bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2022‒06‒12
thirteen papers selected by
the Merkel lab
Ludwig-Maximilians University
  1. Liposomal siRNA Delivery in Papillary Thyroid Carcinoma Cells.
  2. Optimal combination of cationic lipid and phospholipid in cationic liposomes for gene knockdown in breast cancer cells and mouse lung using siRNA lipoplexes.
  3. Lipid nanoparticle formulations for optimal RNA-based topical delivery to murine airways.
  4. Interleukin-12 Plasmid DNA Delivery by N-[(2-Hydroxy-3-trimethylammonium)propyl]chitosan-Based Nanoparticles.
  5. Advances in the polymeric delivery of nucleic acid vaccines.
  6. Novel nanocarriers for silencing anti-phagocytosis CD47 marker in acute myeloid leukemia cells.
  7. Emerging approaches for enabling RNAi therapeutics.
  8. BSA-PEI Nanoparticle Mediated Efficient Delivery of CRISPR/Cas9 into MDA-MB-231 Cells.
  9. Optimal self-assembly of lipid nanoparticles (LNP) in a ring micromixer.
  10. Lipid nanoparticle delivery of unmodified mRNAs encoding multiple monoclonal antibodies targeting poxviruses in rabbits.
  11. RNAi Based Therapeutics and Novel RNA Bioengineering Technologies.
  12. Construction of nanocarriers based on nucleic acids and their applications in nanobiology delivery systems.
  13. Mucus-permeable polymyxin B-hyaluronic acid/ poly (lactic-co-glycolic acid) nanoparticle platform for the nebulized treatment of lung infections.
  1. Methods Mol Biol. 2022 ;2534 121-133
    Liposomal siRNA Delivery in Papillary Thyroid Carcinoma Cells.
    Farhadul Islam, Yaoqi Zhou, Alfred K Lam.
      The discovery of RNA interference (RNAi) has opened a new strategy in cancer therapy, especially by silencing target genes. Pharmacologically it can be achieved by introducing of small (19-21 base pairs) dsRNA molecules known as small interfering RNA (siRNA) targeting interested genes. siRNA mediated gene has been widely investigated for its utility in treating various diseases including cancer. However, the systemic delivery of interested siRNA via non-viral methods remains a major challenge with large numbers of polymeric and liposomal systems being tested. The most effective methods involving cationic liposomes delivery to cells. Nonetheless, systemic delivery of siRNA via cationic lipid particles is often poor due to rapid uptake by reticuloendothelial organs, resulting in decreased delivery of these particles to the site of interest. Polyethylene glycol (PEG) has been used in siRNA-liposomes formulation to minimize reticuloendothelial uptake. Also, PEGylation permits the accumulation of the liposomes-loaded siRNA at the tumor sites with defective vasculatures such as enhanced permeability and retention phenomena. Thus, a simple method to prepare stable PEGylated siRNA-loaded lipid particles could provide better systemic delivery system in treating various cancers, including papillary thyroid carcinoma. Here we illustrate a simple protocol for the formulation of siRNA-loaded lipid particles by hydration of freeze-dried matrix (HFDM) method for effective delivery of target specific siRNA to papillary thyroid carcinoma cells.
    Keywords:  Liposome; Papillary thyroid carcinoma; RNAi; Systemic delivery; siRNA
    DOI:  https://doi.org/10.1007/978-1-0716-2505-7_9
  2. Mol Med Rep. 2022 Aug;pii: 253. [Epub ahead of print]26(2):
    Optimal combination of cationic lipid and phospholipid in cationic liposomes for gene knockdown in breast cancer cells and mouse lung using siRNA lipoplexes.
    Yoshiyuki Hattori, Min Tang, Satomi Torii, Kana Tomita, Ayane Sagawa, Nodoka Inoue, Reo Yamagishi, Kei-Ichi Ozaki.
