bims-novged 2024-02-04 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2024–02–04
twelve papers selected by
the Merkel lab, Ludwig-Maximilians University

Microfluidic platform enables shear-less aerosolization of lipid nanoparticles for messenger RNA inhalation.
Biodistribution of Therapeutic Small Interfering RNAs Delivered with Lipid-Substituted Polyethylenimine-Based Delivery Systems.
A careful look at lipid nanoparticle characterization: analysis of benchmark formulations for encapsulation of RNA cargo size gradient.
Pulmonary RNA Interference against Acute Lung Injury Mediated by Mucus- and Cell-penetrating Nanocomplexes.
Enhancing Gene Therapy through Ultradeformable Vesicles for Efficient siRNA Delivery.
Self-assembled redox-responsive BRD4 siRNA nanoparticles: fomulation and its in vitro delivery in gastric cancer cells.
Lipid nanoparticle composition for adjuvant formulation modulates disease after influenza virus infection in QIV vaccinated mice.
Membrane-Cloaked Nanoparticles for RNA Interference of β-Catenin in Triple-Negative Breast Cancer.
pH-Dependent Lyotropic Liquid Crystalline Mesophase and Ionization Behavior of Phytantriol-Based Ionizable Lipid Nanoparticles.
Multifunctional siRNA/ferrocene/cyclodextrin nanoparticles for enhanced chemodynamic cancer therapy.
Hydrophobicity Tuning of Cationic Polyaspartamide Derivatives for Enhanced Antisense Oligonucleotide Delivery.
The Constitutional Isomerism of One-Component Ionizable Amphiphilic Janus Dendrimers Orchestrates the Total and Targeted Activities of mRNA Delivery.

bioRxiv. 2024 Jan 20. pii: 2024.01.17.576136. [Epub ahead of print]

Microfluidic platform enables shear-less aerosolization of lipid nanoparticles for messenger RNA inhalation.

Jeonghwan Kim, Antony Jozic, Elissa Bloom, Brian Jones, Michael Marra, Namratha Turuvekere Vittala Murthy, Yulia Eygeris, Gaurav Sahay.

Leveraging the extensive surface area of the lungs for gene therapy, inhalation route offers distinct advantages for delivery. Clinical nebulizers that employ vibrating mesh technology are the standard choice for converting liquid medicines into aerosols. However, they have limitations when it comes to delivering mRNA through inhalation, including severe damage to nanoparticles due to shearing forces. Here, we introduce a novel microfluidic aerosolization platform (MAP) that preserves the structural and physicochemical integrity of lipid nanoparticles, enabling safe and efficient mRNA delivery to the respiratory system. Our results demonstrated the superiority of the novel MAP over the conventional vibrating mesh nebulizer, as it avoided problems such as particle aggregation, loss of mRNA encapsulation, and deformation of nanoparticle morphology. Notably, aerosolized nanoparticles generated by the microfluidic device led to enhanced transfection efficiency across various cell lines. In vivo experiments with mice that inhaled these aerosolized nanoparticles revealed successful, lung-specific mRNA transfection without observable signs of toxicity. This pioneering MAP represents a significant advancement for the pulmonary gene therapy, enabling precise and effective delivery of aerosolized nanoparticles.

DOI: https://doi.org/10.1101/2024.01.17.576136
Mol Pharm. 2024 Jan 30.

Biodistribution of Therapeutic Small Interfering RNAs Delivered with Lipid-Substituted Polyethylenimine-Based Delivery Systems.

Luis C Morales, Amarnath Rajendran, Aysha Ansari, Remant Kc, Mohammad Nasrullah, Kitipong Kiti, Panadda Yotsomnuk, Marianna Kulka, Daniel Nisakar Meenakshi Sundaram, Hasan Uludağ.

