bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2023–05–07
twenty-six papers selected by
the Merkel lab, Ludwig-Maximilians University



  1. J Mater Chem B. 2023 May 02.
      The efficacy of messenger RNA (mRNA)-based vaccines or therapies relies on delivery vehicles that can transport them into the cytosol of cells. Lipid nanoparticles (LNPs) are the most clinically advanced carrier for mRNA. The chemical structure of an ionizable lipid is critical for the delivery efficiency of the LNPs. Herein, we synthesize a new ionizable lipid containing fluorinated alkyl chains (F-L319) and evaluate its mRNA delivery efficiency compared to its hydrocarbon counterpart (L319). While LNPs formulated with F-L319 alone showed decreased mRNA encapsulation and delivery efficiencies in comparison to the L319-LNP, we found that combining the appropriate ratios of F-L319 and L319 as hybrid ionizable lipids in LNPs (hybrid-LNPs) greatly enhanced mRNA delivery efficiency both in vitro and in vivo. Upon intravenous injection, the hybrid-LNP showed targeted mRNA expression in the spleen. Mechanistic studies indicate that the enhanced mRNA delivery of the hybrid-LNP is attributed to both improved mRNA encapsulation and cellular uptake. Collectively, fluorination of ionizable lipids represents a promising strategy to improve the delivery efficiency of LNPs.
    DOI:  https://doi.org/10.1039/d3tb00516j
  2. Acta Pharm Sin B. 2023 Apr;13(4): 1429-1437
      Evasion of apoptosis is a hallmark of cancer, attributed in part to overexpression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2). In a variety of cancer types, including lymphoma, Bcl-2 is overexpressed. Therapeutic targeting of Bcl-2 has demonstrated efficacy in the clinic and is the subject of extensive clinical testing in combination with chemotherapy. Therefore, the development of co-delivery systems for Bcl-2 targeting agents, such as small interfering RNA (siRNA), and chemotherapeutics, such as doxorubicin (DOX), holds promise for enabling combination cancer therapies. Lipid nanoparticles (LNPs) are a clinically advanced nucleic acid delivery system with a compact structure suitable for siRNA encapsulation and delivery. Inspired by ongoing clinical trials of albumin-hitchhiking doxorubicin prodrugs, here we developed a DOX-siRNA co-delivery strategy via conjugation of doxorubicin to the surface of siRNA-loaded LNPs. Our optimized LNPs enabled potent knockdown of Bcl-2 and efficient delivery of DOX into the nucleus of Burkitts' lymphoma (Raji) cells, leading to effective inhibition of tumor growth in a mouse model of lymphoma. Based on these results, our LNPs may provide a platform for the co-delivery of various nucleic acids and DOX for the development of new combination cancer therapies.
    Keywords:  Bcl-2; Chemotherapy; Doxorubicin; Lipid nanoparticles; Lymphoma; siRNA delivery
    DOI:  https://doi.org/10.1016/j.apsb.2022.07.011
  3. Acta Pharm Sin B. 2023 Apr;13(4): 1400-1428
      Emerging therapies based on localized delivery of siRNA to lungs have opened up exciting possibilities for treatment of different lung diseases. Localized delivery of siRNA to lungs has shown to result in severalfold higher lung accumulation than systemic route, while minimizing non-specific distribution in other organs. However, to date, only 2 clinical trials have explored localized delivery of siRNA for pulmonary diseases. Here we systematically reviewed recent advances in the field of pulmonary delivery of siRNA using non-viral approaches. We firstly introduce the routes of local administration and analyze the anatomical and physiological barriers towards effective local delivery of siRNA in lungs. We then discuss current progress in pulmonary delivery of siRNA for respiratory tract infections, chronic obstructive pulmonary diseases, acute lung injury, and lung cancer, list outstanding questions, and highlight directions for future research. We expect this review to provide a comprehensive understanding of current advances in pulmonary delivery of siRNA.
    Keywords:  Acute lung injury; Injection routes; Lung cancer; Non-viral delivery; Particle pharmacokinetics; Pulmonary inflammation; Respiratory tract infections; siRNA
    DOI:  https://doi.org/10.1016/j.apsb.2022.07.010
  4. Nat Biomed Eng. 2023 May 01.
      Developing safe and effective nanoparticles for the delivery of messenger RNA (mRNA) is slow and expensive, partly due to the lack of predictive power of in vitro screening methods and the low-throughput nature of in vivo screening. While DNA barcoding and batch analysis present methods for increasing in vivo screening throughput, they can also result in incomplete or misleading measures of efficacy. Here, we describe a high-throughput and accurate method for the screening of pooled nanoparticle formulations within the same animal. The method uses liquid chromatography with tandem mass spectrometry to detect peptide barcodes translated from mRNAs in nanoparticle-transfected cells. We show the method's applicability by evaluating a library of over 400 nanoparticle formulations with 384 unique ionizable lipids using only nine mice to optimize the formulation of a biodegradable lipid nanoparticle for mRNA delivery to the liver. Barcoding lipid nanoparticles with peptide-encoding mRNAs may facilitate the rapid development of nanoparticles for mRNA delivery to specific cells and tissues.
