes of mRNA vaccines against the coronavirus disease 2019 (COVID-19) and small interfering (siRNA) therapeutics. However, further developments are still required to reduce toxicity, improve stability and biodistribution of mRNA-LNPs (lipid nanoparticles). Here, we show a rational combinatorial approach to select the best formulation based on a new cationic lipid molecule (IM21.7c), which includes an imidazolium polar head. The study allowed us to select the optimal 5 lipids composition for in vivo mRNA delivery. IM21.7c based mRNA-LNPs measuring <100 nm had high encapsulation efficiency, protected mRNA from degradation, and exhibited sustained release kinetics for effective in vitro transfection. Most interestingly the biodistribution was significantly different from other clinically approved LNPs, with increased targeting to the lung. Further studies are now required to expand the possible applications of these new molecules.
    Keywords:  Cationic lipid; Vaccines; imidazolium-based lipid; in vivo delivery; lipid nanoparticles (LNPs); mRNA delivery; transfection
  3. Mol Med Rep. 2023 Oct;pii: 180. [Epub ahead of print]28(4):
      Previously, using three types of cationic lipids, the effect of phospholipids in liposomal formulations on gene-knockdown efficacy was determined after in vitro and in vivo transfection with small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) containing various cationic lipids and phospholipids. In the present study, six other types of cationic lipids, namely N,N-dimethyl-N-tetradecyltetradecan-1-aminium bromide, N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), 2-[bis{2-(tetradecanoyloxy)ethyl}amino]-N,N,N-trimethyl-2-oxoethan-1-aminium chloride (DC-6-14), 1,2-di-O-octadecenyl-3-trimethylammonium propane chloride (DOTMA), 1,2-distearoyl-3-trimethylammonium-propane chloride (DSTAP) and 1,2-dioleoyl-3-dimethylammonium-propane were selected, and the effect of phospholipids in liposomal formulations containing each cationic lipid on gene-knockdown was evaluated. A total of 30 types of cationic liposomes composed of each cationic lipid with phosphatidylethanolamine containing unsaturated or saturated diacyl chains (C14, C16 or C18) were prepared. Regardless of the type of cationic lipid, the inclusion of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in the liposomal formulations resulted in injectable size of siRNA lipoplexes after mixing of siRNA and cationic liposomes. Transfection of their lipoplexes with luciferase (Luc) siRNA into human breast cancer MCF-7-Luc cells stably expressing Luc led to a strong knockdown of Luc. Furthermore, the systemic injection of siRNA lipoplexes composed of DC-1-16, DC-6-14, DOTMA or DSTAP with DOPE resulted in siRNA accumulation in the lungs. Significant gene-knockdown was observed in the lungs of mice following the systemic injection of siRNA lipoplexes containing DC-1-16 and DOPE. Cationic liposomes composed of DC-1-16 and DOPE serve as potential carriers for in vitro and in vivo siRNA transfection.
    Keywords:  cationic liposome; gene-knockdown; lung; phospholipid; small interfering RNA delivery
  4. Biomaterials. 2023 Aug 14. pii: S0142-9612(23)00287-9. [Epub ahead of print]301 122279
      Efficient and safe delivery of vulnerable mRNA is a long-standing challenge for the broad application of the emerging mRNA-based therapeutics. Herein, a combinatorial library containing 119 novel lipids was constructed via sequential aza-Michael addition reactions of arylates and varying amines to tackle the ongoing challenge in mRNA delivery. Through in vitro screening of the lipid library on IGROV 1 cells, we identified several synthetic lipids with superior mRNA delivery efficacy. The delivery capability of these lipids was verified by the potent expression of luciferase in BALB/c mice upon intravenous administration of luciferase-encoding mRNA lipid nanoparticles (LNPs). Further investigations on the structure-activity relationship revealed that lipids with branched hydrophobic tails were better at delivering mRNA than those containing linear tails at the similar total number of carbons. In comparison to linear tails, the branched tails endowed LNPs with less inner hydrophobicity, fewer surface charges, and proper stability, which benefits the cellular uptake of LNPs and the intracellular trafficking of mRNA, thus improves the delivery efficacy of mRNA. The therapeutical potential of the lead LNPs was evaluated by delivering ovalbumin (OVA)-encoding mRNA to mice bearing B16-OVA melanoma tumors. The results demonstrated that the administration of OVA mRNA LNPs significantly activated CD8+ T cells in tumor microenvironment and substantially prohibited the growth of the aggressive B16-OVA tumors. The robust antitumor efficacy highlights the great potential of these LNPs in cancer immunotherapy.