  Small interfering RNAs (siRNAs) have emerged as a powerful tool to manipulate gene expression in vitro. However, their potential therapeutic application encounters significant challenges, such as degradation in vivo, limited cellular uptake, and restricted biodistribution, among others. This study evaluates the siRNA delivery efficiency of three different lipid-substituted polyethylenimine (PEI)-based carriers, named Leu-Fect A-C, to different organs in vivo, including xenograft tumors, when injected into the bloodstream of mice. The siRNA analysis was undertaken by stem-loop RT-PCR, followed by qPCR or digital droplet PCR. Formulating siRNAs with a Leu-Fect series of carriers generated nanoparticles that effectively delivered the siRNAs into K652 and MV4-11 cells, both models of leukemia. The Leu-Fect carriers were able to successfully deliver BCR-Abl and FLT3 siRNAs into leukemia xenograft tumors in mice. All three carriers demonstrated significantly enhanced siRNA delivery into organs other than the liver, including the xenograft tumors. Preferential biodistribution of siRNAs was observed in the lungs and spleen. Among the delivery systems, Leu-Fect A exhibited the highest biodistribution into organs. In conclusion, lipid-substituted PEI-based delivery systems offer improvements in addressing pharmacokinetic challenges associated with siRNA-based therapies, thus opening avenues for their potential translation into clinical practice.

Keywords:  biodistribution; lipopolymer; nanoparticle; pharmacokinetics; siRNA

DOI:  https://doi.org/10.1021/acs.molpharmaceut.3c01077
Sci Rep. 2024 01 29. 14(1): 2403

A careful look at lipid nanoparticle characterization: analysis of benchmark formulations for encapsulation of RNA cargo size gradient.

Gretchen B Schober, Sandra Story, Dev P Arya.

With the recent success of lipid nanoparticle (LNP) based SARS-CoV-2 mRNA vaccines, the potential for RNA therapeutics has gained widespread attention. LNPs are promising non-viral delivery vectors to protect and deliver delicate RNA therapeutics, which are ineffective and susceptible to degradation alone. While food and drug administration (FDA) approved formulations have shown significant promise, benchmark lipid formulations still require optimization and improvement. In addition, the translatability of these formulations for several different RNA cargo sizes has not been compared under the same conditions. Herein we analyze "gold standard" lipid formulations for encapsulation efficiency of various non-specific RNA cargo lengths representing antisense oligonucleotides (ASO), small interfering RNA (siRNA), RNA aptamers, and messenger RNA (mRNA), with lengths of 10 bases, 21 base pairs, 96 bases, 996 bases, and 1929 bases, respectively. We evaluate encapsulation efficiency as the percentage of input RNA encapsulated in the final LNP product (EEinput%), which shows discrepancy with the traditional calculation of encapsulation efficiency (EE%). EEinput% is shown to be < 50% for all formulations tested, when EE% is consistently > 85%. We also compared formulations for LNP size (Z-average) and polydispersity index (PDI). LNP size does not appear to be strongly influenced by cargo size, which is a counterintuitive finding. Thoughtful characterization of LNPs, in parallel with consideration of in vitro or in vivo behavior, will guide design and optimization for better understanding and improvement of future RNA therapeutics.

DOI: https://doi.org/10.1038/s41598-024-52685-1
Acta Biomater. 2024 Jan 28. pii: S1742-7061(24)00044-8. [Epub ahead of print]

Pulmonary RNA Interference against Acute Lung Injury Mediated by Mucus- and Cell-penetrating Nanocomplexes.

Wenxin Zhang, Jingrui Shen, Jialong Liang, Chenglong Ge, Yang Zhou, Lichen Yin, Yong Ji.