    DOI:  https://doi.org/10.1038/s41551-023-01030-4
  5. Acta Pharm Sin B. 2023 Apr;13(4): 1348-1357
      Messenger RNA (mRNA) has drawn much attention in the medical field. Through various treatment approaches including protein replacement therapies, gene editing, and cell engineering, mRNA is becoming a potential therapeutic strategy for cancers. However, delivery of mRNA into targeted organs and cells can be challenging due to the unstable nature of its naked form and the low cellular uptake. Therefore, in addition to mRNA modification, efforts have been devoted to developing nanoparticles for mRNA delivery. In this review, we introduce four categories of nanoparticle platform systems: lipid, polymer, lipid-polymer hybrid, and protein/peptide-mediated nanoparticles, together with their roles in facilitating mRNA-based cancer immunotherapies. We also highlight promising treatment regimens and their clinical translation.
    Keywords:  Cancer immunotherapy; Lipid nanoparticles; Lipid–polymer hybrid nanoparticles; Messenger RNA; Polymeric nanoparticles; Protein/peptide-mediated nanoparticles; mRNA delivery
    DOI:  https://doi.org/10.1016/j.apsb.2023.03.001
  6. Nanomedicine (Lond). 2023 Apr 26.
      Aims: Achieving an effective biocompatible system for siRNAs delivery to the tumor site remains a significant challenge. Materials & methods: Selenium nanoparticles (SeNPs) modified by chitosan (CS) and hyaluronic acid (HA) were fabricated for PLK1 siRNAs (siPLK1) delivery to the bladder cancer cells. The HA-CS-SeNP@siPLK1 efficacy was evaluated using in vitro and in vivo models. Results: HA-CS-SeNP@siPLK1 was selectively internalized into T24 cells through clathrin-mediated endocytosis. Treatment with HA-CS-SeNP@siPLK1 successfully silenced the PLK1 gene, inhibited cell proliferation and induced cell cycle arrest in vitro. HA-CS-SeNP@siPLK1 could also inhibit tumor growth in vivo without causing systemic toxicity. Conclusion: Our results suggest that HA-CS-SeNPs may provide a good vehicle for delivering siPLK1 to the bladder tumor site.
    Keywords:  PLK1; bladder cancer; chitosan; gene therapy; selenium nanoparticles; siRNA delivery
    DOI:  https://doi.org/10.2217/nnm-2022-0198
  7. J Cancer Res Ther. 2023 Apr;19(Supplement): S278-S284
       Background: The poor response of metastatic osteo- and chondrosarcomas to chemotherapy could be the result of multidrug resistance (MDR), which may be overcome through the use of small interfering RNA (siRNA). However, several methodologic questions remain unresolved.
    Aims: To test the toxicity of three commonly used siRNA transfection reagents and apply the least toxic reagent to investigate the siRNA-induced MDR1 mRNA knockdown.
    Methods: The toxicity of TransIT-TKO, Lipofectamine 2000, and X-tremeGENE siRNA transfection reagents was investigated on osteosarcoma (MG-63) and chondrosarcoma (SW1353) cell lines. The toxicity was measured at 4 and 24 hours using a MTT toxicity assay. The least toxic transfection reagent was applied to investigate the siRNA-induced MDR1 mRNA knockdown effect using qRT-PCR. Furthermore, five housekeeping genes were assessed in the BestKeeper software to obtain mRNA expression normalization.
    Results: Lipofectamine 2000 was the least toxic transfection reagent, reducing the cell viability only in chondrosarcoma 24 hours following exposure to the highest dose. In contrast, TransIT-TKO and X-tremeGENE transfection reagents displayed a significant reduction in cell viability in both chondrosarcoma after 4 hours and in osteosarcoma after 24 hours. Significant MDR1 mRNA silencing of over 80% was achieved in osteo- and chondrosarcoma using Lipofectamine at a final siRNA concentration of 25 nM. No significant dose response was observed in knockdown efficiency in either Lipofectamine or siRNA concentration.
    Conclusion: Lipofectamine 2000 was the least toxic transfection reagent in osteo- and chondrosarcoma. Successful siRNA-induced MDR1 mRNA silencing of over 80% was achieved.
