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1
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Mustafa R, Diorio D, Harper M, Punihaole D. Revealing two distinct molecular binding modes in polyethyleneimine-DNA polyplexes using infrared spectroscopy. SOFT MATTER 2025; 21:4192-4200. [PMID: 40326406 PMCID: PMC12053835 DOI: 10.1039/d5sm00213c] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/01/2025] [Accepted: 04/23/2025] [Indexed: 05/07/2025]
Abstract
In this study, we use infrared spectroscopy to investigate the molecular binding modes of DNA with linear and branched polyethylenimine (LPEI and BPEI). PEI-based polymers are widely studied as non-viral gene delivery vectors, but their low transfection efficiency limits their clinical success. One key factor affecting their performance is how they bind DNA as it directly impacts the packaging, protection, and release of the cargo in cells. While PEI-DNA binding has traditionally been viewed through the lens of electrostatics, computational models suggest additional binding mechanisms may be involved. Our findings reveal that LPEI and BPEI exhibit two distinct molecular binding modes, which influence DNA packaging into polyplexes. Identifying these binding modes provides critical insights into polymer complexation mechanisms to nucleic acids that can guide the rational design of more efficient and versatile PEI-based gene delivery systems.
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Affiliation(s)
- Rusul Mustafa
- Department of Chemistry, University of Vermont, 82 University Place, Burlington, Vermont, USA.
| | - Danielle Diorio
- Department of Chemistry, University of Vermont, 82 University Place, Burlington, Vermont, USA.
| | - Madeline Harper
- Department of Chemistry, University of Vermont, 82 University Place, Burlington, Vermont, USA.
| | - David Punihaole
- Department of Chemistry, University of Vermont, 82 University Place, Burlington, Vermont, USA.
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2
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Kissi-Twum AA, Pionek K, Loeb DD. The HBV variant CpF97L supports the secretion of pgRNA-containing virions at a level much greater than WT HBV. J Virol 2025; 99:e0010025. [PMID: 40231820 PMCID: PMC12090816 DOI: 10.1128/jvi.00100-25] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2025] [Accepted: 03/21/2025] [Indexed: 04/16/2025] Open
Abstract
Viruses in the Hepadnaviridae family, including hepatitis B virus (HBV), replicate their double-stranded DNA (dsDNA) genomes through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA), in the viral capsid within an infected cell. In the cell, capsids containing pgRNA, single-stranded DNA (ssDNA), and dsDNA are present. However, capsids containing dsDNA (referred to as mature genomes) are preferentially secreted in virions while only small amounts of capsids with pgRNA and ssDNA (referred to as immature genomes) are enveloped and secreted. The naturally occurring HBV core protein variant, CpF97L, is an exception; HBV CpF97L secretes high levels of ssDNA-containing virions in addition to dsDNA virions. We asked whether HBV CpF97L is capable of secreting pgRNA-containing virions as well. We found that HBV CpF97L secretes high levels of pgRNA-containing virions compared to wild-type (WT) HBV when reverse transcription was inhibited by entecavir or by the Y63F change in P protein. We detected pgRNA virions in Huh7 and HepG2 cell lines, indicating that RNA virion secretion was independent of the cell line used in virion propagation. More importantly, pgRNA virions were detected when dsDNA virions were synthesized as well. Our findings suggest that the capsids of CpF97L are constitutively matured, allowing for virions with immature genomes (ssDNA and pgRNA) to be secreted in addition to dsDNA virions.IMPORTANCEFinding a cure for hepatitis B is critical, as over 250 million people live with a hepatitis B virus (HBV) infection. HBV replicates through a series of nascent RNA and DNA intermediates in capsids, resulting in the secretion of a DNA virion to propagate the infection. HBV infections have been managed with nucleos(t)ide analogs (NAs), which terminate DNA synthesis during replication. During NA treatment, DNA levels plummet, RNA-containing capsids accumulate in infected cells and are secreted, albeit inefficiently, as virions. RNA virions in serum have therefore been proposed to be used as an indicator for covalently closed circular DNA (cccDNA) (HBV's minichromosome in hepatocytes) to determine patients who can be withdrawn from NAs without virological rebound. However, it is unknown if RNA virions are efficiently secreted by the frequent HBV variants that secrete high levels of ssDNA-containing virions, as these will lead to an erroneous overestimate of the cccDNA reservoir; hence, the need for our study.
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Affiliation(s)
- Abena Adomah Kissi-Twum
- McArdle Laboratory for Cancer Research, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA
- Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Karolyn Pionek
- McArdle Laboratory for Cancer Research, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Daniel D. Loeb
- McArdle Laboratory for Cancer Research, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA
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3
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O'Neal P, Washington K, Estes B, Chandran PL. A Facile and Versatile Platform for Cytosolic Delivery of Proteins in Nanoshells of DNA or RNA: Packaging Options in Multiplexed Delivery. Biomacromolecules 2025; 26:3084-3103. [PMID: 40310684 DOI: 10.1021/acs.biomac.5c00149] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/03/2025]
Abstract
Polyethylenimine (PEI) polymers are used to compact DNA into nanoparticles for delivery into cells. We have shown that PEI-mannose polymers compact DNA into nanoshell-like particles, which can load proteins as well. Here we show that these DNA containers are uniquely versatile for scavenging proteins, irrespective of size, charge, and hydrophobicity from dilute solutions. The number of DNA containers for loading proteins can be controlled independently of the protein loading per container by changing the amounts of DNA and protein in solution. This provides control of the fraction of cells receiving the payload and the relative amounts of DNA and protein per cell. The proteins released inside cells retain enzymatic activity. The proposed technology provides a new way to approach protein delivery by hitchhiking proteins within a facile and well-established DNA-delivery mechanism and by utilizing sugar biophysics to load a wide range of proteins in a single-step process.
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Affiliation(s)
- Pilar O'Neal
- Department of Chemical Engineering, College of Engineering and Architecture, Howard University, 2300 sixth St. NW, Washington D.C. 20059, United States
| | - Kareem Washington
- Department of Genetics, College of Medicine, Howard University, 520 W St. NW, Washington D.C. 20059, United States
| | - Bram Estes
- Department of Protein Therapeutics, Amgen Inc, One Amgen Center Drive, Thousand Oaks, California 91320, United States
| | - Preethi L Chandran
- Department of Chemical Engineering, College of Engineering and Architecture, Howard University, 2300 sixth St. NW, Washington D.C. 20059, United States
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4
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Xing Y, Zhou L, Chen Y, Zhang Q, Wu X, Gan Z, Wu K, Jiang D, Wei S, Chen H. A dual-peptides and specific promoter-modified nano gene delivery system for myocardial hypertrophy treatment. Int J Biol Macromol 2025; 311:143759. [PMID: 40319986 DOI: 10.1016/j.ijbiomac.2025.143759] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2024] [Revised: 04/15/2025] [Accepted: 04/30/2025] [Indexed: 05/07/2025]
Abstract
Cardiac hypertrophy represents the heart's adaptive response to physiological or pathological stimuli, functioning to alleviate ventricular wall stress and preserve cardiac function and efficiency. However, pathological hypertrophy usually progresses to heart failure. Gene therapy, in contrast to conventional chemotherapeutic drugs, has the ability to impact cardiac hypertrophy directly, while the lack of adequate vectors limits its application. Gemini surfactants (GS) have been proven to be effective for transfection in vivo in earlier research. To diminish the natural liver targeting of GS nanoparticles, a dual-targeted myocardial biomimetic GS nanocomplex gene delivery system with functional peptides TAT and PCM modified and synergized with cardiac-specific promoter chicken cardiac troponin T promoter (cTnT) is designed in this study. Bioluminescence imaging reveals the utility of targeting hypertrophy myocardium, resulting in low localization in the liver upon systemic administration. Biochemical indicators, echocardiography, gross morphology and histology all indicate that GS-nanocomplexes attenuate ISO-induced cardiac hypertrophy. RNA sequencing results reflect different uptake pathways for different GS nanocomplexes, and the investigation of cellular uptake under various endocytosis inhibitors demonstrate that clathrin-mediated endocytosis (CME) serves as the primary endocytic pathway for GS-pDNA uptake and caveolin-mediated endocytosis (CVME) serves as the primary endocytic pathway for GS-pDNA-TP-RBCM uptake. The endocytic pathway for nanocomplexes is confirmed by CAV-1 silencing. In summary, this research presents a dual myocardium-targeted biomimetic GS nanocomplex for gene delivery for cardiomyocytes. The in vivo and in vitro targeting ability, good biocompatibility and helpful therapeutic efficacy for cardiac hypertrophy are verified, and the uptake mechanism and intracellular transport pathway of GS nanocomplex are revealed. This innovative approach provides a promising therapeutic strategy for the treatment of cardiac hypertrophy.
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Affiliation(s)
- Yangchen Xing
- College of Pharmacy, Chongqing Medical University, Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China
| | - Lu Zhou
- College of Pharmacy, Chongqing Medical University, Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China
| | - Yuxin Chen
- College of Pharmacy, Chongqing Medical University, Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China
| | - Qianyu Zhang
- College of Pharmacy, Chongqing Medical University, Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China
| | - Xianwei Wu
- College of Pharmacy, Chongqing Medical University, Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China
| | - Zongjie Gan
- College of Pharmacy, Chongqing Medical University, Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China
| | - Kexin Wu
- College of Pharmacy, Chongqing Medical University, Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China; College of Pharmaceutical Sciences, Zhejiang University, Yuhangtang Road, Xihu District, Hangzhou 310012, PR China
| | - Dongjun Jiang
- College of Pharmacy, Chongqing Medical University, Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China
| | - Shiqi Wei
- College of Pharmacy, Chongqing Medical University, Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China
| | - Huali Chen
- College of Pharmacy, Chongqing Medical University, Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China.
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5
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Su WC, Lieu R, Fu Y, Kempen T, Yu Z, Zhang K, Chen T, Fan Y. A platform method for simultaneous quantification of lipid and nucleic acid components in lipid nanoparticles. J Chromatogr A 2025; 1746:465788. [PMID: 39987694 DOI: 10.1016/j.chroma.2025.465788] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2024] [Revised: 02/14/2025] [Accepted: 02/16/2025] [Indexed: 02/25/2025]
Abstract
Nucleic acid-based medicines have achieved significant advancements in recent years, with lipid nanoparticles (LNPs) being a pivotal platform for their delivery. However, the complexity of LNP presents significant challenges, requiring analytical methods to identify and quantify individual components to guide formulation development and ensure quality and safety. Current approaches often perform nucleic acid and lipid analysis separately and focus on a single type of formulation, highlighting the need for a simple platform method that can be applied to diverse formulations. We present a platform ion-pair reversed-phase HPLC method with UV and charged aerosol detection (CAD) to simultaneously separate and quantify lipid and nucleic acid components in LNPs. The method separated and quantified 12 lipid species and three types of nucleic acids (antisense oligonucleotide, single-guide RNA, and mRNA), covering a broad range of therapeutic cargoes. Notably, this can be achieved for the first time by one HPLC run with one-step facile sample preparation. Specifically, we used a simple buffer containing Triton and heparin to enable the single-step, simultaneous extraction of both nucleic acid and lipid components from LNPs, achieving quantification recoveries of 90-110 %. We further applied this method and addressed process and quality control challenges of LNPs, including the recovery rate of individual LNP components after purification and simultaneous quantification of co-loaded, different nucleic acid species for potential gene editing applications. This new platform method offers a robust and widely applicable tool to assess the quality of lipid-based nucleic acid therapies.
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Affiliation(s)
- Wan-Chih Su
- Synthetic Molecule Pharmaceutical Sciences, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
| | - Raymond Lieu
- Synthetic Molecule Pharmaceutical Sciences, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA
| | - Yige Fu
- Synthetic Molecule Pharmaceutical Sciences, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA
| | - Trevor Kempen
- Synthetic Molecule Pharmaceutical Sciences, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA
| | - Zhixin Yu
- Synthetic Molecule Pharmaceutical Sciences, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA
| | - Kelly Zhang
- Synthetic Molecule Pharmaceutical Sciences, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA
| | - Tao Chen
- Synthetic Molecule Pharmaceutical Sciences, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
| | - Yuchen Fan
- Synthetic Molecule Pharmaceutical Sciences, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
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6
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Montaño-González PA, Bravo-Lozano LM, Chevance S, Dole F, Rosselgong J, Loyer P, Tranchimand S, Chapel JP, Gauffre F, Schatz C, Bravo-Anaya LM. Interactions between PEI and biological polyanions and the ability of glycosaminoglycans in destabilizing PEI/peGFP-C3 polyplexes for genetic material release. Int J Biol Macromol 2025; 301:140351. [PMID: 39880239 DOI: 10.1016/j.ijbiomac.2025.140351] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2024] [Revised: 12/24/2024] [Accepted: 01/24/2025] [Indexed: 01/31/2025]
Abstract
The lack of understanding of polyplexes stability and their dissociation mechanisms, allowing the release of DNA, is currently a major limitation in non-viral gene delivery. One proposed mechanism for DNA-based polyplexes dissociation is based on the electrostatic interactions between polycations and biological polyanions, such as glycosaminoglycans (GAGs). This work aimed at investigating whether GAGs such as heparin, chondroitin sulphate and hyaluronic acid promote the dissociation of PEI/DNA polyplexes. We studied the electrostatic complexation between branched poly(ethyleneimine) (b-PEI25) and polyanions (model DNA and GAGs) through conductivity and ζ-potential measurements. The formation of b-PEI25/polyanion polyplexes through electrostatic interactions was analyzed in depth, providing key insights into charge stoichiometry, morphology, thermodynamics and physicochemical characteristics. The stability of polyplexes was tested in the presence of the different GAGs. Heparin was found to be the only polyanion capable of releasing peGFP-C3 plasmid from polyplexes, complexing stoichiometrically with the free b-PEI25 in excess, before releasing the plasmid. The ability of GAGs to disrupt polyplexes and release DNA was correlated with the thermodynamic characteristics of b-PEI25/polyanions complexation. Our findings indicate that heparin's strong interaction with PEI and its high charge density, compared to other GAGs and polyanions, are pivotal in determining complex stability and promoting DNA release.