  Trans-mucosal delivery of anti-inflammatory siRNA into alveolar macrophages represents a promising modality for the treatment of acute lung injury (ALI). However, its therapeutic efficacy is often hurdled by the lack of effective carriers that can simultaneously overcome the mucosal barrier and cell membrane barrier. Herein, we developed mucus/cell membrane dual-penetrating, macrophage-targeting polyplexes which enabled efficient intratracheal delivery of TNF-α siRNA (siTNF-α) to attenuate pulmonary inflammation against lipopolysaccharide (LPS)-induced ALI. P-G@Zn, a cationic helical polypeptide bearing both guanidine and zinc dipicolylamine (Zn-DPA) side charged groups, was designed to condense siTNF-α and promote macrophage internalization due to its helicity-dependent membrane activity. Coating of the polyplexes with charge-neutralizing carboxylated mannan (Man-COOH) greatly enhanced the mucus penetration potency due to shielding of the electrostatic adhesive interactions with the mucus, and it cooperatively enabled active targeting to alveolar macrophages to potentiate the intracellular delivery efficiency of siTNF-α. As such, intratracheally administered Man-COOH/P-G@Zn/siTNF-α polyplexes provoked notable TNF-α silencing by ∼75% in inflamed lung tissues at 500 μg siRNA/kg, and demonstrated potent anti-inflammatory performance to treat ALI. This study provides an effective tool for the synchronized trans-mucosal delivery of siRNA into macrophages, and the unique properties of the polyplexes render remarkable potentials for anti-inflammatory therapy against ALI. STATEMENT OF SIGNIFICANCE: siRNA-mediated anti-inflammatory management of acute lung injury (ALI) is greatly challenged by the insufficient delivery across the mucus layer and cell membrane. To address such critical issue, mucus/cell membrane dual-penetrating, macrophage-targeting polyplexes are herein developed, which are comprised of an outer shell of carboxylated mannan (Man-COOH) and an inner nanocore formed by TNF-α siRNA (siTNF-α) and a cationic helical polypeptide P-G@Zn. Man-COOH coating endowed the polyplexes with high mucus-penetrating capability and macrophage-targeting ability, while P-G@Zn bearing both guanidine and zinc dipicolylamine afforded potent siTNF-α condensation capacity and high intracellular delivery efficiency with reduced cytotoxicity. Intratracheally administered polyplexes solicit pronounced TNF-α silencing and anti-inflammatory efficiencies in ALI mice. This study renders an effective example for overcoming the multiple barriers against trans-mucosal delivery of siRNA into macrophages, and holds profound potentials for gene therapy against ALI.

Keywords:  Acute lung injury (ALI); cell penetrating α-helical polypeptide; macrophage targeting; mucus penetration; siRNA delivery

DOI:  https://doi.org/10.1016/j.actbio.2024.01.032
Pharm Nanotechnol. 2024 Jan 26.

Enhancing Gene Therapy through Ultradeformable Vesicles for Efficient siRNA Delivery.

Chintan Aundhia, Nirmal Shah, Chitrali Talele, Aarti Zanwar, Mamta Kumari, Sapana Patil.

  Gene therapy is a revolutionary approach aimed at treating various diseases by manipulating the expression of specific genes. The composition and formulation of ultra-deformable vesicles play a crucial role in determining their properties and performance as siRNA delivery vectors. In the development of ultra-deformable vesicles for siRNA delivery, careful lipid selection and optimization are crucial for achieving desirable vesicle characteristics and efficient siRNA encapsulation and delivery. The stratum corneum acts as a protective barrier, limiting the penetration of molecules, including siRNA, into the deeper layers of the skin. Ultradeformable vesicles offer a promising solution to overcome this barrier and facilitate efficient siRNA delivery to target cells in the skin. The stratum corneum, the outermost layer of the skin, acts as a significant barrier to the penetration of siRNA.These engineering approaches enable the production of uniform and well-defined vesicles with enhanced deformability and improved siRNA encapsulation efficiency. Looking ahead, advancements in ultra-deformable vesicle design and optimization, along with continued exploration of combination strategies and regulatory frameworks, will further drive the field of ultra-deformable vesicle-based siRNA delivery.

Keywords:  Drug delivery systems; Gene therapy; Lipid-based vesicle; Nanocarriers; Ultradeformable vesicles; siRNA delivery

DOI:  https://doi.org/10.2174/0122117385271654231215064542
J Chemother. 2024 Jan 31. 1-15

Self-assembled redox-responsive BRD4 siRNA nanoparticles: fomulation and its in vitro delivery in gastric cancer cells.

Mengying Zhang, Zhonghua An, Yiming Jiang, Meijiao Wei, Xiangbo Li, Yifan Wang, Hongbo Wang, Yanling Gong.