    Keywords:  Chondrosarcoma; MDR1; housekeeping genes; mRNA silencing; osteosarcoma; siRNA transfection reagents
    DOI:  https://doi.org/10.4103/jcrt.JCRT_565_19
  8. Mol Ther Methods Clin Dev. 2023 Jun 08. 29 271-283
      Many diseases, especially cancer, are caused by the abnormal expression of non-coding microRNAs (miRNAs), which regulate gene expression, leading to the development of miRNA-based therapeutics. Synthetic miRNA inhibitors have shown promising efficacy in blocking the activity of aberrant miRNAs that are upregulated in disease-specific pathologies. On the other hand, miRNAs that aid in preventing certain diseases and are reduced in expression in the disease state need different strategies. To tackle this, miRNA mimics, which mimic the activity of endogenous miRNAs, can be delivered for those miRNAs downregulated in different disease states. However, the delivery of miRNA mimics remains a challenge. Here, we report a cationic polylactic-co-glycolic acid (PLGA)-poly-L-histidine delivery system to deliver miRNA mimics. We chose miR-34a mimics as a proof of concept for miRNA delivery. miR-34a-loaded PLGA-poly-L-histidine nanoparticles (NPs) were formulated and biophysically characterized to analyze the structural properties of miRNA mimic-loaded NPs. In vitro efficacy was determined by investigating miR-34a and downstream target levels and performing cell viability and apoptosis assays. We confirmed in vivo efficacy through prolonged survival of miR-34a NP-treated A549-derived xenograft mice treated intratumorally. The results of these studies establish PLGA-poly-L-histidine NPs as an effective delivery system for miRNA mimics for treating diseases characterized by downregulated miRNAs.
    Keywords:  PLGA; PLGA-poly-L-histidine; miR-34a mimic; miRNA mimic; miRNA mimic delivery
    DOI:  https://doi.org/10.1016/j.omtm.2023.03.015
  9. Nanotechnology. 2023 May 02.
      Acute lung injury (ALI) can be induced by various injury factors, which is closely related to the inflammatory reaction and cellular ferroptosis reported recently. Glutathione peroxidase (GPX4) palys an important role in the inflammatory reaction, which also is the core regulatory protein of ferroptosis. Up-regulation of GPX4 can be helpful to inhibit the cellular ferroptosis and inflammatory reaction to treat ALI. mPEI/pGPX4 gene therapeutic system based on mannitol-modified polyethyleneimine (mPEI) was constructed. Compared with PEI/pGPX4 nanoparticles using commoditized gene vector PEI 25k, mPEI/pGPX4 nanoparticles achieved caveolae-mediated endocytosis and improved the gene therapeutic effect. mPEI/pGPX4 nanoparticles could up-regulate the gene expression of GPX4, inhibit inflammatory reaction and the cellular ferroptosis, thereby alleviating the acute lung injury in vitro and in vivo. The finding indicated that gene therapy with pGPX4 is a potential therapeutic system for the effective treatment of acute lung injury.
    Keywords:  GPX4; acute lung injury; ferroptosis; gene delivery; gene therapeutic system; plasmid DNA
    DOI:  https://doi.org/10.1088/1361-6528/acd198
  10. Acta Pharm Sin B. 2023 Apr;13(4): 1383-1399
      Exosome is an excellent vesicle for in vivo delivery of therapeutics, including RNAi and chemical drugs. The extremely high efficiency in cancer regression can partly be attributed to its fusion mechanism in delivering therapeutics to cytosol without endosome trapping. However, being composed of a lipid-bilayer membrane without specific recognition capacity for aimed-cells, the entry into nonspecific cells can lead to potential side-effects and toxicity. Applying engineering approaches for targeting-capacity to deliver therapeutics to specific cells is desirable. Techniques with chemical modification in vitro and genetic engineering in cells have been reported to decorate exosomes with targeting ligands. RNA nanoparticles have been used to harbor tumor-specific ligands displayed on exosome surface. The negative charge reduces nonspecific binding to vital cells with negatively charged lipid-membrane due to the electrostatic repulsion, thus lowering the side-effect and toxicity. In this review, we focus on the uniqueness of RNA nanoparticles for exosome surface display of chemical ligands, small peptides or RNA aptamers, for specific cancer targeting to deliver anticancer therapeutics, highlighting recent advances in targeted delivery of siRNA and miRNA that overcomes the previous RNAi delivery roadblocks. Proper understanding of exosome engineering with RNA nanotechnology promises efficient therapies for a wide range of cancer subtypes.