      Formulation of cationic liposomes is a key factor that determine the gene knockdown efficiency by cationic liposomes/siRNA complexes (siRNA lipoplexes). Here, to determine the optimal combination of cationic lipid and phospholipid in cationic liposomes for in vitro and in vivo gene knockdown using siRNA lipoplexes, three types of cationic lipid were used, namely 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB) and 11-[(1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino]-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12). Thereafter, 30 types of cationic liposome composed of each cationic lipid with phosphatidylcholine or phosphatidylethanolamine containing saturated or unsaturated dialkyl chains (C14, C16, or C18) were prepared. The inclusion of phosphatidylethanolamine containing unsaturated and long dialkyl chains with DOTAP- or DDAB-based cationic liposomes induced strong luciferase gene knockdown in human breast cancer MCF-7-Luc cells stably expressing luciferase gene. Furthermore, the inclusion of phosphatidylcholine or phosphatidylethanolamine containing saturated and short dialkyl chains or unsaturated and long dialkyl chains into TC-1-12-based cationic liposomes resulted in high gene knockdown efficacy. When cationic liposomes composed of DDAB/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), TC-1-12/DOPE and TC-1-12/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine were used, significant gene knockdown occurred in the lungs of mice following systemic injection of siRNA lipoplexes. Overall, the present findings indicated that optimal phospholipids in cationic liposome for in vitro and in vivo siRNA transfection were affected by the types of cationic lipid used.
    Keywords:  cationic liposome; gene knockdown; lung; phospholipid; siRNA delivery
    DOI:  https://doi.org/10.3892/mmr.2022.12769
  3. Eur J Pharm Sci. 2022 Jun 07. pii: S0928-0987(22)00119-1. [Epub ahead of print] 106234
    Lipid nanoparticle formulations for optimal RNA-based topical delivery to murine airways.
    A Tam, J Kulkarni, K An, L Li, D R Dorscheid, G K Singhera, P Bernatchez, Gsd Reid, Kyt Chan, D Witzigmann, P R Cullis, D D Sin, C J Lim.
      INTRODUCTION: Lipid nanoparticles (LNP) have been successfully used as a platform technology for delivering nucleic acids to the liver. To broaden the application of LNPs in targeting non-hepatic tissues, we developed LNP-based RNA therapies (siRNA or mRNA) for the respiratory tract. Such optimized LNP systems could offer an early treatment strategy for viral respiratory tract infections such COVID-19.METHODS: We generated a small library of six LNP formulations with varying helper lipid compositions and characterized their hydrodynamic diameter, size distribution and cargo entrapment properties. Next, we screened these LNP formulations for particle uptake and evaluated their potential for transfecting mRNA encoding green fluorescence protein (GFP) or SARS-CoV2 nucleocapsid-GFP fusion reporter gene in a human airway epithelial cell line in vitro. Following LNP-siGFP delivery, GFP protein knockdown efficiency was assessed by flow cytometry to determine %GFP+ cells and median fluorescence intensity (MFI) for GFP. Finally, lead LNP candidates were validated in Friend leukemia virus B (FVB) male mice via intranasal delivery of an mRNA encoding luciferase, using in vivo bioluminescence imaging.
    RESULTS: Dynamic light scattering revealed that all LNP formulations contained particles with an average diameter of <100 nm and a polydispersity index of <0.2. Human airway epithelial cell lines in culture internalized LNPs with differential GFP transfection efficiencies (73-97%). The lead formulation LNP6 entrapping GFP or Nuc-GFP mRNA demonstrated the highest transfection efficiency (97%). Administration of LNP-GFP siRNA resulted in a significant reduction of GFP protein expression. For in vivo studies, intranasal delivery of LNPs containing helper lipids (DSPC, DOPC, ESM or DOPS) with luciferase mRNA showed significant increase in luminescence expression in nasal cavity and lungs by at least 10 times above baseline control.
    CONCLUSION: LNP formulations enable the delivery of RNA payloads into human airway epithelial cells, and in the murine respiratory system; they can be delivered to nasal mucosa and lower respiratory tract via intranasal delivery. The composition of helper lipids in LNPs crucially modulates transfection efficiencies in airway epithelia, highlighting their importance in effective delivery of therapeutic products for airways diseases.
    Keywords:  airway epithelial cells; lipid nanoparticles; mRNA; nasal cavity; siRNA
    DOI:  https://doi.org/10.1016/j.ejps.2022.106234
  4. Polymers (Basel). 2022 May 27. pii: 2176. [Epub ahead of print]14(11):
    Interleukin-12 Plasmid DNA Delivery by N-[(2-Hydroxy-3-trimethylammonium)propyl]chitosan-Based Nanoparticles.