  With the development of newer biomarkers in the diagnosis of gastric cancer (GC), therapeutic targets are emerging and molecular-targeted therapy is in progress RNA interference has emerged as a promising method of gene targeting therapy. However, naked small interfering RNA (siRNA) is unstable and susceptible to degradation, so employing vectors for siRNA delivery is the focus of our research. Therefore, we developed LMWP modified PEG-SS-PEI to deliver siRNA targeting BRD4 (L-NPs/siBRD4) for GC therapy. L-NPs/siBRD4 were prepared by electrostatic interaction and characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The release characteristics, cellular uptake and intracellular localization were also investigated. The in vitro anticancer activity of the prepared nanoparticles was analysed by MTT, Transwell invasion and wound healing assay. Quantitative real time-polymerase chain reaction (qRT-PCR) and Western blot were used to detect the effect of gene silencing. The results showed that the optimal N/P was 30 and the prepared L-NPs/siBRD4 uniformly distributed in the system with a spherical and regular shape. L-NPs/siBRD4 exhibited an accelerated release in GSH-containing media from 12h to 24h. The uptake of L-NPs/siBRD4 was enhanced and mainly co-localized in the lysosomes. After 6h incubation, LMWP modified PEG-SS-PEI helped siRNA escape from the lysosomes and diffused into the cytoplasm. L-NPs/siBRD4 significantly inhibited the proliferation, migration and invasion of cells. This might be related with the silence of BRD4, then inhibition of PI3K/Akt and c-Myc. Our results demonstrate that L-NPs/siBRD4 are a novel delivery system with anticancer, which may provide a more effective strategy for GC treatment.

Keywords:  BRD4; LMWP; cell-penetrating peptides; disulfide; gastric cancer; nanoparticles; siRNA

DOI:  https://doi.org/10.1080/1120009X.2024.2308980
bioRxiv. 2024 Jan 15. pii: 2024.01.14.575599. [Epub ahead of print]

Lipid nanoparticle composition for adjuvant formulation modulates disease after influenza virus infection in QIV vaccinated mice.

Sonia Jangra, Alexander Lamoot, Gagandeep Singh, Gabriel Laghlali, Yong Chen, Tingting Yz, Adolfo García-Sastre, Bruno G De Geest, Michael Schotsaert.

Adjuvants can enhance vaccine effectiveness of currently licensed influenza vaccines. We tested influenza vaccination in a mouse model with two adjuvants: Sendai virus derived defective interfering (SDI) RNA, a RIG-I agonist, and an amphiphilic imidazoquinoline (IMDQ-PEG-Chol), TLR7/8 adjuvant. The negatively charged SDI RNA was formulated into lipid nanoparticles (LNPs) facilitating the direct delivery of a RIG-I agonist to the cytosol. We have previously tested SDI and IMDQ-PEG-Chol as standalone and combination adjuvants for influenza and SARS-CoV-2 vaccines. Here we tested two different ionizable lipids, K-Ac7-Dsa and S-Ac7-Dog, for LNP formulations. The adjuvanticity of IMDQ-PEG-Chol with and without empty or SDI-loaded LNPs was validated in a licensed vaccine setting (quadrivalent influenza vaccine or QIV) against H1N1 influenza virus, showing robust induction of antibody titres and T cell responses. Depending on the adjuvant combination and LNP lipid composition (K-Ac7-Dsa or S-Ac7-Dog lipids), humoral and cellular vaccine responses could be tailored towards type 1 or type 2 host responses with specific cytokine profiles that correlated with protection during viral infection. The extent of protection conferred by different vaccine/LNP/adjuvant combinations was examined against challenge with the vaccine-matching strain of H1N1 influenza A virus. Groups that received either LNP formulated with SDI, IMDQ-PEG-Chol or both showed very low levels of viral replication in their lungs at five days post virus infection. LNP ionizable lipid composition as well as loading (empty versus SDI) also skewed host responses to infection, as reflected in the cytokine and chemokine levels in lungs of vaccinated animals upon infection. These studies show the potential of LNPs as adjuvant delivery vehicles for licensed vaccines and illustrate the importance of LNP composition for subsequent host responses to infection, an important point of consideration for vaccine safety.

DOI: https://doi.org/10.1101/2024.01.14.575599
ACS Biomater Sci Eng. 2024 Feb 02.