    Keywords:  Chemical drug delivery; Endolysosome trapping; Exosome engineering; RNA interference; RNA nanotechnology; Targeted delivery
    DOI:  https://doi.org/10.1016/j.apsb.2022.11.019
  11. Int J Pharm. 2023 May 02. pii: S0378-5173(23)00432-5. [Epub ahead of print] 123012
      To develop a combinatorial artificial-neural-network design-of-experiment (ANN-DOE) model, the effect of ionizable lipid, an ionizable lipid-to-cholesterol ratio, N/P ratio, flow rate ratio (FRR), and total flow rate (TFR) on the outcome responses of mRNA-LNP vaccine were evaluated using a definitive screening design (DSD) and machine learning (ML) algorithms. Particle size (PS), PDI, zeta potential (ZP), and encapsulation efficiency (EE) of mRNA-LNP were optimized within a defined constraint (PS 40-100 nm, PDI≤0.30, ZP≥(±)0.30 mV, EE≥70%), fed to ML algorithms (XGBoost, bootstrap forest, support vector machines, k-nearest neighbors, generalized regression-Lasso, ANN) and prediction was compared to ANN-DOE model. Increased FRR decreased the PS and increased ZP, while increased TFR increased PDI and ZP. Similarly, DOTAP and DOTMA produced higher ZP and EE. Particularly, a cationic ionizable lipid with an N/P ratio ≥6 provided a higher EE. ANN showed better predictive ability (R2=0.7269-0.9946), while XGBoost demonstrated better RASE (0.2833-2.9817). The ANN-DOE model outperformed both optimized ML models by R2=1.21% and RASE=43.51% (PS prediction), R2=0.23% and RASE=3.47% (PDI prediction), R2=5.73% and RASE=27.95% (ZP prediction), and R2=0.87% and RASE=36.95% (EE prediction), respectively, which demonstrated that ANN-DOE model was superior in predicting the bioprocess compared to independent models.
    Keywords:  XGBoost; artificial-neural-network design-of-experiment; lipid nanoparticle (LNP); machine learning; messenger RNA; support vector machines
    DOI:  https://doi.org/10.1016/j.ijpharm.2023.123012
  12. J Biomed Mater Res B Appl Biomater. 2023 May 03.
      The goal of the study was to estimate transfection efficacy and drug release in function of the PEG derivative in cationic liposomes and lipoplexes in both 2D and 3D in vitro models as well as in a mouse model (in vivo). For this purpose, cationic PEGylated nanocarriers based on OrnOrnGlu(C16 H33 )2 lipopeptides were fabricated and characterized. The nanocarriers were loaded with DNA plasmid pGL3 or with siRNA targeting 5'-UTR region of Hepatitis C virus, and their transfection efficacies were studied by luciferase test or by PCR technique, respectively. The pGL3-lipoplexes containing PEG derivative b (6 mol % PEG) were selected as the most promising nanocarriers for further in vivo study. In vitro cytotoxicity assay of the pGL3-lipoplexes with the PEG derivative b showed 2- and 1.5-fold enhancements of IC50 levels for HEK293T and HepG2 cells, respectively. Accumulation of the liposomes in the cells was studied by confocal microscopy using both 2D (monolayer culture) and 3D (multicellular spheroids) in vitro models. The PEGylated liposomes were found to penetrate cells more slowly than unmodified ones (without PEG). Thus, maximum liposomes in the HEK293T cells was observed after 1 and 3 h in the case of 2D and 3D in vitro models, respectively. Biodistribution study in mice showed that the PEGylated lipoplexes containing the PEG derivative b were eliminated from the bloodstream more slowly, namely with the doubled half-life time, than unmodified ones. Thus, the enhanced transfection efficacy and prolonged drug release of the PEGylated lipoplexes containing the optimal PEG derivative was demonstrated. This approach could be promising for development of novel siRNA-based drugs.
    Keywords:  3D in vitro model; DNA plasmid; PEG derivative; PEGylation; biodistribution study; lipoplexes; liposomes; multicellular spheroids; siRNA delivery; transfection
    DOI:  https://doi.org/10.1002/jbm.b.35259
  13. Biomaterials. 2023 Apr 26. pii: S0142-9612(23)00141-2. [Epub ahead of print]298 122133
      Liver fibrosis is a chronic disease without effective treatment in the clinic. Gene editing systems such as the well-known CRISPR/Cas9 have shown great potential in the biomedical field. However, the delivery of the ribonucleoprotein is challenging due to the unstable RNA probe and the requirement for the entrance to the nucleus. Recently, a structure-guided endonuclease (SGN) has been reported as an effective gene-editing system composed of a nuclease and stable DNA probes, which can regulate the protein expression by targeting specific mRNA outside the nucleus. Here, we conjugated the SGN to a nanomicelle as the delivery system. In the resulting material, the chance of the collision between the endonuclease and the probe was raised due to the confinement of the two components within the 40-nm nanomicelle, thus the mRNA can be cleaved immediately after being captured by the probe, resulting in a space-induced nucleotide identification-cleavage acceleration effect. The delivery system was used to treat liver fibrosis via the co-delivery of SGN and a drug rosiglitazone to the hepatic stellate cells, which separately downregulated the expression of tissue inhibitor of metalloprotease-1 and inactivated the hepatic stellate cells. The system successfully reversed the liver fibrosis in mice through the bidirectional regulatory that simultaneously promoted the degradation and inhibited the production of the collagen, demonstrating the great potency of the SGN system as gene medicine.