    Ali Dehshahri, Bahman Khalvati, Zahra Taheri, Farshad Safari, Reza Mohammadinejad, Abolfazl Heydari.
      Cationic polysaccharides are capable of forming polyplexes with nucleic acids and are considered promising polymeric gene carriers. The objective of this study was to evaluate the transfection efficiency and cytotoxicity of N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan salt (HTCS), a quaternary ammonium derivative of chitosan (CS), which benefits from non-ionizable positive charges. In this work, HTCS with a full quaternization of amino groups and a molar mass of 130,000 g·mol-1 was synthesized to use for delivery of a plasmid encoding the interleukin-12 (IL-12) gene. Thus, a polyplex based on HTCS and the IL-12 plasmid was prepared and then was characterized in terms of particle size, zeta potential, plasmid condensation ability, and protection of the plasmid against enzymatic degradation. We showed that HTCS was able to condense the IL-12 plasmid by the formation of polyplexes in the range of 74.5 ± 0.75 nm. The level of hIL-12 production following the transfection of the cells with HTCS polyplexes at a C/P ratio of 8:1 was around 4.8- and 2.2-fold higher than with CS and polyethylenimine polyplexes, respectively. These findings highlight the role of HTCS in the formation of polyplexes for the efficient delivery of plasmid DNA.
    Keywords:  gene delivery; interleukin-12; nanoparticles; polyplex; quaternized chitosan
    DOI:  https://doi.org/10.3390/polym14112176
  5. Theranostics. 2022 ;12(9): 4081-4109
    Advances in the polymeric delivery of nucleic acid vaccines.
    Gang Chen, Bowen Zhao, Elena F Ruiz, Fuwu Zhang.
      Nucleic acid vaccines, especially messenger RNA (mRNA) vaccines, display unique benefits in the current COVID-19 pandemic. The application of polymeric materials as delivery carriers has greatly promoted nucleic acid vaccine as a promising prophylactic and therapeutic strategy. The inherent properties of polymeric materials render nucleic acid vaccines with excellent in vivo stability, enhanced biosafety, specific cellular uptake, endolysosomal escape, and promoted antigen expression. Although polymeric delivery of nucleic acid vaccines has progressed significantly in the past decades, clinical translation of polymer-gene vaccine systems still faces insurmountable challenges. This review summarizes the diverse polymers and their characterizations and representative formulations for nucleic acid vaccine delivery. We also discussed existing problems, coping strategies, and prospect relevant to applications of nucleic acid vaccines and polymeric carriers. This review highlights the rational design and development of polymeric vaccine delivery systems towards meeting the goals of defending serious or emerging diseases.
    Keywords:  cellular immunity; gene delivery; humoral immunity; nucleic acid vaccine; polymer
    DOI:  https://doi.org/10.7150/thno.70853
  6. Colloids Surf B Biointerfaces. 2022 Jun 01. pii: S0927-7765(22)00292-2. [Epub ahead of print]217 112609
    Novel nanocarriers for silencing anti-phagocytosis CD47 marker in acute myeloid leukemia cells.
    Eman M Hassan, Shan Zou.
      Acute myeloid leukemia (AML), a malignant disorder of Hematopoietic stem cells, can escape immunosurveillance by over expression of the cluster of differentiation 47 (CD47) marker, which functions as an inhibitory signal, suppressing phagocytosis by binding to signal regulatory protein α (SIRPα) on macrophages. AML is treated mainly by chemotherapy, which has drastic side effects and poor outcomes for the patients. Most AML patients develop drug resistance, so other methods to treat AML are highly required. Small interfering RNA (siRNA) is considered as an antitumor therapeutic due to its ability to silence genes associated with the overexpressed cancer markers and subsequently re-sensitize cancer cells. However, delivering siRNA into cells faces challenges, and the development of an effective delivery system is desired for successful silencing at the gene level. Herein, we report the usage of different formulations of graphene oxide (GO) as carriers for the delivery of CD47_siRNA (siRNA against CD47) into AML cells in vitro. The polyethylene glycol (PEG) and dendrimers (PAMAM) modified GO with small flake sizes achieved the highest silencing efficiency of the anti-phagocytosis marker CD47 gene, resulted CD47 protein down-regulation in AML cells. Moreover, the concentration at which the GO-based formulations was used has shown no cytotoxicity in AML cells or normal blood cells, which could be used to screen potential drugs for targeted gene therapy in AML.