Membrane-Cloaked Nanoparticles for RNA Interference of β-Catenin in Triple-Negative Breast Cancer.

Mackenzie A Scully, Ruth Wilhelm, Dana E Wilkins, Emily S Day.

  There is an outstanding need for targeted therapies for triple-negative breast cancer (TNBC), an aggressive breast cancer subtype. Since TNBC's rapid growth and metastasis are driven by hyperactive Wnt signaling, suppressing the key-pathway mediator β-catenin through RNA interference may improve patient outcomes. However, small interfering ribonucleic acid (siRNA) molecules require a carrier to elicit targeted gene silencing. Here, we show that 4T1 cancer cell membrane wrapped poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) can deliver siRNA into TNBC cells, silence β-catenin expression, and reduce the cells' tumorigenic qualities. Compared to unwrapped and nontargeted NPs, the cancer cell membrane wrapped nanoparticles (CCNPs) exhibit dramatically improved uptake by TNBC cells versus breast epithelial cells and greater gene silencing at mRNA and protein levels. Congruently, β-catenin siRNA-loaded CCNPs significantly activate senescence in 2D cultured TNBC cells and reduce proliferation in 3D spheroids. This work advances the development of nucleic acid carriers for targeted RNA interference therapy.

Keywords:  Wnt signaling; biomimetic; gene regulation; membrane-wrapped; nanomedicine; oncology; siRNA; targeted therapy

DOI:  https://doi.org/10.1021/acsbiomaterials.4c00160
Small. 2024 Jan 31. e2309200

pH-Dependent Lyotropic Liquid Crystalline Mesophase and Ionization Behavior of Phytantriol-Based Ionizable Lipid Nanoparticles.

Haitao Yu, Brendan Dyett, Nigel Kirby, Xudong Cai, Mohamad El Mohamad, Steven Bozinovski, Calum J Drummond, Jiali Zhai.

  Self-assembled lipid nanoparticles (LNPs), serving as essential nanocarriers in recent COVID-19 mRNA vaccines, provide a stable and versatile platform for delivering a wide range of biological materials. Notably, LNPs with unique inverse mesostructures, such as cubosomes and hexosomes, are recognized as fusogenic nanocarriers in the drug delivery field. This study delves into the physicochemical properties, including size, lyotropic liquid crystalline mesophase, and apparent pKa of LNPs with various lipid components, consisting of two ionizable lipids (ALC-0315 and SM-102) used in commercial COVID-19 mRNA vaccines and a well-known inverse mesophase structure-forming helper lipid, phytantriol (PT). Two partial mesophase diagrams are generated for both ALC-0315/PT LNPs and SM-102/PT LNPs as a function of two factors, ionizable lipid ratio (α, 0-100 mol%) and pH condition (pH 3-11). Furthermore, the impact of different LNP stabilizers (Pluronic F127, Pluronic F108, and Tween 80) on their pH-dependent phase behavior is evaluated. The findings offer insights into the self-assembled mesostructure and ionization state of the studied LNPs with potentially enhanced endosomal escape ability. This research is relevant to developing innovative next-generation LNP systems for delivering various therapeutics.

Keywords:  ALC-0315; SM-102; cubosome; hexosome; ionizable lipid; lipid nanoparticles; lyotropic liquid crystals; phytantriol

DOI:  https://doi.org/10.1002/smll.202309200
Nanoscale. 2024 Feb 01.

Multifunctional siRNA/ferrocene/cyclodextrin nanoparticles for enhanced chemodynamic cancer therapy.

Gowtham Raj, D S Vasudev, Sarah Christopher, Anupama Babulal, P Harsha, Soumakanya Ram, Mehul Tiwari, Markus Sauer, Reji Varghese.