    Keywords:  Gene editing; Liver fibrosis; Nanocarrier; Ribonucleoprotein; mRNA
    DOI:  https://doi.org/10.1016/j.biomaterials.2023.122133
  14. ACS Nano. 2023 Apr 25.
      Since glioblastomas (GBMs) are radioresistant malignancies and most GBM recurrences occur in radiotherapy, increasing the effectiveness of radiotherapy by gene-silencing has recently attracted attention. However, the difficulty in precisely tuning the composition and RNA loading in nanoparticles leads to batch-to-batch variations of the RNA therapeutics, thus significantly restricting their clinical translation. Here, we bioengineer bacteriophage Qβ particles with a designed broccoli light-up three-way junction (b-3WJ) RNA scaffold (contains two siRNA/miRNA sequences and one light-up aptamer) packaging for the silencing of genes in radioresistant GBM cells. The in vitro results demonstrate that the cleavage of de novo designed b-3WJ RNA by Dicer enzyme can be easily monitored in real-time using fluorescence microscopy, and the TrQβ@b-3WJLet-7gsiEGFR successfully knocks down EGFR and IKKα simultaneously and thereby inactivates NF-κB signaling to inhibit DNA repair. Delivery of TrQβ@b-3WJLet-7gsiEGFR through convection-enhanced delivery (CED) infusion followed by 2Gy X-ray irradiation demonstrated that the median survival was prolonged to over 60 days compared with the 2Gy X-ray irradiated group (median survival: 31 days). Altogether, the results of this study could be critical for the design of RNAi-based genetic therapeutics, and CED infusion serves as a powerful delivery system for promoting radiotherapy against GBMs without evidence of systemic toxicity.
    Keywords:  3WJ RNAs; RNAi therapeutics; brain tumors; gene silencing; radioresistance; virus-like particles (VLPs)
    DOI:  https://doi.org/10.1021/acsnano.3c01102
  15. J Control Release. 2023 Apr 28. pii: S0168-3659(23)00296-1. [Epub ahead of print]
      MiRNA-based gene therapy as a novel targeted therapy has yielded promising results in experimental cancer treatment, however, the inefficient delivery of miRNA to target tissues has limited its application in vivo. Here a unique dual-membrane-camouflaged miRNA21 antagomir delivery nanoplatform (M@NPs/miR21) with immune escape and homologous targeting properties was constructed by cancer cell membrane and macrophage membrane. Different from the single-cell membrane camouflage strategy, the dual-membrane camouflage miRNA21 antagomir delivery nanoplatform based on modification of CD47 protein with immune escape signal and galectin-3 protein with tumor cell aggregation enables efficient, safe and targeted therapy for colon cancer and lung metastases. Camouflaged with the dual-cell membrane, the "Trojan horse" like "pseudo-tumor cell" and/or "pseudo-macrophage" (M@NPs/miR21) carried the target gene miR21 antagomir to the tumor site and showed significant anti-tumor properties at the periphery and the core of subcutaneous tumor tissues. In addition, M@NPs/miR21 was more likely to penetrate dense tumor tissues and function within the tumor mass than NPs/miR21 without membrane coating. M@NPs/miR21 can deliver miR21 antagomir into MC38 cancer cells and tumor tissues, promote tumor apoptosis, and regulate the expression of Bcl2 and Ki67. Moreover, the M@NPs/miR21 gene delivery system not only can effectively inhibit the progression of subcutaneous tumors and lung metastases, but also showed minimal toxicity and good biosafety, making this delivery system particularly attractive for future translational research.
    Keywords:  Dual-membrane camouflage; Gene delivery system; Homologous targeting; Immune escape; cancer therapy
    DOI:  https://doi.org/10.1016/j.jconrel.2023.04.046
  16. Mol Pharm. 2023 May 04.
      Nanomedicine represents a promising way to devise better drug delivery systems (DDSs), and the development of cell/tissue-based lipid carriers is a promising strategy. In this study, the author proposes the concept of reconstituted lipid nanoparticles (rLNPs) and offers a facile preparation method. The results demonstrated that the preparation of ultrasmall (∼20 nm) rLNPs can be highly reproducible from both cells (a mouse breast cancer cell line, 4T1) and tissue (mouse liver tissue). As a selected model platform, rLNPs derived from mouse liver tissue can be further labeled with imaging molecules (indocyanine green and coumarin 6) and modified with targeting moiety (biotin). Moreover, rLNPs were proved to be highly biocompatible and able to load various drugs, such as doxorubicin hydrochloride (Dox) and curcumin (Cur). Most importantly, Dox-loaded rLNPs (rLNPs/Dox) exerted good in vitro and in vivo anticancer performances. Therefore, rLNPs might be a potential versatile carrier for the construction of different DDSs and treatment of a variety of diseases.