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Affiliation(s)
| | | | - Soizic Chevance
- Univ Rennes, CNRS, ISCR (Institut des Sciences Chimiques de Rennes), UMR 6226, F-35000 Rennes, France
| | - François Dole
- Centre de Recherche Paul Pascal (CRPP), UMR CNRS 5031, Université de Bordeaux, 33600 Pessac, France
| | - Julien Rosselgong
- Univ Rennes, CNRS, ISCR (Institut des Sciences Chimiques de Rennes), UMR 6226, F-35000 Rennes, France
| | - Pascal Loyer
- Univ Rennes, Inserm, INRAE, Institut NUMECAN, UMR-A 1341, UMR-S 1317, Plateforme SynNanoVect, F-35000 Rennes, France
| | - Sylvain Tranchimand
- Univ Rennes, École Nationale Supérieure de Chimie de Rennes, CNRS, ISCR, UMR 6226, F-35000 Rennes, France
| | - Jean-Paul Chapel
- Centre de Recherche Paul Pascal (CRPP), UMR CNRS 5031, Université de Bordeaux, 33600 Pessac, France
| | - Fabienne Gauffre
- Univ Rennes, CNRS, ISCR (Institut des Sciences Chimiques de Rennes), UMR 6226, F-35000 Rennes, France
| | - Christophe Schatz
- Univ Bordeaux, Bordeaux INP, LCPO, CNRS, UMR 5629, F-33000 Pessac, France
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7
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Xiong S, Sun M, Zhang Y, Kong PR, Gan L, Gao L, Xu K, Wu HY, Zhu DY, Lin YH, Li R, Luo CX. Astrocytic BEST1 can serve as a target for functional recovery after ischemic stroke. Mol Ther 2025:S1525-0016(25)00196-0. [PMID: 40119514 DOI: 10.1016/j.ymthe.2025.03.022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2024] [Revised: 12/23/2024] [Accepted: 03/17/2025] [Indexed: 03/24/2025] Open
Abstract
Solid evidence from animal experiments supported the concept of peri-infarct tonic inhibition. Related drug targets have the potential to be translated for clinical stroke treatment. Recently, we reported the contribution of neuronal bestrophin-1 (BEST1)-mediated glutamate release to acute ischemic damage exacerbation in rodents. Now, we found a switch of abnormal BEST1 expression and function from neurons to astrocytes in the peri-infarct cortex following astrocytic activation. Excessive GABA was released through astrocytic BEST1 channel during the subacute phase of stroke, leading to sustained tonic inhibition. Astrocyte-specific knockdown of BEST1 promoted motor functional recovery, depending on reduced tonic inhibition. Moreover, we prepared self-assembled nanoparticles encapsulating siBest1 (SNP-siBest1), which displayed high brain accumulation and long circulation and knocked down astrocytic BEST1 effectively and safely. Systemic treatment with SNP-siBest1 after ischemic stroke showed a therapeutic effect in mice. Therefore, BEST1 is a potential target for stroke therapy from acute to subacute phase, and selective BEST1 blockers beyond nanoparticles are worth developing.
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Affiliation(s)
- Shuai Xiong
- Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Meng Sun
- Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Ying Zhang
- Department of Pharmaceutics, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Pei-Ran Kong
- Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Lu Gan
- Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Ling Gao
- Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Ke Xu
- Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Hai-Yin Wu
- Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China; Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166, China
| | - Dong-Ya Zhu
- Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China; Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166, China
| | - Yu-Hui Lin
- Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China.
| | - Rui Li
- Department of Pharmaceutics, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China.
| | - Chun-Xia Luo
- Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China; Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166, China; The Key Laboratory of Modern Toxicology of Ministry of Education, Nanjing Medical University, Nanjing 211166, China.
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8
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da Silva R, Viana VE, Avila LA, Zotti MJ, Smagghe G, Junior AM, Camargo ER, Fajardo AR. Advances on polymeric nanocarriers for sustainable agriculture: Enhancing dsRNA/siRNA delivery to combat agricultural pests. Int J Biol Macromol 2024; 282:137000. [PMID: 39476891 DOI: 10.1016/j.ijbiomac.2024.137000] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2024] [Revised: 10/25/2024] [Accepted: 10/26/2024] [Indexed: 11/03/2024]
Abstract
The application of exogenous RNA for gene-silencing strategies has gained significant traction in agriculture, offering a highly efficient and eco-friendly alternative to conventional plant protection methods. This success has been driven by advances in biotechnology, from the design of long double-stranded RNA (dsRNA) and small interfering RNA (siRNA) molecules to the development of nanocarrier systems that address the challenge of RNA delivery into plant cells. In particular, polymer-based nanocarriers have emerged as a promising solution for enhancing the stability and delivery efficiency of RNA molecules. This review provides a comprehensive overview of the current state of research on the use of polymeric nanocarriers in RNA interference (RNAi) systems for crop protection. It examines key technological developments that have enabled the effective delivery of dsRNA/siRNA to target organisms, with a focus on the unique advantages polymers offer as carriers. Recent studies highlight significant progress in the preparation, characterization, and application of polymeric nanocarriers for RNA encapsulation and delivery. The review also explores the environmental and health challenges posed by these technologies, emphasizing the need for sustainable approaches in their development. Specifically, the production of nanocarriers must adhere to the principles of green chemistry, prioritizing chemical modification routes that reduce harmful residues, such as toxic solvents. Finally, this paper discusses both the current challenges and future prospects of using polymer-based nanocarriers in sustainable agriculture, offering critical insights into their potential to transform crop protection through RNAi technologies.
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Affiliation(s)
- Renata da Silva
- Laboratory of Technology and Development of Composites and Polymer Materials (LaCoPol), Federal University of Pelotas (UFPel), Pelotas, RS, Brazil
| | - Vívian E Viana
- Department of Crop Protection, Federal University of Pelotas (UFPel), Pelotas, RS, Brazil
| | - Luis A Avila
- Department of Plant and Soil Sciences, Mississippi State University, Mississippi State, MS 39762, USA
| | - Moisés J Zotti
- Department of Crop Protection, Molecular Entomology, Federal University of Pelotas (UFPel), Pelotas, RS, Brazil
| | - Guy Smagghe
- Department of Plants and Crops, Ghent University, Ghent, Belgium; Institute of Entomology, Guizhou University, Guiyang, China; Cellular and Molecular Life Sciences, Department of Biology, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Aldo Merotto Junior
- Graduate Group in Plant Science, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
| | - Edinalvo R Camargo
- Department of Crop Protection, Federal University of Pelotas (UFPel), Pelotas, RS, Brazil
| | - André R Fajardo
- Laboratory of Technology and Development of Composites and Polymer Materials (LaCoPol), Federal University of Pelotas (UFPel), Pelotas, RS, Brazil.
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9
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Qiao H, Chen J, Dong M, Shen J, Yan S. Nanocarrier-Based Eco-Friendly RNA Pesticides for Sustainable Management of Plant Pathogens and Pests. NANOMATERIALS (BASEL, SWITZERLAND) 2024; 14:1874. [PMID: 39683262 DOI: 10.3390/nano14231874] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/01/2024] [Revised: 11/18/2024] [Accepted: 11/20/2024] [Indexed: 12/18/2024]
Abstract
The production of healthy agricultural products has increased the demand for innovative and sustainable plant protection technologies. RNA interference (RNAi), described as post-transcriptional gene silencing, offers great opportunities for developing RNA pesticides for sustainable disease and pest control. Compared with traditional synthesized pesticides, RNA pesticides possess many advantages, such as strong targeting, good environmental compatibility, and an easy development process. In this review, we systematically introduce the development of RNAi technology, highlight the advantages of RNA pesticides, and illustrate the challenges faced in developing high-efficiency RNA pesticides and the benefits of nanocarriers. Furthermore, we introduce the process and mechanism of nanocarrier-mediated RNAi technology, summarize the applications of RNA pesticides in controlling plant pathogens and pests, and finally outline the current challenges and future prospects. The current review provides theoretical guidance for the in-depth research and diversified development of RNA pesticides, which can promote the development and practice of nanocarrier-mediated RNAi.
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Affiliation(s)
- Heng Qiao
- Department of Plant Biosecurity, College of Plant Protection, China Agricultural University, Beijing 100193, China
| | - Jingyi Chen
- Department of Plant Biosecurity, College of Plant Protection, China Agricultural University, Beijing 100193, China
| | - Min Dong
- Department of Plant Biosecurity, College of Plant Protection, China Agricultural University, Beijing 100193, China
| | - Jie Shen
- Department of Plant Biosecurity, College of Plant Protection, China Agricultural University, Beijing 100193, China
| | - Shuo Yan
- Department of Plant Biosecurity, College of Plant Protection, China Agricultural University, Beijing 100193, China
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10
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McCormick K, Moreno Herrero J, Haas H, Fattah S, Heise A, O’Brien FJ, Cryan SA. Optimizing the Delivery of mRNA to Mesenchymal Stem Cells for Tissue Engineering Applications. Mol Pharm 2024; 21:1662-1676. [PMID: 38504417 PMCID: PMC10988554 DOI: 10.1021/acs.molpharmaceut.3c00898] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2023] [Revised: 02/29/2024] [Accepted: 03/01/2024] [Indexed: 03/21/2024]
Abstract
Messenger RNA (mRNA) represents a promising therapeutic tool in the field of tissue engineering for the fast and transient production of growth factors to support new tissue regeneration. However, one of the main challenges to optimizing its use is achieving efficient uptake and delivery to mesenchymal stem cells (MSCs), which have been long reported as difficult-to-transfect. The aim of this study was to systematically screen a range of nonviral vectors to identify optimal transfection conditions for mRNA delivery to MSCs. Furthermore, for the first time, we wanted to directly compare the protein expression profile from three different types of mRNA, namely, unmodified mRNA (uRNA), base-modified mRNA (modRNA), and self-amplifying mRNA (saRNA) in MSCs. A range of polymer- and lipid-based vectors were used to encapsulate mRNA and directly compared in terms of physicochemical properties as well as transfection efficiency and cytotoxicity in MSCs. We found that both lipid- and polymer-based materials were able to successfully condense and encapsulate mRNA into nanosized particles (<200 nm). The overall charge and encapsulation efficiency of the nanoparticles was dependent on the vector type as well as the vector:mRNA ratio. When screened in vitro, lipid-based vectors proved to be superior in terms of mRNA delivery to MSCs cultured in a 2D monolayer and from a 3D collagen-based scaffold with minimal effects on cell viability, thus opening the potential for scaffold-based mRNA delivery. Modified mRNA consistently showed the highest levels of protein expression in MSCs, demonstrating 1.2-fold and 5.6-fold increases versus uRNA and saRNA, respectively. In summary, we have fully optimized the nonviral delivery of mRNA to MSCs, determined the importance of careful selection of the mRNA type used, and highlighted the strong potential of mRNA for tissue engineering applications.
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Affiliation(s)
- Katie McCormick
- Tissue
Engineering Research Group, Department of Anatomy and Regenerative
Medicine, RCSI, Dublin D02 YN77, Ireland
- Science
Foundation Ireland Advance Materials and Bioengineering Research Centre, Dublin D02 W9K7, Ireland
| | | | | | - Sarinj Fattah
- Tissue
Engineering Research Group, Department of Anatomy and Regenerative
Medicine, RCSI, Dublin D02 YN77, Ireland
- School
of Pharmacy and Biomolecular Sciences, RCSI, Dublin D02 YN77, Ireland
| | - Andreas Heise
- Science
Foundation Ireland Advance Materials and Bioengineering Research Centre, Dublin D02 W9K7, Ireland
- Dept.
of Chemistry, RCSI, Dublin D02 YN77, Ireland
- Science
Foundation Ireland Centre for Research in Medical Devices, Galway H91 W2TY, Ireland
| | - Fergal J. O’Brien
- Tissue
Engineering Research Group, Department of Anatomy and Regenerative
Medicine, RCSI, Dublin D02 YN77, Ireland
- Science
Foundation Ireland Advance Materials and Bioengineering Research Centre, Dublin D02 W9K7, Ireland
- Science
Foundation Ireland Centre for Research in Medical Devices, Galway H91 W2TY, Ireland
- Trinity
Centre for Biomedical Engineering, Trinity
College Dublin, Dublin D02 R590, Ireland
| | - Sally-Ann Cryan
- Tissue
Engineering Research Group, Department of Anatomy and Regenerative
Medicine, RCSI, Dublin D02 YN77, Ireland
- Science
Foundation Ireland Advance Materials and Bioengineering Research Centre, Dublin D02 W9K7, Ireland
- Science
Foundation Ireland Centre for Research in Medical Devices, Galway H91 W2TY, Ireland
- Trinity
Centre for Biomedical Engineering, Trinity
College Dublin, Dublin D02 R590, Ireland
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11
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Yuan C, Chang S, Zhang C, Dong D, Ding J, Mahdavian AR, Hu Z, Sun L, Tan S. Post cross-linked ROS-responsive poly(β-amino ester)-plasmid polyplex NPs for gene therapy of EBV-associated nasopharyngeal carcinoma. J Mater Chem B 2024; 12:3129-3143. [PMID: 38451208 DOI: 10.1039/d3tb02926c] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/08/2024]
Abstract
Nasopharyngeal carcinoma (NPC) is one of the most common tumors in South China and Southeast Asia and is thought to be associated with Epstein-Barr virus (EBV) infection. Downregulation of latent membrane protein 1 (LMP1) encoded by EBV can reduce the expression of NF-κB and PI3K, induce apoptosis, and inhibit the growth of EBV-related NPC. For targeted cleavage of the Lmp1 oncogene via the CRISPR/Cas9 gene editing system, a post cross-linked ROS-responsive poly(β-amino ester) (PBAE) polymeric vector was developed for the delivery of CRISPR/Cas9 plasmids both in vitro and in vivo. After composition optimization, the resultant polymer-plasmid polyplex nanoparticles (NPs) showed a diameter of ∼230 nm and a zeta potential of 22.3 mV with good stability. Compared with the non-cross-linked system, the cross-linked NPs exhibited efficient and quick cell uptake, higher transfection efficiency in EBV-positive C666-1 cells (53.5% vs. 40.6%), more efficient gene editing ability against the Mucin2 model gene (Muc2) (17.9% vs. 15.4%) and Lmp1 (8.5% vs. 5.6%), and lower intracellular reactive oxygen species (ROS) levels. The NPs achieved good tumor penetration and tumor growth inhibition in the C666-1 xenograft tumor model via Lmp1 cleavage, indicating their potential for gene therapy of EBV-related NPC.