    Keywords:  Cancer immunotherapy; Gene therapy; Lipid; Nanoparticle; mRNA delivery
  5. Sci Transl Med. 2023 Aug 16. 15(709): eabq0603
      An inhalable platform for messenger RNA (mRNA) therapeutics would enable minimally invasive and lung-targeted delivery for a host of pulmonary diseases. Development of lung-targeted mRNA therapeutics has been limited by poor transfection efficiency and risk of vehicle-induced pathology. Here, we report an inhalable polymer-based vehicle for delivery of therapeutic mRNAs to the lung. We optimized biodegradable poly(amine-co-ester) (PACE) polyplexes for mRNA delivery using end-group modifications and polyethylene glycol. These polyplexes achieved high transfection of mRNA throughout the lung, particularly in epithelial and antigen-presenting cells. We applied this technology to develop a mucosal vaccine for severe acute respiratory syndrome coronavirus 2 and found that intranasal vaccination with spike protein-encoding mRNA polyplexes induced potent cellular and humoral adaptive immunity and protected susceptible mice from lethal viral challenge. Together, these results demonstrate the translational potential of PACE polyplexes for therapeutic delivery of mRNA to the lungs.
  6. Bioact Mater. 2024 Jan;31 1-17
      Endothelial cell dysfunction occurs in a variety of acute and chronic pulmonary diseases including pulmonary hypertension, viral and bacterial pneumonia, bronchopulmonary dysplasia, and congenital lung diseases such as alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). To correct endothelial dysfunction, there is a critical need for the development of nanoparticle systems that can deliver drugs and nucleic acids to endothelial cells with high efficiency and precision. While several nanoparticle delivery systems targeting endothelial cells have been recently developed, none of them are specific to lung endothelial cells without targeting other organs in the body. In the present study, we successfully solved this problem by developing non-toxic poly(β-amino) ester (PBAE) nanoparticles with specific structure design and fluorinated modification for high efficiency and specific delivery of nucleic acids to the pulmonary endothelial cells. After intravenous administration, the PBAE nanoparticles were capable of delivering non-integrating DNA plasmids to lung microvascular endothelial cells but not to other lung cell types. IVIS whole body imaging and flow cytometry demonstrated that DNA plasmid were functional in the lung endothelial cells but not in endothelial cells of other organs. Fluorination of PBAE was required for lung endothelial cell-specific targeting. Hematologic analysis and liver and kidney metabolic panels demonstrated the lack of toxicity in experimental mice. Thus, fluorinated PBAE nanoparticles can be an ideal vehicle for gene therapy targeting lung microvascular endothelium in pulmonary vascular disorders.
    Keywords:  Gene delivery; Lung microvascular endothelium; Poly (β-amino) esters nanoparticle; Specific targeting
  7. Macromol Biosci. 2023 Aug 12. e2300238
      Cancer development and progression of cancer is closely associated with the activation of oncogenes and loss of tumor suppressor genes. Nucleic acid drugs (e.g., siRNA, mRNA, and DNA) have been widely used for cancer therapy due to their specific ability to regulate the expression of any cancer-associated genes. However, nucleic acid drugs are negatively charged biomacromolecules that are susceptible to serum nucleases and could not cross cell membrane. Therefore, specific delivery tools are required to facilitate the intracellular delivery of nucleic acid drugs. In the past few decades, a variety of nanoparticles (NPs) have been designed and developed for nucleic acid delivery and cancer therapy. In particular, the polymeric NPs in response to the abnormal redox status in cancer cells have garnered much more attention as their potential in redox-triggered nanostructure dissociation and rapid intracellular release of nucleic acid drugs. In this review, we briefly introduce the important genes or signaling pathways regulating the abnormal redox status in cancer cells and systemically summarize the recent development of redox-responsive NPs for nucleic acid delivery and cancer therapy. We also discuss the future development of NPs-mediated nucleic acid delivery and their challenges in clinical translation. This article is protected by copyright. All rights reserved.
    Keywords:  Polymeric Nanoparticles; cancer therapy; delivery; nucleic acid; redox-responsive
  8. Nat Rev Clin Oncol. 2023 Aug 16.
      Harnessing mRNA-lipid nanoparticles (LNPs) to treat patients with cancer has been an ongoing research area that started before these versatile nanoparticles were successfully used as COVID-19 vaccines. Currently, efforts are underway to harness this platform for oncology therapeutics, mainly focusing on cancer vaccines targeting multiple neoantigens or direct intratumoural injections of mRNA-LNPs encoding pro-inflammatory cytokines. In this Review, we describe the opportunities of using mRNA-LNPs in oncology applications and discuss the challenges for successfully translating the findings of preclinical studies of these nanoparticles into the clinic. We critically appraise the potential of various mRNA-LNP targeting and delivery strategies, considering physiological, technological and manufacturing challenges. We explore these approaches in the context of the potential clinical applications best suited to each approach and highlight the obstacles that currently need to be addressed to achieve these applications. Finally, we provide insights from preclinical and clinical studies that are leading to this powerful platform being considered the next frontier in oncology treatment.