    Keywords:  drug delivery systems; reconstituted lipid nanoparticles; solvent diffusion method
    DOI:  https://doi.org/10.1021/acs.molpharmaceut.2c01033
  17. J Math Biol. 2023 Apr 30. 86(5): 86
      A compartment model for an in-host liquid nanoparticle delivered mRNA vaccine is presented. Through non-dimensionalisation, five timescales are identified that dictate the lifetime of the vaccine in-host: decay of interferon gamma, antibody priming, autocatalytic growth, antibody peak and decay, and interleukin cessation. Through asymptotic analysis we are able to obtain semi-analytical solutions in each of the time regimes which allows us to predict maximal concentrations and better understand parameter dependence in the model. We compare our model to 22 data sets for the BNT162b2 and mRNA-1273 mRNA vaccines demonstrating good agreement. Using our analysis, we estimate the values for each of the five timescales in each data set and predict maximal concentrations of plasma B-cells, antibody, and interleukin. Through our comparison, we do not observe any discernible differences between vaccine candidates and sex. However, we do identify an age dependence, specifically that vaccine activation takes longer and that peak antibody occurs sooner in patients aged 55 and greater.
    Keywords:  In-host modelling; MRNA-based vaccines; Model reduction; Vaccines; Waning immunity
    DOI:  https://doi.org/10.1007/s00285-023-01919-3
  18. Biomed Pharmacother. 2023 Apr 27. pii: S0753-3322(23)00578-4. [Epub ahead of print]163 114789
      There is growing evidence showing that single administration of immunotherapeutic agents has limited efficacy in a number of cancer patients mainly due to tumor heterogeneity and immunosuppressive tumor microenvironment. In this study, a novel nanoparticle-based strategy was applied to achieve efficient tumor-targeted therapy by combining chemotherapeutic agents, i.e., doxorubicin (Dox) and melittin (Mel), with an immune checkpoint inhibitor (PD-L1 DsiRNA). The proposed nanoparticle was prepared by the formation of a complex between Mel and PD-L1 DsiRNA (Dicer-substrate short-interfering RNA), followed by the loading of Dox. The surface of the resultant particles (DoxMel/PD-L1 DsiRNA) was then modified with hyaluronic acid (HA) to increase their stability and distribution. In addition, HA can also act as a tumor-targeting agent through binding to its receptor CD44 on the surface of cancer cells. We demonstrated that the surface engineering of DoxMel/PD-L1 DsiRNA with HA significantly enhances its specificity towards breast cancer cells. Moreover, we observed a noticeable reduction in PD-L1 expression together with a synergistic effect of Dox and Mel on killing cancer cells and inducing immunogenic cell death, leading to significantly diminished tumor growth in 4T1-breast tumor bearing Balb/c mice, improved survival rate and extensive infiltration of immune cells including cytotoxic T cells into the tumor microenvironment. Safety analysis revealed that there is no significant toxicity associated with the developed nanoparticle. All in all, the proposed targeted combination treatment strategy can be considered as a useful method to reduce cancer-associated mortality.
    Keywords:  Breast cancer; Cancer chemotherapy; Cancer immunotherapy; Nanoparticle; Targeted delivery
    DOI:  https://doi.org/10.1016/j.biopha.2023.114789
  19. Chem Commun (Camb). 2023 May 05.
      To ensure specificity of small interfering RNAs (siRNAs), the antisense strand must be selected by the RNA-induced silencing complex (RISC). We have previously demonstrated that a 5'-morpholino-modified nucleotide at the 5'-end of the sense strand inhibits its interaction with RISC ensuring selection of the desired antisense strand. To improve this antagonizing binding property even further, a new set of morpholino-based analogues, Mo2 and Mo3, and a piperidine analogue, Pip, were designed based on the known structure of Argonaute2, the slicer enzyme component of RISC. Sense strands of siRNAs were modified with these new analogues, and the siRNAs were evaluated in vitro and in mice for RNAi activity. Our data demonstrated that Mo2 is the best RISC inhibitor among the modifications tested and that it effectively mitigates sense strand-based off-target activity of siRNA.
    DOI:  https://doi.org/10.1039/d3cc01143g
  20. Nature. 2023 May 02.
      Messenger RNA (mRNA) vaccines are being used to contain COVID-19 (1, 2, 3), but still suffer from the critical limitation of mRNA instability and degradation, which is a major obstacle in the storage, distribution, and efficacy of the vaccine products (4). Previous work showed that increasing secondary structure lengthens mRNA half-life, which, together with optimal codons, improves protein expression (5). Therefore, a principled mRNA design algorithm must optimize both structural stability and codon usage. However, due to synonymous codons, the mRNA design space is prohibitively large (e.g., ~10632 candidates for the SARS-CoV-2 Spike protein), which poses insurmountable computational challenges. Here we provide a simple and unexpected solution using a classical concept in computational linguistics, where finding the optimal mRNA sequence is akin to identifying the most likely sentence among similar sounding alternatives (6). Our algorithm LinearDesign takes only 11 minutes for the Spike protein, and can jointly optimize stability and codon usage. On both COVID-19 and varicella-zoster virus mRNA vaccines, LinearDesign substantially improves mRNA half-life and protein expression, and dramatically increases antibody titer by up to 128× in vivo, compared to the codon-optimization benchmark. This surprising result reveals the great potential of principled mRNA design, and enables the exploration of previously unreachable but highly stable and efficient designs. Our work is a timely tool not only for vaccines but also for mRNA medicine encoding all therapeutic proteins (e.g., monoclonal antibodies and anti-cancer drugs (7, 8)).