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Affiliation(s)
- Caiyan Yuan
- School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
- The First Hospital of Nanchang, Nanchang 330008, China
| | - Shuangyan Chang
- Department of Obstetrics and Gynecology, Zhongnan Hospital of Wuhan University, Donghu 169th Road, Wuchang District, Wuhan 430062, Hubei, China.
| | - Chong Zhang
- School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
| | - Dirong Dong
- Department of Obstetrics and Gynecology, Zhongnan Hospital of Wuhan University, Donghu 169th Road, Wuchang District, Wuhan 430062, Hubei, China.
| | - Jiahui Ding
- School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
| | - Ali Reza Mahdavian
- Polymer Science Department, Iran Polymer and Petrochemical Institute, Tehran 14967, Iran
| | - Zheng Hu
- Department of Obstetrics and Gynecology, Zhongnan Hospital of Wuhan University, Donghu 169th Road, Wuchang District, Wuhan 430062, Hubei, China.
| | - Lili Sun
- Department of Obstetrics and Gynecology, Zhongnan Hospital of Wuhan University, Donghu 169th Road, Wuchang District, Wuhan 430062, Hubei, China.
| | - Songwei Tan
- School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
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12
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Yang S, Wu Y, Zhong W, Chen R, Wang M, Chen M. GSH/pH Dual Activatable Cross-linked and Fluorinated PEI for Cancer Gene Therapy Through Endogenous Iron De-Hijacking and in Situ ROS Amplification. ADVANCED MATERIALS (DEERFIELD BEACH, FLA.) 2024; 36:e2304098. [PMID: 37689975 DOI: 10.1002/adma.202304098] [Citation(s) in RCA: 33] [Impact Index Per Article: 33.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Revised: 08/14/2023] [Indexed: 09/11/2023]
Abstract
Ferroptosis-related cancer therapy is limited by insufficient Fe2+ /Fe3+ redox pair and hydrogen peroxide (H2 O2 ) for producing lethal hydroxyl radicals (·OH). Although exogenous iron or ROS-producing drugs can enhance ferroptosis, exploiting endogenous iron (labile iron pool, LIP) stored in ferritin and promoting ROS generation may be safer. Herein, a metal/drug-free nanomedicine is developed for responsive LIP release and H2 O2 generation on the mitochondria membranes, amplifying hydroxyl radical production to enhance ferroptosis-mediated antitumor effects. A glutathione(GSH)/pH dual activatable fluorinated and cross-linked polyethyleneimine (PEI) with dialdehyde polyethylene glycol layer nanocomplex loaded with MTS-KR-SOD (Mitochondria-targeting-sequence-KillerRed-Superoxide Dismutase) and CRISPR/Cas9-CA IX (Carbonic anhydrase IX (CA IX)) plasmids (FP@MC) are developed for enhanced ferroptosis through endogenous iron de-hijacking and in situ ROS amplification. Two plasmids are constructed to knockdown CA IX and translate KillerRed-SOD recombinant protein specifically on mitochondria membranes, respectively. The CA IX knockdown acidifies the intracellular environment, leading the release of LIP from ferritin as a "flare" to initiate endogenous chemodynamic therapy. Meanwhile, MTS-KR-SOD generates H2 O2 when irradiated by a 590 nm laser to assist chemodynamic therapy, leading to ROS amplification for mitochondria damage and lipid peroxide accumulation. The combined therapeutic effects aggravate cancer ferroptosis and suppress tumor growth, providing a new paradigm for amplifying ROS and iron ions to promote ferroptosis-related cancer therapy.
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Affiliation(s)
- Suleixin Yang
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau SAR, 999078, China
| | - Yi Wu
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau SAR, 999078, China
| | - Wenzhao Zhong
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau SAR, 999078, China
| | - Ruie Chen
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau SAR, 999078, China
| | - Meilin Wang
- MoE Frontiers Science Center for Precision Oncology, University of Macau, Macau SAR, 999078, China
| | - Meiwan Chen
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau SAR, 999078, China
- MoE Frontiers Science Center for Precision Oncology, University of Macau, Macau SAR, 999078, China
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13
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Casper J, Schenk SH, Parhizkar E, Detampel P, Dehshahri A, Huwyler J. Polyethylenimine (PEI) in gene therapy: Current status and clinical applications. J Control Release 2023; 362:667-691. [PMID: 37666302 DOI: 10.1016/j.jconrel.2023.09.001] [Citation(s) in RCA: 91] [Impact Index Per Article: 45.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2023] [Revised: 08/24/2023] [Accepted: 09/01/2023] [Indexed: 09/06/2023]
Abstract
Polyethlyenimine (PEI) was introduced 1995 as a cationic polymer for nucleic acid delivery. PEI and its derivatives are extensively used in basic research and as reference formulations in the field of polymer-based gene delivery. Despite its widespread use, the number of clinical applications to date is limited. Thus, this review aims to consolidate the past applications of PEI in DNA delivery, elucidate the obstacles that hinder its transition to clinical use, and highlight potential prospects for novel iterations of PEI derivatives. The present review article is divided into three sections. The first section examines the mechanism of action employed by PEI, examining fundamental aspects of cellular delivery including uptake mechanisms, release from endosomes, and transport into the cell nucleus, along with potential strategies for enhancing these delivery phases. Moreover, an in-depth analysis is conducted concerning the mechanism underlying cellular toxicity, accompanied with approaches to overcome this major challenge. The second part is devoted to the in vivo performance of PEI and its application in various therapeutic indications. While systemic administration has proven to be challenging, alternative localized delivery routes hold promise, such as treatment of solid tumors, application as a vaccine, or serving as a therapeutic agent for pulmonary delivery. In the last section, the outcome of completed and ongoing clinical trials is summarized. Finally, an expert opinion is provided on the potential of PEI and its future applications. PEI-based formulations for nucleic acid delivery have a promising potential, it will be an important task for the years to come to introduce innovations that address PEI-associated shortcomings by introducing well-designed PEI formulations in combination with an appropriate route of administration.
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Affiliation(s)
- Jens Casper
- Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
| | - Susanne H Schenk
- Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
| | - Elahehnaz Parhizkar
- Department of Pharmaceutics, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Pascal Detampel
- Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
| | - Ali Dehshahri
- Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
| | - Jörg Huwyler
- Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
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14
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Liu S, Su C, Zhang D, Song Z, Wang X, Wang J, Yuan X. Construction of a Delivery Platform for Vaccine Based on Modified Nanotubes: Sustainable Prevention against Plant Viral Disease, Simplified Preparation Method, and Protection of Plasmid. ACS APPLIED MATERIALS & INTERFACES 2023; 15:44541-44553. [PMID: 37672476 DOI: 10.1021/acsami.3c09168] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/08/2023]
Abstract
Control of plant viral diseases through cross-protection conferred by an attenuated vaccine is an important strategy for plant protection. However, the mutated site of an attenuated vaccine may not be stably inherited, while viruses have evolved efficient repair mechanisms for the maintenance of genomic integrity. Here, the wide host range and broad selection of mutation sites in cucumber mosaic virus (CMV) enabled construction of an attenuated vaccine through insertional mutation of the CMV 2b protein. CMV-R2E was stably inherited in tobacco for more than 10 generations and had a high relative control efficacy of CMV. Then, the use of polyetherimide (PEI)-modified functionalized carboxylated single-walled carbon nanotubes (PSWNTs) was investigated for vaccine delivery to address the problems of poor stability, complex procedure on field application, and exacting storage conditions with Agrobacterium inoculation. After co-incubating at a 1:300 ratio for 30 min, the vaccine and PSWNTs combined to form pCMV-R2E@PSWNTs, which resulted in a significant increase in the average height of the nanoparticles from 6.56 to 72.34 nm. The relative control efficacy of pCMV-R2E@PSWNTs to CMV was found to be 90.37%. Furthermore, the protective effect of PSWNTs on plasmids was investigated under various environmental conditions and the potential plant toxicity of pCMV-R2E@PSWNTs was assessed, providing a theoretical basis for field application of the vaccine nano-delivery system. A highly effective, stable viral vaccine for plants was thus developed and combined with nanocarriers to address the problems of field application. This approach has the potential to enable wider use of attenuated vaccines for sustainable prevention against plant viral disease in the field.
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Affiliation(s)
- Shanshan Liu
- Pest Integrated Management Key Laboratory of China Tobacco, Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao 266100, P. R. China
| | - Chenyu Su
- Pest Integrated Management Key Laboratory of China Tobacco, Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao 266100, P. R. China
| | - Deping Zhang
- China Tobacco Guangxi Industrial Co., Ltd., Nanning 530000, P. R. China
| | - Zhanfeng Song
- China Tobacco Guangxi Industrial Co., Ltd., Nanning 530000, P. R. China
| | - Xinwei Wang
- Pest Integrated Management Key Laboratory of China Tobacco, Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao 266100, P. R. China
| | - Jie Wang
- Pest Integrated Management Key Laboratory of China Tobacco, Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao 266100, P. R. China
| | - Xuefeng Yuan
- Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Tai'an 271000, P. R. China
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15
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Malkawi WI, Laird NZ, Phruttiwanichakun P, Mohamed E, Elangovan S, Salem AK. Application of Lyophilized Gene-Delivery Formulations to Dental Implant Surfaces: Non-Cariogenic Lyoprotectant Preserves Transfection Activity of Polyplexes Long-Term. J Pharm Sci 2023; 112:83-90. [PMID: 36372226 PMCID: PMC9772140 DOI: 10.1016/j.xphs.2022.11.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2022] [Revised: 11/04/2022] [Accepted: 11/04/2022] [Indexed: 11/13/2022]
Abstract
Titanium is the metal of choice for dental implants because of its biocompatibility and ability to merge with human bone tissue. Despite the great success rate of dental implants, early and late complications occur. Coating titanium dental implant surfaces with polyethyleneimine (PEI)-plasmid DNA (pDNA) polyplexes improve osseointegration by generating therapeutic protein expression at the implantation site. Lyophilization is an approach for stabilizing polyplexes and extending their shelf life; however, most lyoprotectants are sugars that can aid bacterial growth in the peri-implant environment. In our research, we coated titanium surfaces with polyplex solutions containing varying amounts of lyoprotectants. We used two common lyoprotectants (sucrose and polyvinylpyrrolidone K30) and showed for the first time that sucralose (a sucrose derivative used as an artificial sweetener) might act as a lyoprotectant for polyplex solutions. Human embryonic kidney (HEK) 293T cells were used to quantify the transfection efficiency and cytotoxicity of the polyplex/lyoprotectant formulations coating titanium surfaces. Polyplexes that were lyophilized in the presence of a lyoprotectant displayed both preserved particle size and high transfection efficiencies. Polyplexes lyophilized in 2% sucralose have maintained transfection efficacy for three years. These findings suggest that modifying dental implants with lyophilized polyplexes might improve their success rate in the clinic.
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Affiliation(s)
- Walla I Malkawi
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, 52242, United States
| | - Noah Z Laird
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, 52242, United States
| | - Pornpoj Phruttiwanichakun
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, 52242, United States
| | - Esraa Mohamed
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, 52242, United States
| | - Satheesh Elangovan
- Department of Periodontics, College of Dentistry and Dental Clinics, The University of Iowa, Iowa City, IA, 52242, United States
| | - Aliasger K Salem
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, 52242, United States.
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16
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Mohamed MA, Yan L, Shahini A, Rajabian N, Jafari A, Andreadis ST, Wu Y, Cheng C. Well-Defined pH-Responsive Self-Assembled Block Copolymers for the Effective Codelivery of Doxorubicin and Antisense Oligonucleotide to Breast Cancer Cells. ACS APPLIED BIO MATERIALS 2022; 5:4779-4792. [PMID: 36170623 DOI: 10.1021/acsabm.2c00464] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
The worldwide steady increase in the number of cancer patients motivates the development of innovative drug delivery systems for combination therapy as an effective clinical modality for cancer treatment. Here, we explored a design concept based on poly(ethylene glycol)-b-poly(2-(dimethylamino)ethyl methacrylate)-b-poly(2-hydroxyethyl methacrylate-formylbenzoic acid) [PEG-b-PDMAEMA-b-P(HEMA-FBA)] for the dual delivery of doxorubicin (DOX) and GTI2040 (an antisense oligonucleotide for ribonucleotide reductase inhibition) to MCF-7 breast cancer cells. PEG-b-PDMAEMA-b-PHEMA, the precursor copolymer, was prepared through chain extensions from a PEG-based macroinitiator via two consecutive atom transfer radical polymerization (ATRP) steps. Then, it was modified at the PHEMA block with 4-formylbenzoic acid (FBA) to install reactive aldehyde moieties. A pH-responsive polymer-drug conjugate (PDC) was obtained by conjugating DOX to the polymer structure via acid-labile imine linkages, and subsequently self-assembled in an aqueous solution to form DOX-loaded self-assembled nanoparticles (DOX-SAN) with a positively charged shell. DOX-SAN condensed readily with negatively charged GTI2040 to form GTI2040/DOX-SAN nanocomplexes. Gel-retardation assay confirmed the affinity between GTI2040 and DOX-SAN. The GTI2040/DOX-SAN nanocomplex at N/P ratio of 30 exhibited a volume-average hydrodynamic size of 136.4 nm and a zeta potential of 21.0 mV. The pH-sensitivity of DOX-SAN was confirmed by the DOX release study based on the significant cumulative DOX release at pH 5.5 relative to pH 7.4. Cellular uptake study demonstrated favorable accumulation of GTI2040/DOX-SAN inside MCF-7 cells compared with free GTI2040/DOX. In vitro cytotoxicity study indicated higher therapeutic efficacy of GTI2040/DOX-SAN relative to DOX-SAN alone because of the downregulation of the R2 protein of ribonucleotide reductase. These outcomes suggest that the self-assembled pH-responsive triblock copolymer is a promising platform for combination therapy, which may be more effective in combating cancer than individual therapies.