    Keywords:  Graphene oxide (GO); Nano-conjugate; Small interfering RNA (siRNA); Surface modification; knockdown efficiency
    DOI:  https://doi.org/10.1016/j.colsurfb.2022.112609
  7. Chem Asian J. 2022 Jun 11.
    Emerging approaches for enabling RNAi therapeutics.
    Argha Mario Mallick, Archana Tripathi, Sukumar Mishra, Asmita Mukherjee, Chiranjit Dutta, Ananya Chatterjee, Rituparna Sinha Roy.
      RNA interference (RNAi) is a primitive evolutionary mechanism developed to escape incorporation of foreign genetic material. siRNA has been instrumental in achieving the therapeutic potential of RNAi by theoretically silencing any gene of interest in a reversible and sequence-specific manner. Extrinsically administered siRNA generally needs a delivery vehicle to span across different physiological barriers and load into the RISC complex in the cytoplasm in its functional form to show its efficacy. This review discusses the designing principles and examples of different classes of delivery vehicles that have proved to be efficient in RNAi therapeutics. We also briefly discuss the role of RNAi therapeutics in genetic and rare diseases, epigenetic modifications, immunomodulation and combination modality to inch closer in creating a personalized therapy for metastatic cancer. At the end, we present, strategies and look into the opportunities to develop efficient delivery vehicles for RNAi which can be translated into clinics.
    Keywords:  siRNA, gene silencing, drug delivery, endosomal escape, lipids, polymers, peptides
    DOI:  https://doi.org/10.1002/asia.202200451
  8. Mol Biotechnol. 2022 Jun 07.
    BSA-PEI Nanoparticle Mediated Efficient Delivery of CRISPR/Cas9 into MDA-MB-231 Cells.
    Hossein Rahimi, Kasra Arbabi Zaboli, Jose Thekkiniath, Seyed Hossein Mousavi, Behrooz Johari, Mohammad Reza Hashemi, Hamed Nosrati, David Goldschneider, Agnes Bernet, Hossein Danafar, Saeed Kaboli.
      The discovery of bacterial-derived Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has revolutionized genome engineering and gene therapy due to its wide range of applications. One of the major challenging issues in CRISPR/Cas system is the lack of an efficient, safe, and clinically suitable delivery of the system's components into target cells. Here, we describe the development of polyethylenimine coated-bovine serum albumin nanoparticles (BSA-PEI NPs) for efficient delivery of CRISPR/Cas9 system in both DNA (px458 plasmid) and ribonucleoprotein (RNP) forms into MDA-MB-231 human breast cancer cell line. Our data showed that synthesized BSA-PEI (BP) NPs delivered plasmid px458 at concentrations of 0.15, 0.25, and 0.35 µg/µl with efficiencies of approximately 29.7, 54.8, and 84.1% into MDA-MB-231 cells, respectively. Our study demonstrated that Cas9/sgRNA RNP complex efficiently (~ 92.6%) delivered by BSA-PEI NPs into the same cells. Analysis of toxicity and biocompatibility of synthesized NPs on human red blood cells, MDA-MB-231 cells, and mice showed that the selected concentration (28 µg/µl) of BSA-PEI NPs for transfection had no remarkable toxicity effects. Thus, obtained results suggest BSA-PEI NPs as one of the most promising carrier for delivering CRISPR/Cas9 to target cells.
    Keywords:  BSA-PEI; CRISPR/Cas9; Delivery; Nanoparticles
    DOI:  https://doi.org/10.1007/s12033-022-00514-z
  9. Sci Rep. 2022 Jun 08. 12(1): 9483
    Optimal self-assembly of lipid nanoparticles (LNP) in a ring micromixer.