  9. Int J Pharm. 2023 Aug 14. pii: S0378-5173(23)00739-1. [Epub ahead of print] 123319
      The emergence of SARS-CoV-2 in Wuhan, China in 2019 has had a profound impact on humanity in every facet. While vaccines against this viral pathogen have been approved a year later, limitations to this therapeutic intervention persist, such as drug sensitivity to transportation and storage conditions, as well as significant financial losses from non-injected resuspended vials. Our research delves into the effects of thermal denaturation (4 - 40°C) and light irradiation (720 and 10460 kJ/m2) on the mRNA-based vaccines BNT162b2 from BioNTech/Pfizer and mRNA-1273 from Moderna. We also investigated vaccine stability following incubation in syringes to simulate potential interactions with silicon oil. By assaying the effects of these stressors via biochemical and biophysical methods, we aim to elucidate the physicochemical properties, integrity, and stability of these mRNA-based vaccines. Furthermore, the incorporation of a fluorophore into both vaccines allowed us to monitor their localization within cells and assess their capacity to evade vesicular transport mechanisms, thus evaluating the differences between the two formulations. A comprehensive understanding of the aforementioned attributes can enable the establishment of optimal storage and manipulation conditions for these vaccines, thereby ensuring their safe and efficacious application while minimizing the waste of functional and safe therapeutic agents.
    Keywords:  light stress; mRNA-based COVID-19 Vaccines; physicochemical stability; solid lipid nanoparticles; thermal stress
  10. J Clin Pharmacol. 2023 Aug 17.
      Small interfering RNAs (siRNAs) represent a new class of drugs with tremendous potential for battling previously undruggable diseases. After nearly two decades of efforts in addressing the problems of the poor drug profile of naked unmodified siRNAs, this new modality has finally come to fruition, with five agents (patisiran, givosiran, lumasiran, inclisiran, and vutrisiran) being approved since 2018 and many others in the different phases of clinical development. Unlike small-molecule drugs and protein therapeutics, siRNAs have different sizes, distinct mechanism of action, different physicochemical and pharmacological properties, and accordingly a unique PK/PD relationship. To support the continuous development of siRNA, it is important to have a thorough and deep understanding of the PK/PD and clinical pharmacology related features of siRNAs. Since most of the current siRNA products are conjugated by N-acetylgalactosamine (GalNAc), this review focuses on the PK/PD and clinical pharmacology of GalNAc-conjugated siRNAs, including their absorption, distribution, metabolism, excretion (ADME) properties, PK/PD models, drug-drug interactions, clinical pharmacology in special populations, and safety evaluation. In addition, necessary background information related to the development of siRNA as a therapeutic modality, including its mechanism of action, the advantages of siRNAs, the problems of naked siRNAs, as well as the strategies used to enhance the clinical utility of siRNA, have also been covered. The goal of this review is to serve as a "primer" on siRNA PK/PD, and I hope the readers, especially those who have limited background on siRNA therapeutics, will have a fundamental understanding on siRNA PK/PD after reading this review. This article is protected by copyright. All rights reserved.
    Keywords:  Clinical pharmacology of siRNAs; GalNAc-conjugated siRNAs; Givosiran; Inclisiran; Lumasiran; Vutrisiran; siRNA PK/PD; siRNA pharmacometrics modeling
  11. Methods Mol Biol. 2023 ;2709 191-202
      The protocol described in this chapter allows for acquiring topography images of RNA-based nanoring structures and assessing their dynamic properties using atomic force microscopy (AFM) imaging. AFM is an indispensable tool for characterization of nucleic acid-based nanostructures with the exceptional capability of observing complexes in the range of a few nanometers. This method can visualize structural characteristics and evaluate differences between individual structurally different RNA nanorings. Due to the highly resolved AFM topography images, we introduce an approach that allows to distinguish the differences in the dynamic behavior of RNA nanoparticles not amenable to other experimental techniques. This protocol describes in detail the preparation procedures of RNA nanostructures, AFM imaging, and data analysis.
    Keywords:  Atomic force microscopy; Flexibility analysis; Mechanical stability; Mica surface modification; RNA nanoparticle; Topography imaging