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Affiliation(s)
- Mohamed Alaa Mohamed
- Department of Chemical and Biological Engineering, University at Buffalo, the State University of New York, Buffalo, New York 14260, United States.,Chemistry Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt
| | - Lingyue Yan
- Department of Biomedical Engineering, University at Buffalo, the State University of New York, Buffalo, New York 14260, United States
| | - Aref Shahini
- Department of Chemical and Biological Engineering, University at Buffalo, the State University of New York, Buffalo, New York 14260, United States
| | - Nika Rajabian
- Department of Chemical and Biological Engineering, University at Buffalo, the State University of New York, Buffalo, New York 14260, United States
| | - Amin Jafari
- Department of Chemical and Biological Engineering, University at Buffalo, the State University of New York, Buffalo, New York 14260, United States
| | - Stelios T Andreadis
- Department of Chemical and Biological Engineering, University at Buffalo, the State University of New York, Buffalo, New York 14260, United States.,Department of Biomedical Engineering, University at Buffalo, the State University of New York, Buffalo, New York 14260, United States.,Center of Excellence in Bioinformatics and Life Sciences, Buffalo, New York 14263, United States.,Cell, Gene and Tissue Engineering (CGTE) Center, Buffalo, New York 14263, United States
| | - Yun Wu
- Department of Biomedical Engineering, University at Buffalo, the State University of New York, Buffalo, New York 14260, United States.,Cell, Gene and Tissue Engineering (CGTE) Center, Buffalo, New York 14263, United States
| | - Chong Cheng
- Department of Chemical and Biological Engineering, University at Buffalo, the State University of New York, Buffalo, New York 14260, United States
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17
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Costa RR, Reis RL, Pashkuleva I. Glycosaminoglycans as polyelectrolytes: implications in bioactivity and assembly of biomedical devices. INTERNATIONAL MATERIALS REVIEWS 2022; 67:765-795. [DOI: 10.1080/09506608.2022.2026860] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/04/2021] [Accepted: 12/16/2021] [Indexed: 01/04/2025]
Affiliation(s)
- Rui R. Costa
- 3B's Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Barco, Portugal
- ICVS/3B's, PT Government Associated Laboratory, Braga, Portugal
| | - Rui L. Reis
- 3B's Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Barco, Portugal
- ICVS/3B's, PT Government Associated Laboratory, Braga, Portugal
| | - Iva Pashkuleva
- 3B's Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Barco, Portugal
- ICVS/3B's, PT Government Associated Laboratory, Braga, Portugal
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18
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Abstract
Despite an extensive theoretical and numerical background, the translocation ratchet mechanism, which is fundamental for the transmembrane transport of biomolecules, has never been experimentally reproduced at the nanoscale. Only the Sec61 and bacterial type IV pilus pores were experimentally shown to exhibit a translocation ratchet mechanism. Here we designed a synthetic translocation ratchet and quantified its efficiency as a nanopump. We measured the translocation frequency of DNA molecules through nanoporous membranes and showed that polycations at the trans side accelerated the translocation in a ratchet-like fashion. We investigated the ratchet efficiency according to geometrical and kinetic parameters and observed the ratchet to be only dependent on the size of the DNA molecule with a power law [Formula: see text]. A threshold length of 3 kbp was observed, below which the ratchet did not operate. We interpreted this threshold in a DNA looping model, which quantitatively explained our results.
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19
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Akbari E, Ajdary S, Ardakani EM, Agi E, Milani A, Seyedinkhorasani M, Khalaj V, Bolhassani A. Immunopotentiation by linking Hsp70 T-cell epitopes to Gag-Pol-Env-Nef-Rev multiepitope construct and increased IFN-gamma secretion in infected lymphocytes. Pathog Dis 2022; 80:6608937. [PMID: 35704612 DOI: 10.1093/femspd/ftac021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2022] [Revised: 06/03/2022] [Accepted: 06/13/2022] [Indexed: 11/12/2022] Open
Abstract
Therapeutic human immunodeficiency virus (HIV) vaccines can boost the anti-HIV host immunity to control viral replication and eliminate viral reservoirs in the absence of anti-retroviral therapy. In this study, two computationally designed multiepitope Gag-Pol-Env-Nef-Rev and Hsp70-Gag-Pol-Env-Nef-Rev constructs harboring immunogenic and highly conserved HIV T cell epitopes were generated in E. coli as polypeptide vaccine candidates. Furthermore, the multiepitope gag-pol-env-nef-rev and hsp70-gag-pol-env-nef-rev DNA vaccine constructs were prepared and complexed with MPG cell-penetrating peptide. The immunogenicity of the multiepitope constructs were evaluated using the homologous and heterologous prime/boost strategies in mice. Moreover, the secretion of IFN-γ was assessed in infected lymphocytes in vitro. Our data showed that the homologous polypeptide regimens could significantly induce a mixture of IgG1 and IgG2a antibody responses, activate T cells to secret IFN-γ, IL-5, IL-10, and generate Granzyme B. Moreover, IFN-γ secretion was significantly enhanced in single-cycle replicable (SCR) HIV-1 virions-infected splenocytes in these groups compared to uninfected splenocytes. The linkage of heat shock protein 70 (Hsp70) epitopes to Gag-Pol-Env-Nef-Rev polypeptide in the homologous regimen increased significantly cytokines and Granzyme B levels, and IFN-γ secretion in virions-infected splenocytes. Briefly, both designed constructs in the homologous regimens can be used as a promising vaccine candidate against HIV infection.
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Affiliation(s)
- Elahe Akbari
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.,Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
| | - Soheila Ajdary
- Department of Immunology, Pasteur Institute of Iran, Tehran, Iran
| | | | - Elnaz Agi
- Iranian Comprehensive Hemophilia Care Center, Tehran, Iran
| | - Alireza Milani
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
| | | | - Vahid Khalaj
- Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
| | - Azam Bolhassani
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
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20
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Construction and application of star polycation nanocarrier-based microRNA delivery system in Arabidopsis and maize. J Nanobiotechnology 2022; 20:219. [PMID: 35525952 PMCID: PMC9077854 DOI: 10.1186/s12951-022-01443-4] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Accepted: 04/25/2022] [Indexed: 11/15/2022] Open
Abstract
Background MicroRNA (miRNA) plays vital roles in the regulation of both plant architecture and stress resistance through cleavage or translation inhibition of the target messenger RNAs (mRNAs). However, miRNA-induced gene silencing remains a major challenge in vivo due to the low delivery efficiency and instability of miRNA, thus an efficient and simple method is urgently needed for miRNA transformation. Previous researches have constructed a star polycation (SPc)-mediated transdermal double-stranded RNA (dsRNA) delivery system, achieving efficient dsRNA delivery and gene silencing in insect pests. Results Here, we tested SPc-based platform for direct delivery of double-stranded precursor miRNA (ds-MIRNA) into protoplasts and plants. The results showed that SPc could assemble with ds-MIRNA through electrostatic interaction to form nano-sized ds-MIRNA/SPc complex. The complex could penetrate the root cortex and be systematically transported through the vascular tissue in seedlings of Arabidopsis and maize. Meanwhile, the complex could up-regulate the expression of endocytosis-related genes in both protoplasts and plants to promote the cellular uptake. Furthermore, the SPc-delivered ds-MIRNA could efficiently increase mature miRNA amount to suppress the target gene expression, and the similar phenotypes of Arabidopsis and maize were observed compared to the transgenic plants overexpressing miRNA. Conclusion To our knowledge, we report the first construction and application of star polycation nanocarrier-based platform for miRNA delivery in plants, which explores a new enable approach of plant biotechnology with efficient transformation for agricultural application. Graphical Abstract ![]()
Supplementary Information The online version contains supplementary material available at 10.1186/s12951-022-01443-4.
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21
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Dekevic G, Tasto L, Czermak P, Salzig D. Statistical experimental designs to optimize the transient transfection of HEK 293 T cells and determine a transfer criterion from adherent cells to larger-scale cell suspension cultures. J Biotechnol 2022; 346:23-34. [DOI: 10.1016/j.jbiotec.2022.01.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Revised: 12/20/2021] [Accepted: 01/15/2022] [Indexed: 02/04/2023]
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22
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Kottaipalayam-Somasundaram SR, Jacob JP, Aiyar B, Merzendorfer H, Nambiar-Veetil M. Chitin metabolism as a potential target for RNAi-based control of the forestry pest Hyblaea puera Cramer (Lepidoptera: Hyblaeidae). PEST MANAGEMENT SCIENCE 2022; 78:296-303. [PMID: 34487617 DOI: 10.1002/ps.6634] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/30/2021] [Accepted: 09/06/2021] [Indexed: 06/13/2023]
Abstract
BACKGROUND Hyblaea puera, commonly known as the teak defoliator, is a serious pest in teak plantations. Despite the availability of control measures, this pest causes losses in yield and quality of timber through voracious feeding. RNA interference (RNAi) is a promising strategy for the control of this pest. Chitin metabolism, which is vital for the growth and development of arthropods, is a potential target for developing RNAi-based insecticides. RESULTS To assess the effects of chitin metabolism inhibition, H. puera larvae were treated with a chitin synthesis inhibitor, diflubenzuron (DFB). DFB treatment caused pupal deformities and disrupted eclosion. Partial gene sequences for three key genes of H. puera chitin metabolism were cloned and sequenced: chitin synthase 1 (HpCHS1), chitinase-h (HpChi-h) and ecdysone receptor (HpEcR). Feeding dsRNA cognate for these three target genes to the first instar of H. puera resulted in mortality and reduction in the corresponding transcript levels as assessed through qRT-PCR. This is the first report of RNAi in this forestry pest. The highest mortality was 45.9%, in response to dsHpEcR treatment; HpChi-h transcripts were the most down-regulated in response to dsHpEcR feeding. DsHpEcR RNAi resulted in growth inhibition and molting arrest. The mortalities were 29.7% and 32.4% for dsHpCHS1 and dsHpChi-h feeding, respectively. CONCLUSION Chitin metabolism could be a potential target for RNAi-based control of H. puera, and HpCHS1, HpChi-h and HpEcR could be suitable target genes. However, the RNAi efficacy needs to be improved through formulations that improve stability and uptake, and employing better delivery strategies. © 2021 Society of Chemical Industry.
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Affiliation(s)
- Sowmiya R Kottaipalayam-Somasundaram
- Plant Biotechnology and Cytogenetics Division, Institute of Forest Genetics and Tree Breeding, Coimbatore, India
- Department Biology/Chemistry, Animal Physiology, University of Osnabrück, Osnabrück, Germany
| | - John P Jacob
- Forest Protection Division, Institute of Forest Genetics and Tree Breeding, Coimbatore, India
| | - Balasubramanian Aiyar
- Plant Biotechnology and Cytogenetics Division, Institute of Forest Genetics and Tree Breeding, Coimbatore, India
| | - Hans Merzendorfer
- Department Biology/Chemistry, Animal Physiology, University of Osnabrück, Osnabrück, Germany
| | - Mathish Nambiar-Veetil
- Plant Biotechnology and Cytogenetics Division, Institute of Forest Genetics and Tree Breeding, Coimbatore, India
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Jerzykiewicz J, Czogalla A. Polyethyleneimine-Based Lipopolyplexes as Carriers in Anticancer Gene Therapies. MATERIALS 2021; 15:ma15010179. [PMID: 35009324 PMCID: PMC8746209 DOI: 10.3390/ma15010179] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/18/2021] [Revised: 12/06/2021] [Accepted: 12/20/2021] [Indexed: 12/12/2022]
Abstract
Recent years have witnessed rapidly growing interest in application of gene therapies for cancer treatment. However, this strategy requires nucleic acid carriers that are both effective and safe. In this context, non-viral vectors have advantages over their viral counterparts. In particular, lipopolyplexes—nanocomplexes consisting of nucleic acids condensed with polyvalent molecules and enclosed in lipid vesicles—currently offer great promise. In this article, we briefly review the major aspects of developing such non-viral vectors based on polyethyleneimine and outline their properties in light of anticancer therapeutic strategies. Finally, examples of current in vivo studies involving such lipopolyplexes and possibilities for their future development are presented.
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Park SY, Yun YH, Park BJ, Seo HI, Chung I. Fabrication and Biological Activities of Plasmid DNA Gene Carrier Nanoparticles Based on Biodegradable l-Tyrosine Polyurethane. Pharmaceuticals (Basel) 2021; 15:ph15010017. [PMID: 35056074 PMCID: PMC8780858 DOI: 10.3390/ph15010017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2021] [Revised: 12/15/2021] [Accepted: 12/22/2021] [Indexed: 12/04/2022] Open
Abstract
Gene therapy is a suitable alternative to chemotherapy due to the complications of drug resistance and toxicity of drugs, and is also known to reduce the occurrence of cellular mutation through the use of gene carriers. In this study, gene carrier nanoparticles with minimal toxicity and high transfection efficiency were fabricated from a biocompatible and biodegradable polymer, l-tyrosine polyurethane (LTU), which was polymerized from presynthesized desaminotyrosyl tyrosine hexyl ester (DTH) and polyethylene glycol (PEG), by using double emulsion and solvent evaporation techniques, resulting in the formation of porous nanoparticles, and then used to evaluate their potential biological activities through molecular controlled release and transfection studies. To assess cellular uptake and transfection efficiency, two model drugs, fluorescently labeled bovine serum albumin (FITC-BSA) and plasmid DNA-linear polyethylenimine (LPEI) complex, were successfully encapsulated in nanoparticles, and their transfection properties and cytotoxicities were evaluated in LX2 as a normal cell and in HepG2 and MCF7 as cancer cells. The morphology and average diameter of the LTU nanoparticles were confirmed using light microscopy, transmission electron microscopy, and dynamic light scattering, while confocal microscopy was used to validate the cellular uptake of FITC-BSA-encapsulated LTU nanoparticles. Moreover, the successful cellular uptake of LTU nanoparticles encapsulated with pDNA-LPEI and the high transfection efficiency, confirmed by gel electrophoresis and X-gal assay transfection, indicated that LTU nanoparticles had excellent cell adsorption ability, facilitated gene encapsulation, and showed the sustained release tendency of genes through transfection experiments, with an optimal concentration ratio of pDNA and LPEI of 1:10. All the above characteristics are ideal for gene carriers designed to transport and release drugs into the cytoplasm, thus facilitating effective gene therapy.
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Affiliation(s)
- Soo-Yong Park
- Department of Polymer Science and Engineering, Pusan National University, Busan 46241, Korea;
| | - Yang H. Yun
- Department of Biomedical Engineering, College of Engineering, The University of Akron, Akron, OH 44325, USA;
| | - Bum-Joon Park
- Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 46241, Korea;
| | - Hyung-Il Seo
- Department of Surgery, Biomedical Research Institute, Pusan National University Hospital, Busan 49241, Korea;
| | - Ildoo Chung
- Department of Polymer Science and Engineering, Pusan National University, Busan 46241, Korea;
- Correspondence:
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Kim J, Kim JY, Kim H, Kim E, Park S, Ryu KH, Lee EG. Increasing Transfection Efficiency of Lipoplexes by Modulating Complexation Solution for Transient Gene Expression. Int J Mol Sci 2021; 22:ijms222212344. [PMID: 34830226 PMCID: PMC8619889 DOI: 10.3390/ijms222212344] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Revised: 11/10/2021] [Accepted: 11/12/2021] [Indexed: 12/16/2022] Open
Abstract
Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.