    DOI:  https://doi.org/10.1038/s41586-023-06127-z
  21. bioRxiv. 2023 Apr 18. pii: 2023.04.17.537186. [Epub ahead of print]
      The liver is known for its remarkable regenerative ability through proliferation of hepatocytes. Yet, during chronic injury or severe hepatocyte death, proliferation of hepatocytes is exhausted. To overcome this hurdle, we propose vascular-endothelial-growth-factor A (VEGFA) as a therapeutic means to accelerate biliary epithelial cell (BEC)-to-hepatocyte conversion. Investigation in zebrafish establishes that blocking VEGF receptors abrogates BEC-driven liver repair, while VEGFA overexpression promotes it. Delivery of VEGFA via non-integrative and safe nucleoside-modified mRNA encapsulated into lipid-nanoparticles (mRNA-LNP) in acutely or chronically injured mouse livers induces robust BEC-to-hepatocyte conversion and reversion of steatosis and fibrosis. In human and murine diseased livers, we further identified VEGFA-receptor KDR-expressing BECs associated with KDR-expressing cell-derived hepatocytes. This defines KDR-expressing cells, most likely being BECs, as facultative progenitors. This study reveals novel therapeutic benefits of VEGFA delivered via nucleoside-modified mRNA-LNP, whose safety is widely validated with COVID-19 vaccines, for harnessing BEC-driven repair to potentially treat liver diseases.
    Highlights: Complementary mouse and zebrafish models of liver injury demonstrate the therapeutic impact of VEGFA-KDR axis activation to harness BEC-driven liver regeneration.VEGFA mRNA LNPs restore two key features of the chronic liver disease in humans such as steatosis and fibrosis.Identification in human cirrhotic ESLD livers of KDR-expressing BECs adjacent to clusters of KDR+ hepatocytes suggesting their BEC origin.KDR-expressing BECs may represent facultative adult progenitor cells, a unique BEC population that has yet been uncovered.
    DOI:  https://doi.org/10.1101/2023.04.17.537186
  22. Rep Biochem Mol Biol. 2023 Jan;11(4): 599-613
       Background: This study aims to prepare high stability chitosan nanoparticles (CNP) and examine the ability of CNP in CpG-ODN delivery when treating allergic mice model.
    Methods: Preparation and characterization of CNP were performed by ionic gelation, dynamic light scattering, and zeta sizer. The CNP cytotoxicity and activation ability of CpG ODN delivered with CNP were tested using a cell counting kit-8 and Quanti blue method. Allergic mice were injected intraperitoneal with 10 ug ovalbumin on day 0 and 7, and then treated with intranasal CpG ODN/CpG ODN, delivered with CNP/CNP, on the third week three times per week for three weeks. The ELISA method measured cytokine and IgE profiles in the allergic mice's plasma and spleen.
    Results: CNP results have sizes 27.73 nm±3.67 dan 188.23 nm±53.47, spherical in shape and non-toxic, and did not alter the NF-κB activation of CpG ODN in RAW-blue cells. The application of CpG ODN delivered by chitosan nanoparticles shows no statistical difference between groups of IFN-γ, IL-10, and IL-13 in Balb/c mice's plasma and spleen, in contrast with IgE level.
    Conclusions: The results showed that using chitosan nanoparticles as a delivery system for CpG ODN has the potency to safely CpG ODN efficacy.
    Keywords:  Allergy; Chitosan nanoparticle; CpG ODN; Immunotherapy; Mice spleen
    DOI:  https://doi.org/10.52547/rbmb.11.4.599
  23. J Inflamm Res. 2023 ;16 1867-1877
       Background: SARS-CoV-2-induced acute lung injury but its nucleocapsid (N) and/or Spike (S) protein involvements in the disease pathology remain elusive.
    Methods: In vitro, the cultured THP-1 macrophages were stimulated with alive SARS-CoV-2 virus at different loading dose, N protein or S protein with/without TICAM2-siRNA, TIRAP-siRNA or MyD88-siRNA. The TICAM2, TIRAP and MyD88 expression in the THP-1 cells after N protein stimulation were determined. In vivo, naïve mice or mice with depletion macrophages were injected with N protein or dead SARS-CoV-2. The macrophages in the lung were analyzed with flow cytometry, and lung sections were stained with H&E or immunohistochemistry. Culture supernatants and serum were harvested for cytokines measurements with cytometric bead array.