    Manon Ripoll, Elian Martin, Mathilde Enot, Oscar Robbe, Chiara Rapisarda, Marie-Claire Nicolai, Aurélie Deliot, Patrick Tabeling, Jean-René Authelin, Mostafa Nakach, Pierre Wils.
      Lipid nanoparticles (LNPs) for RNA and DNA delivery have attracted considerable attention for their ability to treat a broad range of diseases and to vectorize mRNA for COVID vaccines. LNPs are produced by mixing biomolecules and lipids, which self-assemble to form the desired structure. In this domain, microfluidics shows clear advantages: high mixing quality, low-stress conditions, and fast preparation. Studies of LNPs produced in micromixers have revealed, in certain ranges of flow rates, a degradation in performance in terms of size, monodispersity and encapsulation efficiency. In this study, we focus on the ring micromixer, which is well adapted to high throughput. We reveal three regimes, side-by-side, transitional and highly mixed, that control the mixing performance of the device. Furthermore, using cryo-TEM and biochemical analysis, we show that the mixing performances are strongly correlated to the characteristics of the LNPs we produce. We emphasize the importance of the flow-rate ratio and propose a physical criterion based on the onset of temporal instabilities for producing LNPs with optimal characteristics in terms of geometry, monodispersity and encapsulation yield. These criteria are generally applicable.
    DOI:  https://doi.org/10.1038/s41598-022-13112-5
  10. Mol Ther Nucleic Acids. 2022 Jun 14. 28 847-858
    Lipid nanoparticle delivery of unmodified mRNAs encoding multiple monoclonal antibodies targeting poxviruses in rabbits.
    Eric M Mucker, Carolin Thiele-Suess, Patrick Baumhof, Jay W Hooper.
      Poxviruses are a large and complex family of viruses with members such as monkeypox virus and variola virus. The possibility of an outbreak of monkeypox virus (or a related poxvirus) or the misuse of variola virus justifies the development of countermeasures. Furthermore, poxviruses can be a useful surrogate for developing technology involving antibody therapies. In our experiments, we explored the feasibility of utilizing unmodified mRNA that encodes three previously described monoclonal antibodies, c8A, c6C, and c7D11, as countermeasures to smallpox in a relatively large (>3 kg) laboratory animal (rabbits). We confirmed in vitro translation, secretion, and biological activity of mRNA constructs and identified target monoclonal antibody levels from a murine vaccinia virus model that provided a clinical benefit. Individually, we were able to detect c7D11, c8A, and c6C in the serum of rabbits within 1 day of an intramuscular jet injection of lipid nanoparticle (LNP)-formulated mRNA. Injection of a combination of three LNP-formulated mRNA constructs encoding the three different antibodies produced near equivalent serum levels compared with each individual construct administered alone. These data are among the first demonstrating the feasibility of launching multiple antibodies using mRNA constructs in a large, nonrodent species. Based on empirically derived target serum level and the observed decay rate, the antibody levels attained were unlikely to provide protection.
    Keywords:  MT: Delivery strategies; RNA; lipid nanoparticle; monoclonal antibodies; neutralizing antibody; nucleic acid; poxvirus; rabbits
    DOI:  https://doi.org/10.1016/j.omtn.2022.05.025
  11. J Pharmacol Exp Ther. 2022 Jun 09. pii: JPET-MR-2022-001234. [Epub ahead of print]
    RNAi Based Therapeutics and Novel RNA Bioengineering Technologies.
    Gavin M Traber, Ai-Ming Yu.