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Affiliation(s)
- Jaemun Kim
- Department of Bioprocess Engineering, KRIBB School of Biotechnology, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Korea; (J.K.); (J.Y.K.)
- Bioprocess Engineering Center, KRIBB, 30 Yeongudanji-ro Ochang-eup, Cheongwon-gu, Cheongju-si 28116, Korea; (H.K.); (E.K.); (S.P.); (K.-H.R.)
| | - Ji Yul Kim
- Department of Bioprocess Engineering, KRIBB School of Biotechnology, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Korea; (J.K.); (J.Y.K.)
- Bioprocess Engineering Center, KRIBB, 30 Yeongudanji-ro Ochang-eup, Cheongwon-gu, Cheongju-si 28116, Korea; (H.K.); (E.K.); (S.P.); (K.-H.R.)
| | - Hyeonkyeong Kim
- Bioprocess Engineering Center, KRIBB, 30 Yeongudanji-ro Ochang-eup, Cheongwon-gu, Cheongju-si 28116, Korea; (H.K.); (E.K.); (S.P.); (K.-H.R.)
| | - Eunsil Kim
- Bioprocess Engineering Center, KRIBB, 30 Yeongudanji-ro Ochang-eup, Cheongwon-gu, Cheongju-si 28116, Korea; (H.K.); (E.K.); (S.P.); (K.-H.R.)
| | - Soonyong Park
- Bioprocess Engineering Center, KRIBB, 30 Yeongudanji-ro Ochang-eup, Cheongwon-gu, Cheongju-si 28116, Korea; (H.K.); (E.K.); (S.P.); (K.-H.R.)
| | - Kyoung-Hwa Ryu
- Bioprocess Engineering Center, KRIBB, 30 Yeongudanji-ro Ochang-eup, Cheongwon-gu, Cheongju-si 28116, Korea; (H.K.); (E.K.); (S.P.); (K.-H.R.)
| | - Eun Gyo Lee
- Department of Bioprocess Engineering, KRIBB School of Biotechnology, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Korea; (J.K.); (J.Y.K.)
- Bioprocess Engineering Center, KRIBB, 30 Yeongudanji-ro Ochang-eup, Cheongwon-gu, Cheongju-si 28116, Korea; (H.K.); (E.K.); (S.P.); (K.-H.R.)
- Correspondence: ; Tel.: +82-43-240-6633
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26
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Han M, Beon J, Lee JY, Oh SS. Systematic Combination of Oligonucleotides and Synthetic Polymers for Advanced Therapeutic Applications. Macromol Res 2021; 29:665-680. [PMID: 34754286 PMCID: PMC8568687 DOI: 10.1007/s13233-021-9093-5] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Revised: 08/22/2021] [Accepted: 09/25/2021] [Indexed: 11/16/2022]
Abstract
The potential of oligonucleotides is exceptional in therapeutics because of their high safety, potency, and specificity compared to conventional therapeutic agents. However, many obstacles, such as low in vivo stability and poor cellular uptake, have hampered their clinical success. Use of polymeric carriers can be an effective approach for overcoming the biological barriers and thereby maximizing the therapeutic efficacy of the oligonucleotides due to the availability of highly tunable synthesis and functional modification of various polymers. As loaded in the polymeric carriers, the therapeutic oligonucleotides, such as antisense oligonucleotides, small interfering RNAs, microRNAs, and even messenger RNAs, become nuclease-resistant by bypassing renal filtration and can be efficiently internalized into disease cells. In this review, we introduced a variety of systematic combinations between the therapeutic oligonucleotides and the synthetic polymers, including the uses of highly functionalized polymers responding to a wide range of endogenous and exogenous stimuli for spatiotemporal control of oligonucleotide release. We also presented intriguing characteristics of oligonucleotides suitable for targeted therapy and immunotherapy, which can be fully supported by versatile polymeric carriers.
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Affiliation(s)
- Moohyun Han
- Department of Materials Science and Engineering, Pohang University of Science Technology (POSTECH), Pohang, Gyeongbuk, 37673 Korea
| | - Jiyun Beon
- Department of Materials Science and Engineering, Pohang University of Science Technology (POSTECH), Pohang, Gyeongbuk, 37673 Korea
| | - Ju Young Lee
- Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), Ulsan, 44429 Korea
| | - Seung Soo Oh
- Department of Materials Science and Engineering, Pohang University of Science Technology (POSTECH), Pohang, Gyeongbuk, 37673 Korea
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27
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Wang Y, Shahi PK, Wang X, Xie R, Zhao Y, Wu M, Roge S, Pattnaik BR, Gong S. In vivo targeted delivery of nucleic acids and CRISPR genome editors enabled by GSH-responsive silica nanoparticles. J Control Release 2021; 336:296-309. [PMID: 34174352 DOI: 10.1016/j.jconrel.2021.06.030] [Citation(s) in RCA: 54] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2021] [Revised: 06/01/2021] [Accepted: 06/21/2021] [Indexed: 12/11/2022]
Abstract
The rapid development of gene therapy and genome editing techniques brings up an urgent need to develop safe and efficient nanoplatforms for nucleic acids and CRISPR genome editors. Herein we report a stimulus-responsive silica nanoparticle (SNP) capable of encapsulating biomacromolecules in their active forms with a high loading content and loading efficiency as well as a well-controlled nanoparticle size (~50 nm). A disulfide crosslinker was integrated into the silica network, endowing SNP with glutathione (GSH)-responsive cargo release capability when internalized by target cells. An imidazole-containing component was incorporated into the SNP to enhance the endosomal escape capability. The SNP can deliver various cargos, including nucleic acids (e.g., DNA and mRNA) and CRISPR genome editors (e.g., Cas9/sgRNA ribonucleoprotein (RNP), and RNP with donor DNA) with excellent efficiency and biocompatibility. The SNP surface can be PEGylated and functionalized with different targeting ligands. In vivo studies showed that subretinally injected SNP conjugated with all-trans-retinoic acid (ATRA) and intravenously injected SNP conjugated with GalNAc can effectively deliver mRNA and RNP to murine retinal pigment epithelium (RPE) cells and liver cells, respectively, leading to efficient genome editing. Overall, the SNP is a promising nanoplatform for various applications including gene therapy and genome editing.
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Affiliation(s)
- Yuyuan Wang
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA
| | - Pawan K Shahi
- Department of Pediatrics, University of Wisconsin-Madison, Madison, WI 53706, USA; McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Xiuxiu Wang
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA
| | - Ruosen Xie
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA; Department of Materials Science and Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA
| | - Yi Zhao
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA
| | - Min Wu
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA
| | - Seth Roge
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA
| | - Bikash R Pattnaik
- Department of Pediatrics, University of Wisconsin-Madison, Madison, WI 53706, USA; McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Shaoqin Gong
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA; McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Materials Science and Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53715, USA.
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28
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di Leandro L, Giansanti F, Mei S, Ponziani S, Colasante M, Ardini M, Angelucci F, Pitari G, d'Angelo M, Cimini A, Fabbrini MS, Ippoliti R. Aptamer-Driven Toxin Gene Delivery in U87 Model Glioblastoma Cells. Front Pharmacol 2021; 12:588306. [PMID: 33935695 PMCID: PMC8082512 DOI: 10.3389/fphar.2021.588306] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Accepted: 03/16/2021] [Indexed: 11/13/2022] Open
Abstract
A novel suicide gene therapy approach was tested in U87 MG glioblastoma multiforme cells. A 26nt G-rich double-stranded DNA aptamer (AS1411) was integrated into a vector at the 5' of a mammalian codon-optimized saporin gene, under CMV promoter. With this plasmid termed "APTSAP", the gene encoding ribosome-inactivating protein saporin is driven intracellularly by the glioma-specific aptamer that binds to cell surface-exposed nucleolin and efficiently kills target cells, more effectively as a polyethyleneimine (PEI)-polyplex. Cells that do not expose nucleolin at the cell surface such as 3T3 cells, used as a control, remain unaffected. Suicide gene-induced cell killing was not observed when the inactive saporin mutant SAPKQ DNA was used in the (PEI)-polyplex, indicating that saporin catalytic activity mediates the cytotoxic effect. Rather than apoptosis, cell death has features resembling autophagic or methuosis-like mechanisms. These main findings support the proof-of-concept of using PEI-polyplexed APTSAP for local delivery in rat glioblastoma models.
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Affiliation(s)
- Luana di Leandro
- Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy
| | - Francesco Giansanti
- Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy
| | - Sabrina Mei
- Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy
| | - Sara Ponziani
- Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy
| | - Martina Colasante
- Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy
| | - Matteo Ardini
- Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy
| | - Francesco Angelucci
- Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy
| | - Giuseppina Pitari
- Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy
| | - Michele d'Angelo
- Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy
| | - Annamaria Cimini
- Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy
| | | | - Rodolfo Ippoliti
- Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy
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29
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de la Fuente IF, Sawant SS, Tolentino MQ, Corrigan PM, Rouge JL. Viral Mimicry as a Design Template for Nucleic Acid Nanocarriers. Front Chem 2021; 9:613209. [PMID: 33777893 PMCID: PMC7987652 DOI: 10.3389/fchem.2021.613209] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2020] [Accepted: 01/06/2021] [Indexed: 12/11/2022] Open
Abstract
Therapeutic nucleic acids hold immense potential in combating undruggable, gene-based diseases owing to their high programmability and relative ease of synthesis. While the delivery of this class of therapeutics has successfully entered the clinical setting, extrahepatic targeting, endosomal escape efficiency, and subcellular localization. On the other hand, viruses serve as natural carriers of nucleic acids and have acquired a plethora of structures and mechanisms that confer remarkable transfection efficiency. Thus, understanding the structure and mechanism of viruses can guide the design of synthetic nucleic acid vectors. This review revisits relevant structural and mechanistic features of viruses as design considerations for efficient nucleic acid delivery systems. This article explores how viral ligand display and a metastable structure are central to the molecular mechanisms of attachment, entry, and viral genome release. For comparison, accounted for are details on the design and intracellular fate of existing nucleic acid carriers and nanostructures that share similar and essential features to viruses. The review, thus, highlights unifying themes of viruses and nucleic acid delivery systems such as genome protection, target specificity, and controlled release. Sophisticated viral mechanisms that are yet to be exploited in oligonucleotide delivery are also identified as they could further the development of next-generation nonviral nucleic acid vectors.
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Affiliation(s)
| | | | | | | | - Jessica L. Rouge
- Department of Chemistry, University of Connecticut, Storrs, CT, United States
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Sanmartín I, Sendra L, Moret I, Herrero MJ, Aliño SF. Multicompartmental Lipopolyplex as Vehicle for Antigens and Genes Delivery in Vaccine Formulations. Pharmaceutics 2021; 13:pharmaceutics13020281. [PMID: 33669785 PMCID: PMC7922173 DOI: 10.3390/pharmaceutics13020281] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2020] [Revised: 02/17/2021] [Accepted: 02/18/2021] [Indexed: 11/16/2022] Open
Abstract
Vector design and its characterization is an area of great interest in current vaccine research. In this article, we have formulated and characterized a multicompartmental lipopolyplex, which associates multiple liposomes and polyplexes in the same complex. These particles allow the simultaneous delivery of lipid or water-soluble antigens associated with genes to the same cell, in much higher amounts than conventional lipopolyplexes. The vector characterization and optimization were carried out using liposomes with entrapped carboxyfluorescein and adapted electrophoretic assays. Two types of lipopolyplexes (containing hydrophilic or lipophilic antigens) were employed to evaluate their interest in vaccination. The lipopolyplex loaded with an extract of water-soluble melanoma proteins proved to efficiently induce humoral response in murine melanoma model, increasing the levels of IgM and IgG. The specificity of the immune response induced by the lipopolyplex was demonstrated in mice with the lipopolyplex containing the GD3 ganglioside lipid antigen, abundant in melanoma cells. The levels of anti-GD3 IgG increased markedly without modifying the expression of humoral antibodies against other gangliosides.
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Affiliation(s)
- Isaías Sanmartín
- Faculty of Veterinary and Experimental Sciences, Universidad Católica de Valencia, 46001 Valencia, Spain;
- Pharmacology Department, Faculty of Medicine, Universidad de Valencia, 46010 Valencia, Spain; (L.S.); (I.M.); (M.J.H.)
| | - Luis Sendra
- Pharmacology Department, Faculty of Medicine, Universidad de Valencia, 46010 Valencia, Spain; (L.S.); (I.M.); (M.J.H.)
- Pharmacogenetics Unit, Instituto de Investigación Sanitaria La Fe, 46026 Valencia, Spain
| | - Inés Moret
- Pharmacology Department, Faculty of Medicine, Universidad de Valencia, 46010 Valencia, Spain; (L.S.); (I.M.); (M.J.H.)
- Inflammatory Bowel Disease Research Group, Instituto de Investigación Sanitaria La Fe, 46026 Valencia, Spain
| | - María José Herrero
- Pharmacology Department, Faculty of Medicine, Universidad de Valencia, 46010 Valencia, Spain; (L.S.); (I.M.); (M.J.H.)
- Pharmacogenetics Unit, Instituto de Investigación Sanitaria La Fe, 46026 Valencia, Spain
| | - Salvador F. Aliño
- Pharmacology Department, Faculty of Medicine, Universidad de Valencia, 46010 Valencia, Spain; (L.S.); (I.M.); (M.J.H.)