    Results: Alive SARS-CoV-2 virus or N protein but not S protein induced high cytokine releases from macrophages in a time or virus loading dependent manner. MyD88 and TIRAP but not TICAM2 were highly involved in macrophage activation triggered by N protein whilst both inhibited with siRNA decreased inflammatory responses. Moreover, N protein and dead SARS-CoV-2 caused systemic inflammation, macrophage accumulation and acute lung injury in mice. Macrophage depletion in mice decreased cytokines in response to N protein.
    Conclusion: SARS-CoV-2 and its N protein but not S protein induced acute lung injury and systemic inflammation, which was closely related to macrophage activation, infiltration and release cytokines.
    Keywords:  N protein; S protein; SARS-CoV-2; acute lung injury; cytokine; macrophage
    DOI:  https://doi.org/10.2147/JIR.S405722
  24. J Blood Med. 2023 ;14 317-327
      Severe hemophilia is associated with spontaneous, prolonged and recurrent bleeding. Inadequate prevention and treatment of bleeding can lead to serious morbidity and mortality. Due to the limitations of intravenous clotting factor replacement, including the risk of inhibitory antibodies, innovative novel therapies have been developed that have dramatically changed the landscape of hemophilia therapy. Ribonucleic acid interference (RNAi) has brought the opportunity for multiple strategies to manipulate the hemostatic system and ameliorate the bleeding phenotype in severe bleeding disorders. Fitusiran is a RNAi therapeutic that inhibits the expression of the natural anticoagulant serpin antithrombin. Reduction in antithrombin is known to cause thrombosis if coagulation parameters are otherwise normal and can rebalance hemostasis in severe hemophilia. Reports from late stage clinical trials of fitusiran in hemophilia A and B participants, with and without inhibitory antibodies to exogenous clotting factor, have demonstrated efficacy in preventing bleeding events showing promise for a future "universal" prophylactic treatment of individuals with moderate-severe hemophilia.
    Keywords:  RNAi; fitusiran; hemophilia; inhibitor; ribonucleic acid interference; siRNA; small integral RNA
    DOI:  https://doi.org/10.2147/JBM.S390521
  25. Int J Pharm. 2023 Apr 29. pii: S0378-5173(23)00410-6. [Epub ahead of print] 122990
      Intranasal vaccination by directly applying a vaccine dry powder is appealing. However, a method that can be used to transform a vaccine from a liquid to a dry powder and a device that can be used to administer the powder to the desired region(s) of the nasal cavity are critical for successful intranasal vaccination. In the present study, using a model vaccine that contains liposomal monophosphoryl lipid A and QS-21 adjuvant (AdjLMQ) and ovalbumin (OVA) as a model antigen, it was shown that thin-film freeze-drying can be applied to convert the liquid vaccine containing sucrose at a sucrose to lipid ratio of 15:1 (w/w) into dry powders, in the presence or absence of carboxymethyl cellulose sodium salt (CMC) as a mucoadhesive agent. Ultimately, the thin-film freeze-dried AdjLMQ/OVA vaccine powder containing 1.9% (w/w) of CMC (i.e., TFF AdjLMQ/OVA/CMC1.9% powder) was selected for additional evaluation because the TFF AdjLMQ/OVA/CMC1.9% powder was mucoadhesive and maintained the integrity of the antigen and the physical properties of the vaccine. Compared to the TFF AdjLMQ/OVA powder that did not contain CMC, the TFF AdjLMQ/OVA/CMC1.9% powder had a lower moisture content and a higher glass transition temperature. In addition, the TFF AdjLMQ/OVA/CMC1.9% thin films were relatively thicker than the TFF AdjLMQ/OVA thin films without CMC. When sprayed with Aptar Pharma's Unidose Powder Nasal Spray System (UDSP), the TFF AdjLMQ/OVA powder and the TFF AdjLMQ/OVA/CMC1.9% powder generated similar particle size distribution curves, spray patterns, and plume geometries. Importantly, after the TFF AdjLMQ/OVA/CMC1.9% powder was sprayed with the UDSP nasal device, the integrity of the OVA antigen and the AdjLMQ liposomes did not change. Finally, a Taguchi L4 orthogonal array was applied to identify the optimal parameters for using the UDSP device to deliver the TFF AdjLMQ/OVA/CMC1.9% vaccine powder to the middle and lower turbinate and the nasopharynx regions in both adult and child nasal replica casts. Results from this study showed that it is feasible to apply the TFF technology to transform a nasal vaccine candidate from liquid to a dry powder and then use the UDSP nasal device to deliver the TFF vaccine powder to the desired regions in the nasal cavity for intranasal vaccination.
    Keywords:  Vaccine; adult; freeze-drying; nasal; pediatric; powder
    DOI:  https://doi.org/10.1016/j.ijpharm.2023.122990