      RNA interference (RNAi) provides researchers with a versicle means to modulate target gene expression. The major forms of RNAi molecules, genome-derived microRNAs (miRNA) and exogenous small interfering RNAs (siRNA), converge into RNA-induced silencing complexes to achieve post-transcriptional gene regulation. RNAi has proven to be an adaptable and powerful therapeutic strategy where advancements in chemistry and pharmaceutics continue to bring RNAi-based drugs into the clinic. With four siRNA medications already approved by the United States Food and Drug Administration (FDA), several RNAi-based therapeutics continue to advance to clinical trials with functions that closely resemble their endogenous counterparts. Although intended to enhance stability and improve efficacy, chemical modifications may increase risk of off-target effects by altering RNA structure, folding, and biological activity away from their natural equivalents. Novel technologies in development today seek to utilize intact cells to yield true biological RNAi agents that better represent the structures, stabilities, activities, and safety profiles of natural RNA molecules. In this review, we provide an examination of the mechanisms of action of endogenous miRNAs and exogenous siRNAs, the physiological and pharmacokinetic barriers to therapeutic RNA delivery, and a summary of the chemical modifications and delivery platforms in use. We overview the pharmacology of the four FDA approved siRNA medications (patisiran, givosiran, lumasiran, and inclisiran), as well as five siRNAs and several miRNA-based therapeutics currently in clinical trials. Furthermore, we discuss the direct expression and stable carrier-based, in vivo production of novel biological RNAi agents for research and development. Significance Statement In our review, we summarize the major concepts of RNA interreference (RNAi), molecular mechanisms, and current state and challenges of RNAi drug development. We focus our discussion on the pharmacology of FDA-approved RNAi medications and those siRNAs and miRNA-based therapeutics entered the clinical investigations. Novel approaches to producing new true biological RNAi molecules for research and development are highlighted.
    Keywords:  MicroRNA; RNA/siRNA; Regulation - post-transcriptional; cancer; gene regulation; gene therapy; neurodegeneration
    DOI:  https://doi.org/10.1124/jpet.122.001234
  12. Natl Sci Rev. 2022 May;9(5): nwac006
    Construction of nanocarriers based on nucleic acids and their applications in nanobiology delivery systems.
    Yingshu Guo, Xiuping Cao, Xiaofei Zheng, S K Jahir Abbas, Juan Li, Weihong Tan.
      In recent years, nanocarriers based on nucleic acids have emerged as powerful and novel nanocarriers that are able to meet the demand for cancer-cell-specific targeting. Functional dynamics analysis revealed good biocompatibility, low toxicity and programmable structures, and their advantages include controllable size and modifiability. The development of novel hybrids has focused on the distinct roles of biosensing, drug and gene delivery, vaccine transport, photosensitization, counteracting drug resistance and functioning as carriers and logic gates. This review is divided into three parts: (i) DNA nanocarriers, (ii) RNA nanocarriers and (iii) DNA/RNA hybrid nanocarriers and their applications in nanobiology delivery systems. We also provide perspectives on possible future directions for growth in this field.
    Keywords:  biosensing; cell; nanocarriers based on nucleic acids; targeted delivery
    DOI:  https://doi.org/10.1093/nsr/nwac006
  13. J Colloid Interface Sci. 2022 May 21. pii: S0021-9797(22)00905-5. [Epub ahead of print]624 307-319
    Mucus-permeable polymyxin B-hyaluronic acid/ poly (lactic-co-glycolic acid) nanoparticle platform for the nebulized treatment of lung infections.
    Jun Wu, Tianshu Zhai, Jing Sun, Qingsong Yu, Yicheng Feng, Runwei Li, Hao Wang, Qiuhong Ouyang, Tiantian Yang, Qingyuan Zhan, Li Deng, Meng Qin, Fang Wang.
      The aim of this study was to improve the bioavailability of polymyxin B (PMB) in pulmonary nebulized drug delivery. To this end, we developed a nano-delivery system that penetrates the mucus barrier of the lung. Hydrophilic hyaluronic acid (HA) was combined with a water-in-oil system containing a poly (lactic acid)-glycolic acid copolymer of PMB to prepare HA@PLGA-PMB nanoparticles (NPs) with good surface properties. HA@PLGA-PMB NPs with suitable electrical properties, particle size, and good hydrophilicity prevented strong interactions between the NPs and mucus, thereby allowing more drugs to enter deeper into the lung. Compared to the free drug PMB, NPs had more than 2-fold higher mucus penetration efficiency in vitro and better delivery to infected alveolar cells during in vivo nebulization. NPs had better biocompatibility, which further reduced the drug toxicity. More importantly, NPs showed better antimicrobial therapeutic efficacy in the treatment of lung infections in mice. These findings may provide support for the clinical application of nebulized pulmonary antibiotics.
    Keywords:  Hydrophilic; Mucus barrier; Mucus permeability; Sodium hyaluronate; polymyxin B
    DOI:  https://doi.org/10.1016/j.jcis.2022.05.121
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