- Pharmacogenetics Unit, Instituto de Investigación Sanitaria La Fe, 46026 Valencia, Spain
- Clinical Pharmacology Unit, Hospital Universitario y Politécnico La Fe, 46026 Valencia, Spain
- Correspondence: ; Tel.: +34-963-864-972
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31
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Design and fabrication of novel multi-targeted magnetic nanoparticles for gene delivery to breast cancer cells. J Drug Deliv Sci Technol 2021. [DOI: 10.1016/j.jddst.2020.102151] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
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Yan S, Ren BY, Shen J. Nanoparticle-mediated double-stranded RNA delivery system: A promising approach for sustainable pest management. INSECT SCIENCE 2021; 28:21-34. [PMID: 32478473 DOI: 10.1111/1744-7917.12822] [Citation(s) in RCA: 122] [Impact Index Per Article: 30.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/06/2020] [Revised: 04/02/2020] [Accepted: 05/13/2020] [Indexed: 05/10/2023]
Abstract
RNA interference (RNAi) targeting lethal genes in insects has great potential for sustainable crop protection. Compared with traditional double-stranded (ds)RNA delivery systems, nanoparticles such as chitosan, liposomes, and cationic dendrimers offer advantages in delivering dsRNA/small interfering (si)RNA to improve RNAi efficiency, thus promoting the development and practice of RNAi-based pest management strategies. Here, we illustrate the limitations of traditional dsRNA delivery systems, reveal the mechanism of nanoparticle-mediated RNAi, summarize the recent progress and successful applications of nanoparticle-mediated RNAi in pest management, and finally address the prospects of nanoparticle-based RNA pesticides.
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Affiliation(s)
- Shuo Yan
- Department of Entomology and MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, China
| | - Bin-Yuan Ren
- National Agricultural Technology Extension and Service Center, Beijing, China
| | - Jie Shen
- Department of Entomology and MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, China
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Roohizadeh A, Ghaffarinejad A, Salahandish R, Omidinia E. Label-free RNA-based electrochemical nanobiosensor for detection of Hepatitis C. CURRENT RESEARCH IN BIOTECHNOLOGY 2020. [DOI: 10.1016/j.crbiot.2020.11.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
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Zhao Y, Zhao T, Du Y, Cao Y, Xuan Y, Chen H, Zhi D, Guo S, Zhong F, Zhang S. Interaction kinetics of peptide lipids-mediated gene delivery. J Nanobiotechnology 2020; 18:144. [PMID: 33069258 PMCID: PMC7568367 DOI: 10.1186/s12951-020-00707-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2019] [Accepted: 10/09/2020] [Indexed: 11/25/2022] Open
Abstract
BACKGROUND During the course of gene transfection, the interaction kinetics between liposomes and DNA is speculated to play very important role for blood stability, cellular uptake, DNA release and finally transfection efficiency. RESULTS As cationic peptide liposomes exhibited great gene transfer activities both in vitro and in vivo, two peptide lipids, containing a tri-ornithine head (LOrn3) and a mono-ornithine head (LOrn1), were chosen to further clarify the process of liposome-mediated gene delivery in this study. The results show that the electrostatically-driven binding between DNA and liposomes reached nearly 100% at equilibrium, and high affinity of LOrn3 to DNA led to fast binding rate between them. The binding process between LOrn3 and DNA conformed to the kinetics equation: y = 1.663631 × exp (- 0.003427x) + 6.278163. Compared to liposome LOrn1, the liposome LOrn3/DNA lipoplex exhibited a faster and more uniform uptake in HeLa cells, as LOrn3 with a tri-ornithine peptide headgroup had a stronger interaction with the negatively charged cell membrane than LOrn1. The efficient endosomal escape of DNA from LOrn3 lipoplex was facilitated by the acidity in late endosomes, resulting in broken carbamate bonds, as well as the "proton sponge effect" of the lipid. CONCLUSIONS The interaction kinetics is a key factor for DNA transfection efficiency. This work provided insights into peptide lipid-mediated DNA delivery that could guide the development of the next generation of delivery systems for gene therapeutics.
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Affiliation(s)
- Yinan Zhao
- Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Sciences, Dalian Minzu University, Dalian, 116600, China
| | - Tianyi Zhao
- School of Materials Science and Engineering, Zhengzhou University, Zhengzhou, 450001, China
| | - Yanyan Du
- Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Sciences, Dalian Minzu University, Dalian, 116600, China
| | - Yingnan Cao
- Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Sciences, Dalian Minzu University, Dalian, 116600, China
| | - Yang Xuan
- Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Sciences, Dalian Minzu University, Dalian, 116600, China
| | - Huiying Chen
- Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Sciences, Dalian Minzu University, Dalian, 116600, China
| | - Defu Zhi
- Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Sciences, Dalian Minzu University, Dalian, 116600, China
| | - Shutao Guo
- Key Laboratory of Functional Polymer Materials of Ministry of Education, State Key Laboratory of Medicinal Chemical Biology and Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin, 300071, China.
| | - Fangli Zhong
- School of Chemistry and Pharmaceutical Engineering, Jilin Institute of Chemical Technology, Jilin, 132022, China.
| | - Shubiao Zhang
- Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Sciences, Dalian Minzu University, Dalian, 116600, China.
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Dong W, Huang A, Huang J, Wu P, Guo S, Liu H, Qin M, Yang X, Zhang B, Wan M, Zong Y. Plasmid-loadable magnetic/ultrasound-responsive nanodroplets with a SPIO-NP dispersed perfluoropentane core and lipid shell for tumor-targeted intracellular plasmid delivery. Biomater Sci 2020; 8:5329-5345. [PMID: 32793943 DOI: 10.1039/d0bm00699h] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Using ultrasound activating contrast agents to induce sonoporation is a potential strategy for effective lesion-targeted gene delivery. Previous reports have proven that submicron nanodroplets have a better advantage than microbubbles in that they can pass through tumor vasculature endothelial gaps by passive targeting; however, they cannot achieve an adequate dose in tumors to facilitate ultrasound-enhanced gene delivery. Additionally, a few studies focused on delivering macromolecular genetic materials (i.e. overexpression plasmid and CRISPR plasmid) have presented more unique advantages than small-molecular genetic materials (i.e. miRNA mimics, siRNA and shRNA etc.), such as enhancing the expression of target genes with long-term effectiveness. Thereby, we constructed novel plasmid-loadable magnetic/ultrasound-responsive nanodroplets, where superparamagnetic iron oxide nanoparticle dispersed perfluoropentane was encapsulated with lipids to which plasmids could be adhered, and branched polyethylenimine was used to protect the plasmids from enzymolysis. Furthermore, in vitro and in vivo studies were performed to verify the magnetic tumor-targeting ability of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets and focused ultrasound enhanced intracellular plasmid delivery. The plasmid-loadable magnetic/ultrasound-responsive nanodroplets, carrying 16-19 plasmids per droplet, had desirable diameters less than 300 nm, and integrated the merits of excellent magnetic targeting capabilities and phase transition sensitivity to focused ultrasound. Under programmable focused ultrasound exposure, the plasmid-loadable magnetic/ultrasound-responsive nanodroplets underwent a phase-transition into echogenic microbubbles and the subsequent inertial cavitation of the microbubbles achieved an ∼40% in vitro plasmid delivery efficiency. Following intravenous administration, T2-weighted magnet resonance imaging, scanning electron microscopy and inductively coupled plasma optical emission spectrometry of the tumors showed significantly enhanced intratumoral accumulation of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets under an external magnetic field. And a GFP ELISA assay and immunofluorescence staining indicated that focused ultrasound-induced inertial cavitation of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets significantly enhanced the intracellular delivery of plasmids within the tumor after magnet-assisted accumulation, while only lower GFP levels were observed in the tumors on applying focused ultrasound or an external magnet alone. Taken together, utilizing the excellent plasmid-loadable magnetic/ultrasound-responsive nanodroplets combined with magnetism and ultrasound could efficiently deliver plasmids to cancer cells, which could be a potential strategy for macromolecular genetic material delivery in the clinic to treat cancer.
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Affiliation(s)
- Wei Dong
- Key Laboratory of Biomedical Information Engineering of Ministry of Education, Department of Biomedical Engineering, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi, China.
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Dong W, Wu P, Qin M, Guo S, Liu H, Yang X, He W, Bouakaz A, Wan M, Zong Y. Multipotent miRNA Sponge-Loaded Magnetic Nanodroplets with Ultrasound/Magnet-Assisted Delivery for Hepatocellular Carcinoma Therapy. Mol Pharm 2020; 17:2891-2910. [PMID: 32678617 DOI: 10.1021/acs.molpharmaceut.0c00336] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Gene therapy is likely to be the most promising way to tackle cancer, while defects in molecular strategies and delivery systems have led to an impasse in clinical application. Here, it is found that onco-miRNAs of the miR-515 and -449 families were upregulated in hepatocellular carcinoma (HCC), and the sponge targeting miR-515 family had a significant probability to suppress cancer cell proliferation. Then, we constructed non-toxic sponge-loaded magnetic nanodroplets containing 20% C6F14 (SLMNDs-20%) that are incorporated with fluorinated superparamagnetic iron oxide nanoparticles enhancing external magnetism-assisted targeting and enabling a direct visualization of SLMNDs-20% distribution in vivo via magnetic resonance imaging monitoring. SLMNDs-20% could be vaporized by programmable focused ultrasound (FUS) activation, achieving ∼45% in vitro sponge delivery efficiency and significantly enhancing in vivo sponge delivery without a clear apoptosis. Moreover, the sponge-1-carrying SLMNDs-20% could effectively suppress proliferation of xenograft HCC after FUS exposure because sponge-1-suppressing onco-miR-515 enhanced the expression of anti-oncogenes (P21, CD22, TIMP1, NFKB, and E-cadherin) in cancer cells. The current results indicated that ultrasonic cavitation-inducing sonoporation enhanced the intracellular delivery of sponge-1 using SLMNDs-20% after magnetic-assisted accumulation, which was a therapeutic approach to inhibit HCC progression.
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Affiliation(s)
- Wei Dong
- Department of Biomedical Engineering, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China
| | - Pengying Wu
- Department of Biomedical Engineering, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China
| | - Mengfan Qin
- Department of Biomedical Engineering, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China
| | - Shifang Guo
- Department of Biomedical Engineering, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China
| | - Huasheng Liu
- Department of Hematology, The First Affiliated Hospital, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061, China
| | - Xinxing Yang
- Department of Biomedical Engineering, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China.,Department of Ultrasound, The First Affiliated Hospital of AFMU (Xijing Hospital), Air Force Medical University, Xi'an, Shaanxi 710032, China
| | - Wen He
- Department of Biomedical Engineering, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China.,Department of Pharmacy, The First Affiliated Hospital, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061, China
| | - Ayache Bouakaz
- Department of Biomedical Engineering, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China.,Inserm Imaging and Ultrasound, INSERM U930, Imagerie et Cerveau, Université François-Rabelais de Tours, Tours 37000, France
| | - Mingxi Wan
- Department of Biomedical Engineering, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China
| | - Yujin Zong
- Department of Biomedical Engineering, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China
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Gold Nanoparticle-Assisted Virus Formation by Means of the Delivery of an Oncolytic Adenovirus Genome. NANOMATERIALS 2020; 10:nano10061183. [PMID: 32560474 PMCID: PMC7353451 DOI: 10.3390/nano10061183] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/09/2020] [Revised: 06/10/2020] [Accepted: 06/15/2020] [Indexed: 01/14/2023]
Abstract
Oncolytic adenoviruses are a therapeutic alternative to treat cancer based on their ability to replicate selectively in tumor cells. However, their use is limited mainly by the neutralizing antibody (Nab) immune response that prevents repeated dosing. An alternative to facilitate the DNA access to the tumor even in the presence of anti-viral Nabs could be gold nanoparticles able to transfer DNA molecules. However, the ability of these nanoparticles to carry large DNA molecules, such as an oncolytic adenovirus genome, has not been studied. In this work, gold nanoparticles were functionalized with different amounts of polyethylenimine to transfer in a safe and efficient manner a large oncolytic virus genome. Their transfer efficacy and final effect of the oncolytic virus in cancer cells are studied. For each synthesized nanoparticle, (a) DNA loading capacity, (b) complex size, (c) DNA protection ability, (d) transfection efficacy and (e) cytotoxic effect were studied. We observed that small gold nanoparticles (70–80 nm in diameter) protected DNA against nucleases and were able to transfect the ICOVIR-15 oncolytic virus genome encoded in pLR1 plasmid. In the present work, efficient transgene RNA expression, luciferase activity and viral cytopathic effect on cancer cells are reported. These results suggest gold nanoparticles to be an efficient and safe vector for oncolytic adenovirus genome transfer.
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A novel method for removing polyethyleneimine from biopharmaceutical samples: improving assay sensitivity of residual DNA qPCR. Biotechniques 2020; 68:353-358. [PMID: 32228190 DOI: 10.2144/btn-2020-0011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
Polyethyleneimine (PEI) is a flocculent that is widely used in the downstream purification of monoclonal antibodies. It is an in-process residual that is carried through the drug purification process and strongly inhibits residual DNA quantitation by real-time quantitative PCR assay. Very high sample dilutions (e.g., 1:10,000) can overcome the interference of PEI, but at the cost of DNA assay sensitivity. Diluting samples poses a significant risk to the assay sensitivity needed to satisfy regulatory requirements on the quantitation of residual genomic DNA present per dose (i.e., 10 ng/dose). Removing PEI while retaining DNA, by the use of sodium dodecyl sulfate, heparin and/or sarkosyl can overcome the interference of PEI and allow a more accurate quantitation of residual DNA.
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Kurosaki T. [Development of Vaccines with Self-assembled Carriers That Deliver Drugs to Target Organs]. YAKUGAKU ZASSHI 2020; 140:363-368. [PMID: 32115553 DOI: 10.1248/yakushi.19-00174] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
I have developed novel ternary complexes of various vaccines with cationic materials and anionic polymers. Plasmid DNA (pDNA) encoding firefly luciferase was used as a model drug to form adequate ternary complexes. Cationic binary complexes were constructed using pDNA and polyethylenimine, and these binary complexes were coated with various anionic polymers to form ternary complexes. These ternary complexes significantly improved cytotoxicity and aggregation with erythrocytes in comparison to the binary complexes. On the other hand, most of those ternary complexes showed little in vitro transgene efficiency because of their anionic surface charge. γ-Polyglutamic acid (γ-PGA)-ternary complexes, however, demonstrated high in vitro transgene efficiency. After the intravenous administration of γ-PGA-ternary complexes to mice, extremely high gene expression was detected in the marginal zone of the spleen, which is rich in antigen-presenting cells. This spleen-specific phenomenon of γ-PGA-ternary complexes appeared to be suited to DNA vaccines against cancer. I therefore examined the preventive effect of γ-PGA-ternary complexes containing pUb-M, a pDNA encoding melanoma surface antigen, against melanoma-bearing mice. Vaccinations of γ-PGA-ternary complexes into mice significantly suppressed the tumor growth of B16-F10 melanoma cells subcutaneously injected into the mice. In the same manner, vaccinations of γ-PGA-ternary complexes containing ovalbumin (OVA) completely suppressed the growth of E.G7-OVA cells expressing OVA. These results strongly suggest that γ-PGA-ternary complexes are useful in the manufacture of specific tumor vaccines.