The therapeutic outcome of chemodynamic therapy (CDT) is greatly hindered by the presence of oxidative damage repair proteins (MTH1) inside cancer cells. These oxidative damage repair proteins detoxify the action of radicals generated by Fenton or Fenton-like reactions. Hence, it is extremely important to develop a simple strategy for the downregulation of MTH1 protein inside cancer cells along with the delivery of metal ions into cancer cells. A one-pot host-guest supramolecular approach for the codelivery of MTH1 siRNA and metal ions into a cancer cell is reported. Our approach involves the fabrication of an inclusion complex between cationic β-cyclodextrin and a ferrocene prodrug, which spontaneously undergoes amphiphilicity-driven self-assembly to form spherical nanoparticles (NPs) having a positively charged surface. The cationic surface of the NPs was then explored for the loading of MTH1 siRNA through electrostatic interactions. Using HeLa cells as a representative example, efficient uptake of the NPs, delivery of MTH1 siRNA and the enhanced CDT of the nanoformulation are demonstrated. This work highlights the potential of the supramolecular approach as a simple yet efficient method for the delivery of siRNA across the cell membrane for enhanced chemodynamic therapy.

DOI: https://doi.org/10.1039/d3nr06071c
Bioconjug Chem. 2024 Jan 30.

Hydrophobicity Tuning of Cationic Polyaspartamide Derivatives for Enhanced Antisense Oligonucleotide Delivery.

Jongmin Yum, Fadlina Aulia, Keisuke Kamiya, Mao Hori, Nan Qiao, Beob Soo Kim, Mitsuru Naito, Satomi Ogura, Tetsuya Nagata, Takanori Yokota, Satoshi Uchida, Satoshi Obika, Hyun Jin Kim, Kanjiro Miyata.

Various cationic polymers are used to deliver polyplex-mediated antisense oligonucleotides (ASOs). However, few studies have investigated the structural determinants of polyplex functionalities in polymers. This study focused on the polymer hydrophobicity. A series of amphiphilic polyaspartamide derivatives possessing various hydrophobic (R) moieties together with cationic diethylenetriamine (DET) moieties in the side chain (PAsp(DET/R)s) were synthesized to optimize the R moieties (or hydrophobicity) for locked nucleic acid (LNA) gapmer ASO delivery. The gene knockdown efficiencies of PAsp(DET/R) polyplexes were plotted against a hydrophobicity parameter, logD7.3, of PAsp(DET/R), revealing that the gene knockdown efficiency was substantially improved by PAsp(DET/R) with logD7.3 higher than -2.4. This was explained by the increased polyplex stability and improved cellular uptake of ASO payloads. After intratracheal administration, the polyplex samples with a higher logD7.3 than -2.4 induced a significantly higher gene knockdown in the lung tissue compared with counterparts with lower hydrophobicity and naked ASO. These results demonstrate that the hydrophobicity of PAsp(DET/R) is crucial for efficient ASO delivery in vitro and in vivo.

DOI: https://doi.org/10.1021/acs.bioconjchem.3c00456
J Am Chem Soc. 2024 Feb 02.

The Constitutional Isomerism of One-Component Ionizable Amphiphilic Janus Dendrimers Orchestrates the Total and Targeted Activities of mRNA Delivery.

Dipankar Sahoo, Elena N Atochina-Vasserman, Devendra S Maurya, Mahwish Arshad, Srijay S Chenna, Nathan Ona, Jessica A Vasserman, Houping Ni, Drew Weissman, Virgil Percec.

Constitutional isomerism has been previously demonstrated by one of our laboratories to represent a powerful design strategy for the elaboration of complex functional self-organizations. Here we report the design, synthesis, and characterization of 14 positional, skeletal, and functional constitutional isomeric one-component, multifunctional, sequence-defined, amphiphilic ionizable Janus dendrimers (IAJDs). Their coassembly by simple injection with luciferase mRNA (Luc-mRNA) to form dendrimersome nanoparticles (DNPs) was studied. Subsequently, the resulting DNPs were employed to investigate, with screening experiments, the delivery of Luc-mRNA in vivo. Constitutional isomerism was shown to produce changes of up to two orders of magnitude of the total-body luciferase activity and targeted luciferase activity to the spleen and liver, of up to three orders of magnitude difference in targeted luciferase activity to the lungs and up to six orders of magnitude to lymph nodes. These results indicate that constitutional isomerism may represent not only a simple but also an important synthetic strategy that most probably may impact the activity of all components of synthetic vectors used in RNA-based nanomedicine, including in mRNA vaccines and therapeutics.

DOI: https://doi.org/10.1021/jacs.3c13569