  12. Phys Chem Chem Phys. 2023 Aug 14.
      Realizing the potential of nano-hybrid biomaterials in various applications (nanoprobes to drug delivery), special attention has been devoted towards their synthesis and development. Nonetheless, several questions pertaining to the interface chemistry between the constituent entities (biomolecules and organic/inorganic part) of these hybrids, still remain unresolved. Keeping these unsolved issues in mind, the present theoretical investigation focuses on determining the electronic/physicochemical properties and interactions within gold and silver quantum dot-capped single lipid molecules. Quantum dots of varying sizes and shapes have been chosen and then coupled with lipid molecules (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, sodium salt (DMPG)), at the choline/glycerol, carboxylate and phosphate site. It has been identified that Au Qds interact strongly as compared to Ag clusters. In addition to the type, the shape and size of the Qd also influences their attachment with lipids. Among various sites, the phosphate site provides a considerably stronger platform for the coupling of Qds. On the other hand, attachment at the choline site leads to significantly lower interaction energies. The trend noted in interaction energies coincides with the structure-electronic property analysis (interatomic bond distances, charge transfer, PO2- stretching frequencies), which further helps in deducing the nature of interactions. The molecular dynamics simulations performed on selected Qd-lipid complexes established that the Qd interacting with lipids at the phosphate site remains fairly stable at room temperature without undergoing fragmentation into individual components. On the other hand, at the choline site, the Qd-to-lipid coupling is unstable and therefore they experience disintegration at 300 K temperature. Additionally, a unique glycerol-to-phosphate site crossover is evidenced, which reaffirms that the phosphate site is selectively preferred by Qds for binding with lipid molecules.
  13. Methods Mol Biol. 2023 ;2709 3-29
      Molecular dynamics (MD) simulations can be used to investigate the stability and conformational characteristics of RNA nanostructures. However, MD simulations of an RNA nanostructure is computationally expensive due to the size of nanostructure and the number of atoms. Alternatively, MD simulations of RNA motifs can be used to estimate the conformational stability of constructed RNA nanostructure due to their small sizes. In this chapter, we introduce the preparation and MD simulations of two RNA kissing loop (KL) motifs, a linear KL complex and a bent KL complex, and an RNA nanoring. The initial solvated system and topology files of each system will be prepared by two major force fields, AMBER and CHARMM force fields. MD simulations will be performed by NAMD simulation package, which can accept both force fields. In addition, we will introduce the use of the AMBER cpptraj program and visual molecular dynamics (VMD) for data analysis. We will also discuss how MD simulations of two KL motifs can be used to estimate the conformation and stability of RNA nanoring as well as to explain the vibrational characteristics of RNA nanoring.
    Keywords:  AMBER; CHARMM; Molecular dynamics simulations; NAMD; RNA motif; RNA nanostructure
  14. J Liposome Res. 2023 Aug 18. 1-58
      The object of the current study was to develop and evaluate trastuzumab-conjugated Paclitaxel (PTX) and Elacridar (ELA)-loaded PEGylated pH-sensitive liposomes (TPPLs) for site-specific delivery of an anticancer drug. In this study, paclitaxel is used as an anticancer drug which promotes microtubules polymerization and arrest cell cycle progression at mitosis and subsequently leading to cell death. The single use of PTX causes multiple drug resistance (MDR) and results failure of the therapy. Hence, the combination of PTX and P-glycoprotein inhibitor (ELA) are used to achieve maximum therapeutic effects of PTX. Moreover, monoclonal antibody (trastuzumab) is used as ligand for the targeting the drug bearing carriers to BC. Thus, trastuzumab anchored pH-sensitive liposomes bearing PTX and ELA were developed using thin film hydration method and Box-Behnken Design (BBD) for optimizing various formulation variables. The optimized liposomes undergo characterization such as vesicle size, PDI, and zeta potential, which were observed to be 122 ± 2.14 nm, 0.224, and -15.5 mV for PEGylated pH-sensitive liposomes (PEG-Ls) and 134 ± 1.88 nm, 0.238, and -13.98 mV for TPPLs, respectively. The results of the in vitro drug release study of both formulations (PEG-Ls and TPPLs) showed enhanced percentage drug release at an acidic pH 5 as compared to drug release at a physiological pH 7.4. Further, the in vitro cytotoxicity studies were performed in the SK-BR-3 and MDA-MB-231 cell lines. The cellular uptake study of FITC-loaded TPPLs in SK-BR-3 cells showed greater uptake than FITC-loaded PEG-Ls, while in MDA-MB-231 cells there was no significant difference in cell uptake between FITC-loaded TPPLs and FITC-loaded PEG-Ls. Hence, it can be concluded that the HER-2 overexpressing cancer cell line (SK-BR-3) was showing better cytotoxicity and cell uptake of TPPLs than the cells that expressed low levels of HER2 (MDA-MB-231). The in vivo tumor regression study, TPPLs showed significantly more tumor burden reduction i.e. up ∼74% as compared to other liposomes after 28 days. Furthermore, the in vivo studies of TPPLs showed a minimal toxicity profile, minimal hemolysis, higher tumor tissue distribution, and superior antitumor efficacy as compared to other formulations. These studies confirmed that TPPLs are a safe and efficacious treatment for breast cancer.
    Keywords:  Breast cancer; Liposomes; Paclitaxel; Targeted drug delivery system(s); Trastuzumab