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Gao X, Jin Z, Tan X, Zhang C, Zou C, Zhang W, Ding J, Das BC, Severinov K, Hitzeroth II, Debata PR, He D, Ma X, Tian X, Gao Q, Wu J, Tian R, Cui Z, Fan W, Huang Z, Cao C, Bao Y, Tan S, Hu Z. Hyperbranched poly(β-amino ester) based polyplex nanopaticles for delivery of CRISPR/Cas9 system and treatment of HPV infection associated cervical cancer. J Control Release 2020; 321:654-668. [PMID: 32114092 DOI: 10.1016/j.jconrel.2020.02.045] [Citation(s) in RCA: 61] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2019] [Revised: 02/22/2020] [Accepted: 02/27/2020] [Indexed: 12/20/2022]
Abstract
Persistent high-risk HPV infection is the main factor for cervical cancer. HPV E7 oncogene plays an important role in HPV carcinogenesis. Down-regulation of E7 oncogene expression could induce growth inhibition in HPV-positive cells and thus treats HPV related cervical cancer. Here we developed a non-virus gene vector based on poly(amide-amine)-poly(β-amino ester) hyperbranched copolymer (hPPC) for the delivery of CRISPR/Cas9 system to specifically cleave HPV E7 oncogene in HPV-positive cervical cancer cells. The diameter of polyplex nanoparticles (NPs) formed by hPPCs/linear poly(β-amino ester) (PBAE) and plasmids were approximately 300 nm. These hPPCs/PBAE-green fluorescence protein plasmids polyplex NPs showed high transfection efficiency and low toxicity in cells and mouse organs. By cleaving HPV16 E7 oncogene, reducing the expression of HPV16 E7 protein and increasing intracellular retinoblastoma 1 (RB1) amount, hPPCs/PBAE-CRISPR/Cas9 therapeutic plasmids polyplex NPs, especially highly branched hPPC1-plasmids polyplex NPs, exhibited strong growth inhibition of cervical cancer cells in vitro and xenograft tumors in nude mice. Together, the hPPCs/PBAE polyplex NPs to deliver HPV16 E7 targeted CRISPR/Cas9 system in this study could potentially be applied to treat HPV-related cervical cancer.
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Affiliation(s)
- Xueqin Gao
- Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Zhuang Jin
- Department of Obstetrics and Gynecology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China
| | - Xiangyu Tan
- Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China
| | - Chong Zhang
- School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
| | - Chenming Zou
- Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Wei Zhang
- School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
| | - Jiahui Ding
- School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
| | - Bhudev C Das
- Amity Institute of Molecular Medicine & Stem Cell Research, Amity University, Uttar Pradesh, Noida 201313, India
| | - Konstantin Severinov
- Skolkovo Institute of Science and Technology, Skolkovo, Moscow Region 143025, Russia
| | - Inga Isabel Hitzeroth
- Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, South Africa
| | - Priya Ranjan Debata
- Department of Zoology, North Orissa University, Takatpur, Baripada, Odisha 757003, India
| | - Dan He
- Department of Neurology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, Guangdong, China
| | - Xin Ma
- Department of Urology, General Hospital of People's Liberation Army, Beijing 100039, China
| | - Xun Tian
- Department of Obstetrics and Gynecology, Academician expert workstation, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, Hubei, China
| | - Qinglei Gao
- Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China
| | - Jun Wu
- School of Biomedical Engineering, Sun Yat-sen University, Guangzhou 510006, Guangdong, China
| | - Rui Tian
- Department of Obstetrics and Gynecology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China
| | - Zifeng Cui
- Department of Obstetrics and Gynecology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China
| | - Weiwen Fan
- Department of Obstetrics and Gynecology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China
| | - Zhaoyue Huang
- Department of Obstetrics and Gynecology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China
| | - Chen Cao
- Department of Obstetrics and Gynecology, Academician expert workstation, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, Hubei, China
| | - Yuxian Bao
- Generulor Company Bio-X Lab, Guangzhou 510006, Guangdong, China
| | - Songwei Tan
- School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
| | - Zheng Hu
- Department of Obstetrics and Gynecology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China; Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China; Precision Medicine Institute, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.
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Miyazaki M, Obata Y, Abe K, Furusu A, Koji T, Tabata Y, Kohno S. Gene Transfer Using Nonviral Delivery Systems. Perit Dial Int 2020. [DOI: 10.1177/089686080602600603] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
In peritoneal dialysis, loss of peritoneal function is a major factor in treatment failure. The alterations in peritoneal function are related to structural changes in the peritoneal membrane, including peritoneal sclerosis with increased extracellular matrix. Although peritoneal sclerosis is considered reversible to some extent through peritoneal rest, which improves peritoneal function and facilitates morphological changes, there has been no therapeutic intervention and no drug against the development and progression of peritoneal sclerosis. Using recent biotechnological advances in genetic engineering, a strategy based on genetic modification of the peritoneal membrane could be a potential therapeutic maneuver against peritoneal sclerosis and peritoneal membrane failure. Before this gene therapy may be applied clinically, a safe and effective gene delivery system as well as the selection of a gene therapy method must be established. There are presently two kinds of gene transfer vectors: viral and nonviral. Viral vectors are used mainly as a gene delivery system in the field of continuous ambulatory peritoneal dialysis research; however, they have several problems such as immunogenicity and toxicity. On the other hand, nonviral vectors have several advantages over viral vectors. We review here gene transfer using nonviral vector systems in the peritoneum: electroporation, liposomes, and cationized gelatin microspheres. In the field of peritoneal dialysis, gene therapy research using nonviral vectors is presently limited. Improvement in delivery methods together with an intelligent design of targeted genes has brought about large degrees of enhancement in the efficiency, specificity, and temporal control of nonviral vectors.
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Affiliation(s)
- Masanobu Miyazaki
- Second Department of Internal Medicine, Department of Histology, Kyoto, Japan
- Miyazaki-Furukawa Clinic, Nagasaki
| | - Yoko Obata
- Second Department of Internal Medicine, Department of Histology, Kyoto, Japan
| | - Katsushige Abe
- Second Department of Internal Medicine, Department of Histology, Kyoto, Japan
| | - Akira Furusu
- Second Department of Internal Medicine, Department of Histology, Kyoto, Japan
| | - Takehiko Koji
- Cell Biology, Nagasaki University School of Medicine, Kyoto, Japan
| | - Yasuhiko Tabata
- Department of Biomaterials, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | - Shigeru Kohno
- Second Department of Internal Medicine, Department of Histology, Kyoto, Japan
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Conte R, Valentino A, Di Cristo F, Peluso G, Cerruti P, Di Salle A, Calarco A. Cationic Polymer Nanoparticles-Mediated Delivery of miR-124 Impairs Tumorigenicity of Prostate Cancer Cells. Int J Mol Sci 2020; 21:ijms21030869. [PMID: 32013257 PMCID: PMC7038067 DOI: 10.3390/ijms21030869] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2020] [Revised: 01/27/2020] [Accepted: 01/28/2020] [Indexed: 12/13/2022] Open
Abstract
MicroRNAs (miRNAs) play a pivotal role in regulating the expression of genes involved in tumor development, invasion, and metastasis. In particular, microRNA-124 (miR-124) modulates the expression of carnitine palmitoyltransferase 1A (CPT1A) at the post-transcriptional level, impairing the ability of androgen-independent prostate cancer (PC3) cells to completely metabolize lipid substrates. However, the clinical translation of miRNAs requires the development of effective and safe delivery systems able to protect nucleic acids from degradation. Herein, biodegradable polyethyleneimine-functionalized polyhydroxybutyrate nanoparticles (PHB-PEI NPs) were prepared by aminolysis and used as cationic non-viral vectors to complex and deliver miR-124 in PC3 cells. Notably, the PHB-PEI NPs/miRNA complex effectively protected miR-124 from RNAse degradation, resulting in a 30% increase in delivery efficiency in PC3 cells compared to a commercial transfection agent (Lipofectamine RNAiMAX). Furthermore, the NPs-delivered miR-124 successfully impaired hallmarks of tumorigenicity, such as cell proliferation, motility, and colony formation, through CPT1A modulation. These results demonstrate that the use of PHB-PEI NPs represents a suitable and convenient strategy to develop novel nanomaterials with excellent biocompatibility and high transfection efficiency for cancer therapy.
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Affiliation(s)
- Raffaele Conte
- Research Institute on Terrestrial Ecosystems (IRET)—CNR, Via Pietro Castellino 111, 80131 Naples, Italy; (R.C.); (G.P.); (A.D.S.)
| | - Anna Valentino
- Elleva Pharma s.r.l. via P. Castellino, 111 – 80131 Naples, Italy; (A.V.); (F.D.C.)
| | - Francesca Di Cristo
- Elleva Pharma s.r.l. via P. Castellino, 111 – 80131 Naples, Italy; (A.V.); (F.D.C.)
| | - Gianfranco Peluso
- Research Institute on Terrestrial Ecosystems (IRET)—CNR, Via Pietro Castellino 111, 80131 Naples, Italy; (R.C.); (G.P.); (A.D.S.)
| | - Pierfrancesco Cerruti
- Institute for Polymers, Composites and Biomaterials (IPCB-CNR) Via Campi Flegrei, 34, 80078 Pozzuoli (NA), Italy
- Correspondence: (P.C.); (A.C.)
| | - Anna Di Salle
- Research Institute on Terrestrial Ecosystems (IRET)—CNR, Via Pietro Castellino 111, 80131 Naples, Italy; (R.C.); (G.P.); (A.D.S.)
| | - Anna Calarco
- Research Institute on Terrestrial Ecosystems (IRET)—CNR, Via Pietro Castellino 111, 80131 Naples, Italy; (R.C.); (G.P.); (A.D.S.)
- Correspondence: (P.C.); (A.C.)
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Naturally-occurring bacterial cellulose-hyperbranched cationic polysaccharide derivative/MMP-9 siRNA composite dressing for wound healing enhancement in diabetic rats. Acta Biomater 2020; 102:298-314. [PMID: 31751808 DOI: 10.1016/j.actbio.2019.11.005] [Citation(s) in RCA: 52] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2019] [Revised: 10/20/2019] [Accepted: 11/04/2019] [Indexed: 12/16/2022]
Abstract
The anomalous high expression of matrix metalloproteinase 9 (MMP-9) is one important factor that impedes diabetic wound healing. Therefore, inhibition of MMP-9 expression in a diabetic wound could be a feasible method to promote wound healing. In this study, we studied the possibility of self-therapy using wound dressings that contain bacterial cellulose-hyperbranched cationic polysaccharide (BC-HCP) derivatives that encapsulate siRNA (BC-HCP/siMMP-9) and have controlled release properties. Herein, we used four HCPs (Gly-DMAPA, Gly-D4, Amyp-DMAPA, Amyp-D4) as gene carriers. Our results showed that all HCP derivatives were minimally toxic to cells in vitro, while the cationic properties of HCP could be used as a complexation agent for MMP-9 siRNA (siMMP-9). Upon exposure to bacterial cellulose (BC), the BC slowly released HCP/siMMP-9. The released siMMP-9 effectively reduced the gene expression and protein levels of MMP-9 in a human immortalized epithelial cell line (HaCAT) and in diabetic rat wounds. Inhibition of MMP-9 in the wounds of diabetic rats resulted in a significant enhancement of wound healing, suggesting that the BC-HCP/siMMP-9 composite dressing could be used as a safe and effective dressing to promote wound healing in diabetic rats. STATEMENT OF SIGNIFICANCE: In this work, we evaluated the possibility of using bacterial cellulose-hyperbranched cationic polysaccharide derivatives (BC-HCP) as a self-therapeutic wound dressing with siRNA encapsulated and controlled release properties. Our results showed that the BC-HCP/siMMP-9 composite dressing slowly released HCP/siMMP-9. The released siMMP-9 effectively reduced the gene expression and protein level of MMP-9 in human immortalized epithelial cell line and in the wound of diabetic rats. The BC-HCP/siMMP-9 composite dressing promoted diabetic wound healing by the unique nanostructure of BC and by releasing siMMP-9 for specific MMP-9 inhibition. Therefore, it could be used as a safe and effective dressing to promote wound healing in diabetic rats. This is the first evidence on the study of using BC as a dressing composite by encapsulating HCP/siRNA complexes for efficient RNAi gene silencing for better wound healing in diabetic rats.
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Namazi F, Bolhassani A, Sadat SM, Irani S. Delivery of HIV-1 Polyepitope Constructs Using Cationic and Amphipathic Cell Penetrating Peptides into Mammalian Cells. Curr HIV Res 2020; 17:408-428. [DOI: 10.2174/1570162x17666191121114522] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2019] [Revised: 11/06/2019] [Accepted: 11/13/2019] [Indexed: 12/14/2022]
Abstract
Background:
An effective vaccine against human immunodeficiency virus 1 (HIV-1) is
an important global health priority. Despite many efforts in the development of the HIV-1 vaccine,
no effective vaccine has been approved yet. Recently, polyepitope vaccines including several immunogenic
and conserved epitopes of HIV-1 proteins have received special attention.
Methods:
In this study, HIV-1 Nef, Tat, Gp160 and P24 proteins were considered for selection of
immunodominant and conserved epitopes due to their critical roles in the viral life cycle and pathogenesis.
At first, the Nef60-84-Nef126-144-Tat29-49-Gp16030-53-Gp160308-323-P248-151 DNA construct was
designed using in silico studies. Then, the DNA construct was subcloned in pEGFP-N1 and pET-
24a (+) expression vectors and the rNef-Tat-Gp160-P24 polyepitope peptide was generated in E.coli
expression system for in vitro delivery using novel cell-penetrating peptides (CPPs), LDP-NLS and
CyLoP-1, in a non-covalent manner. Also, the HR9 and MPG CPPs were used to transfer the DNA
construct.
Results:
Our results showed that the recombinant polyepitope peptide generated in Rosetta strain
migrated as a clear band of ~31 kDa in SDS-PAGE. The SEM data confirmed the formation of stable
nanoparticles with a size below 250 nm. MTT assay revealed that the complexes did not represent
any considerable cytotoxic effect compared to untreated cells. The results of fluorescence microscopy,
flow cytometry and western blotting indicated that these CPPs successfully delivered polyepitope
constructs into HEK-293T cell line.
Conclusion:
These data suggested that these CPPs can be used as a promising approach for the development
of the HIV-1 vaccine.
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Affiliation(s)
- Fatemeh Namazi
- Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
| | - Azam Bolhassani
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
| | - Seyed Mehdi Sadat
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
| | - Shiva Irani
- Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
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Nademi Y, Tang T, Uludağ H. Membrane lipids destabilize short interfering ribonucleic acid (siRNA)/polyethylenimine nanoparticles. NANOSCALE 2020; 12:1032-1045. [PMID: 31845926 DOI: 10.1039/c9nr08128c] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/10/2023]
Abstract
Cell entry of polymeric nanoparticles (NPs) bearing polynucleotides is an important stage for successful gene delivery. In this work, we addressed the influence of cell membrane lipids on the integrity and configurational changes of NPs composed of short interfering ribonucleic acid (siRNA) and polyethylenimine. We focused on NPs derived from two different PEIs, unmodified low molecular weight PEI and linoleic acid (LA)-substituted PEI, and their interactions with two membrane lipids (zwitterionic 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS)). Our experiments showed that POPS liposomes interacted strongly with both types of NPs, which caused partial dissociation of the NPs. POPC liposomes, however, did not induce any dissociation. Consistent with the experiments, steered molecular dynamics simulations showed a stronger interaction between the NPs and the POPS membrane than between the NPs and the POPC membrane. Lipid substitution on the PEIs enhanced the stability of the NPs during membrane crossing; lipid association between PEIs of the LA-bearing NPs as well as parallel orientation of the siRNAs provided protection against their dissociation (unlike NPs from native PEI). Our observations provide valuable insight into the integrity and structural changes of PEI/siRNA NPs during membrane crossing which will help in the design of more effective carriers for nucleic acid delivery.
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Affiliation(s)
- Yousef Nademi
- Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Canada.
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Sabahi A, Salahandish R, Ghaffarinejad A, Omidinia E. Electrochemical nano-genosensor for highly sensitive detection of miR-21 biomarker based on SWCNT-grafted dendritic Au nanostructure for early detection of prostate cancer. Talanta 2019; 209:120595. [PMID: 31892044 DOI: 10.1016/j.talanta.2019.120595] [Citation(s) in RCA: 48] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2019] [Revised: 11/24/2019] [Accepted: 11/25/2019] [Indexed: 12/19/2022]
Abstract
MicroRNAs (miRNAs) appear as a novel reliable candidate in biomarkers for early diagnosis of cancer. Due to their roles in various types of cancer, their potential as a diagnostic biomarker is getting more attention. Here, a novel electrochemical biosensor for detection of miR-21 was demonstrated, through combining the advantages of electrochemical methods and nanomaterials with the selectivity of oligonucleotides, based on thiolated receptor probe-functionalized dendritic gold nanostructures (den-Au) via the self-assembly monolayer (SAM) process which grafted on the single-wall carbon nanotubes (SWCNTs) platform on the surface of the fluorine-doped tin oxide (FTO) electrode. Cadmium ions (Cd2+) were used as signal units and also signal amplification substance which labeled before on miR-21 target. The oxidation signal of Cd2+ as a signal unit was measured by differential pulse voltammetry (DPV) technique that had a very wide linear relationship with the concentration of miR-21 target (0.01 fmol L-1 to 1 μmol L-1) and low experimental detection limit of 0.01 fmol L-1. Furthermore, fabricated biosensor showed acceptable performance in human serum samples and also good selectivity indiscriminate between the complementary target and non-complementary one, so this nano-genosensor can clinically be used for prostate cancer diagnosis through the detection of miR-21 in human serum samples.
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Affiliation(s)
- Abbas Sabahi
- Research Laboratory of Real Samples Analysis, Faculty of Chemistry, Iran University of Science and Technology (IUST), Tehran, 1684613114, Iran; Electroanalytical Chemistry Research Center, Iran University of Science and Technology (IUST), Tehran, 1684613114, Iran
| | - Razieh Salahandish
- Research Laboratory of Real Samples Analysis, Faculty of Chemistry, Iran University of Science and Technology (IUST), Tehran, 1684613114, Iran; Electroanalytical Chemistry Research Center, Iran University of Science and Technology (IUST), Tehran, 1684613114, Iran
| | - Ali Ghaffarinejad
- Research Laboratory of Real Samples Analysis, Faculty of Chemistry, Iran University of Science and Technology (IUST), Tehran, 1684613114, Iran; Electroanalytical Chemistry Research Center, Iran University of Science and Technology (IUST), Tehran, 1684613114, Iran.
| | - Eskandar Omidinia
- Department of Biochemistry, Genetic and Metabolism Research Group, Pasteur Institute of Iran, Tehran, 13164, Iran.
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Kardani K, Hashemi A, Bolhassani A. Comparison of HIV-1 Vif and Vpu accessory proteins for delivery of polyepitope constructs harboring Nef, Gp160 and P24 using various cell penetrating peptides. PLoS One 2019; 14:e0223844. [PMID: 31671105 PMCID: PMC6822742 DOI: 10.1371/journal.pone.0223844] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2019] [Accepted: 09/30/2019] [Indexed: 02/06/2023] Open
Abstract
To develop an effective therapeutic vaccine against HIV-1, prediction of the most conserved epitopes derived from major proteins using bioinformatics tools is an alternative achievement. The epitope-driven vaccines against variable pathogens represented successful results. Hence, to overcome this hyper-variable virus, we designed the highly conserved and immunodominant peptide epitopes. Two servers were used to predict peptide-MHC-I binding affinity including NetMHCpan4.0 and Syfpeithi servers. The NetMHCIIpan3.2 server was utilized for MHC-II binding affinity. Then, we determined immunogenicity scores and allergenicity by the IEDB immunogenicity predictor and Algpred, respectively. Next, for estimation of toxicity and population coverage, ToxinPred server and IEDB population coverage tool were applied. After that, the MHC-peptide binding was investigated by GalexyPepDock peptide-protein flexible docking server. Finally, two different DNA and peptide constructs containing Nef-Vif-Gp160-P24 and Nef-Vpu-Gp160-P24 were prepared and complexed with four various cell penetrating peptides (CPPs) for delivery into mammalian cells (MPG and HR9 CPPs for DNA delivery, and CyLoP-1 and LDP-NLS CPPs for protein delivery). Our results indicated that the designed DNA and peptide constructs could form non-covalent stable nanoparticles at certain ratios as observed by scanning electron microscope (SEM) and Zetasizer. The flow cytometry results obtained from in vitro transfection of the nanoparticles into HEK-293T cell lines showed that the percentage of GFP expressing cells was about 38.38 ± 1.34%, 25.36% ± 0.30, 54.95% ± 0.84, and 25.11% ± 0.36 for MPG/pEGFP-nef-vif-gp160-p24, MPG/pEGFP-nef-vpu-gp160-p24, HR9/pEGFP-nef-vif-gp160-p24 and HR9/pEGFP-nef-vpu-gp160-p24, respectively. Thus, these data showed that the DNA construct harboring nef-vif-gp160-p24 multi-epitope gene had higher efficiency than the DNA construct harboring nef-vpu-gp160-p24 multi-epitope gene to penetrate into the cells. Moreover, delivery of the recombinant Nef-Vif-Gp160-P24 and Nef-Vpu-Gp160-P24 polyepitope peptides in HEK-293T cells was confirmed as a single band about 32 kDa using western blot analysis. Although, both DNA and peptide constructs could be successfully transported by a variety of CPPs into the cells, but the difference between them in transfection rate will influence the levels of immune responses for development of therapeutic vaccines.
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Affiliation(s)
- Kimia Kardani
- Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Atieh Hashemi
- Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Azam Bolhassani
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
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Zhou M, Jiang N, Fan J, Fu S, Luo H, Su P, Zhang M, Shi H, Zeng J, Huang Y, Li Y, Shen H, Zhang A, Li R. H 7K(R 2) 2-modified pH-sensitive self-assembled nanoparticles delivering small interfering RNA targeting hepatoma-derived growth factor for malignant glioma treatment. J Control Release 2019; 310:24-35. [PMID: 31404556 DOI: 10.1016/j.jconrel.2019.08.010] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2019] [Revised: 08/03/2019] [Accepted: 08/08/2019] [Indexed: 12/12/2022]
Abstract
The lack of effective glioma therapeutics mandates the development of novel treatment strategies. Hepatoma-derived growth factor (HDGF) has been considered as a potential glioma therapeutic target, and its expression level in gliomas is positively related to the malignant grade. Although there are no effective and specific inhibitors against this target, small interfering RNA targeting HDGF (siHDGF)-mediated RNA interference (RNAi) can inhibit the target protein function by knockdown of HDGF expression. However, the application of siHDGF in glioma research and therapy is hampered by the challenge to safe and effective in vivo systemic delivery of siHDGF to gliomas. To address this question, we develop the peptide H7K(R2)2-modified pH-sensitive self-assembled hybrid nanoparticles encapsulating siHDGF (H7K(R2)2-PSNPs (siHDGF)). The acidic glioma microenvironment is beneficial to the membrane penetration of H7K(R2)2-PSNPs and the encapsulated siHDGF. Following systemic administration, H7K(R2)2-PSNPs (siHDGF) can effectively deliver siHDGF into the brain and malignant glioma cells, and therefore can significantly downregulate HDGF expression, inhibit malignant phenotypes of glioma cells, result in reduced tumor volumes and prolonged survival times in nude mice bearing U251 human glioblastoma. Thus, systemic administration of H7K(R2)2-PSNPs (siHDGF) offers an effective way for the targeted delivery of siHDGF and may serve as a practical malignant glioma therapy.
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Affiliation(s)
- Meiling Zhou
- School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Nan Jiang
- School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Junting Fan
- School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Song Fu
- School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Honghua Luo
- School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Ping Su
- School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Mingwan Zhang
- Department of Pharmacy, Tongde Hospital of Zhejiang Province, Hangzhou 310012, China
| | - Huihui Shi
- Kangda College of Nanjing Medical university, Lianyungang 222000, China
| | - Jiaqi Zeng
- School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Yuan Huang
- Department of Pharmacy, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Wuxi 214023, China
| | - Yang Li
- School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
| | - Hong Shen
- Neuro-psychiatric Institute, The Affiliated Brain Hospital of Nanjing Medical University, Nanjing 210029, China
| | - Aixia Zhang
- School of Pharmacy, Nanjing Medical University, Nanjing 211166, China.
| | - Rui Li
- School of Pharmacy, Nanjing Medical University, Nanjing 211166, China.
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Controlling complexation/decomplexation and sizes of polymer-based electrostatic pDNA polyplexes is one of the key factors in effective transfection. Colloids Surf B Biointerfaces 2019; 184:110497. [PMID: 31536938 DOI: 10.1016/j.colsurfb.2019.110497] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2019] [Revised: 08/22/2019] [Accepted: 09/07/2019] [Indexed: 12/20/2022]
Abstract
The delivery of plasmid DNA (pDNA) using polycations has been investigated for several decades; however, obstacles that limit efficient gene delivery still hinder the clinical application of gene therapy. One of the major limiting factors is controlling pDNA binding affinity with polymers to control the complexation and decomplexation of polyplexes. To address this challenge, polycations of α-poly(L-lysine) (APL) and ε-poly(L-lysine) (EPL) were used to prepare variable complexation/decomplexation polyplexes with binding affinities ranging from too tight to too loose and sizes ranging from small to large. APL-EPL/ATP-pDNA polyplexes were also prepared to compare the effects of endosomolytic ATP on complexation/decomplexation and the sizes of polyplexes. The results showed that smaller and tighter polyplexes delivered more pDNA into the cells and into the nucleus than the larger and looser polyplexes. Larger polyplexes exhibited slower cytosolic transport and consequently less nuclear delivery of pDNA than smaller polyplexes. Tighter polyplexes exhibited poor pDNA release in the nucleus, leading to no improvement in transfection efficiency. Thus, polyplexes should maintain a balance between complexation and decomplexation and should have optimal sizes for effective cellular uptake, cytosolic transport, nuclear import, and gene expression. Understanding the effects of complexation/decomplexation and size is important when designing effective polymer-based electrostatic gene carriers.
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50
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Chen Z, Peng Y, Xie X, Feng Y, Li T, Li S, Qin X, Yang H, Wu C, Zheng C, Zhu J, You F, Liu Y. Dendrimer-Functionalized Superparamagnetic Nanobeacons for Real-Time Detection and Depletion of HSP90α mRNA and MR Imaging. Am J Cancer Res 2019; 9:5784-5796. [PMID: 31534519 PMCID: PMC6735378 DOI: 10.7150/thno.36545] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2019] [Accepted: 07/08/2019] [Indexed: 12/18/2022] Open
Abstract
Background & Aims: The use of antisense oligonucleotide-based nanosystems for the detection and regulation of tumor-related gene expression is thought to be a promising approach for cancer diagnostics and therapies. Herein, we report that a cubic-shaped iron oxide nanoparticle (IONC) core nanobeacon is capable of delivering an HSP90α mRNA-specific molecular beacon (HSP90-MB) into living cells and enhancing T2-weighted MR imaging in a tumor model. Methods: The nanobeacons were built with IONC, generation 4 poly(amidoamine) dendrimer (G4 PAMAM), Pluronic P123 (P123) and HSP90-MB labeled with a quencher (BHQ1) and a fluorophore (Alexa Fluor 488). Results: After internalization by malignant cells overexpressing HSP90α, the fluorescence of the nanobeacon was recovered, thus distinguishing cancer cells from normal cells. Meanwhile, MB-mRNA hybridization led to enzyme activity that degraded DNA/RNA hybrids and resulted in downregulation of HSP90α at both the mRNA and protein levels. Furthermore, the T2-weighted MR imaging ability of the nanobeacons was increased after PAMAM and P123 modification, which exhibited good biocompatibility and hemocompatibility. Conclusions: The nanobeacons show promise for applicability to tumor-related mRNA detection, regulation and multiscale imaging in the fields of cancer diagnostics and therapeutics.
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