Mingyu Li, Xiaopeng Ren, Ming Xu, Sitong Dong, Xianzhen Li, Xiaoyi Chen, Conggang Wang, Fan Yang
Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, a functional sugar extensively used in the food industry. However, the lack of safe and efficient heterologous expression systems for SIase has constrained its production and application. In this study, an engineered Bacillus subtilis strain for antibiotic-free SIase production was developed via a food-grade expression system. First, the B. subtilis strain TEA was modified through the CRISPR/Cas9 system, resulting in a mutant strain TEA4, which exhibited enhanced capabilities for recombinant protein expression. For efficient and safe production of SIase, different constitutive and inducible promoters were evaluated. The maltose-inducible promoter Poglv was found to have an extracellular SIase activity of 21.7 U mL-1 in engineered strain TEA4. Subsequent optimization of the culture medium further increased SIase activity to 26.4 U mL-1 during shake flask cultivation. Eventually, using the crude enzyme solution of the engineered strain in biotransformation reactions resulted in a high yield of isomaltulose under high concentrations sucrose, achieving a maximum yield of 83.1%. These findings demonstrated an engineered B. subtilis strain for antibiotic-free SIase production, paving the way for its scale-up industrial production and application.
{"title":"Development of an engineered Bacillus subtilis strain for antibiotic-free sucrose isomerase production","authors":"Mingyu Li, Xiaopeng Ren, Ming Xu, Sitong Dong, Xianzhen Li, Xiaoyi Chen, Conggang Wang, Fan Yang","doi":"10.1002/biot.202400178","DOIUrl":"10.1002/biot.202400178","url":null,"abstract":"<p>Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, a functional sugar extensively used in the food industry. However, the lack of safe and efficient heterologous expression systems for SIase has constrained its production and application. In this study, an engineered <i>Bacillus subtilis</i> strain for antibiotic-free SIase production was developed via a food-grade expression system. First, the <i>B. subtilis</i> strain TEA was modified through the CRISPR/Cas9 system, resulting in a mutant strain TEA4, which exhibited enhanced capabilities for recombinant protein expression. For efficient and safe production of SIase, different constitutive and inducible promoters were evaluated. The maltose-inducible promoter Poglv was found to have an extracellular SIase activity of 21.7 U mL<sup>-1</sup> in engineered strain TEA4. Subsequent optimization of the culture medium further increased SIase activity to 26.4 U mL<sup>-1</sup> during shake flask cultivation. Eventually, using the crude enzyme solution of the engineered strain in biotransformation reactions resulted in a high yield of isomaltulose under high concentrations sucrose, achieving a maximum yield of 83.1%. These findings demonstrated an engineered <i>B. subtilis</i> strain for antibiotic-free SIase production, paving the way for its scale-up industrial production and application.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891304","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jiwon Na, Ali Behboudi, Jiwon Mun, Hoeun Jin, Andrew L. Zydney, Youngbin Baek
Maximizing product yield in biopharmaceutical manufacturing processes is a critical factor in determining the overall cost of goods, especially given the high value of these biological products. However, there has been relatively limited research on the quantitative analysis of protein losses due to adsorption and fouling during the different membrane filtration processes employed in typical downstream operations. This study aims to provide a comprehensive analysis of protein loss in the range of membrane systems used in downstream processing including clarification, virus removal filtration, ultrafiltration/diafiltration for formulation, and final sterile filtration, all using commercially available membranes with three model proteins (bovine serum albumin, human serum albumin, and immunoglobulin G). The correlation between protein loss and various parameters (i.e., protein type, protein concentration, throughput, membrane morphology, and protein removal mechanism) was also investigated. This study provides important insights into the nature of protein loss during membrane processes as well as a methodology for quantifying protein yield loss in bioprocesses.
{"title":"Protein loss during membrane processes in biopharmaceutical manufacturing","authors":"Jiwon Na, Ali Behboudi, Jiwon Mun, Hoeun Jin, Andrew L. Zydney, Youngbin Baek","doi":"10.1002/biot.202400154","DOIUrl":"10.1002/biot.202400154","url":null,"abstract":"<p>Maximizing product yield in biopharmaceutical manufacturing processes is a critical factor in determining the overall cost of goods, especially given the high value of these biological products. However, there has been relatively limited research on the quantitative analysis of protein losses due to adsorption and fouling during the different membrane filtration processes employed in typical downstream operations. This study aims to provide a comprehensive analysis of protein loss in the range of membrane systems used in downstream processing including clarification, virus removal filtration, ultrafiltration/diafiltration for formulation, and final sterile filtration, all using commercially available membranes with three model proteins (bovine serum albumin, human serum albumin, and immunoglobulin G). The correlation between protein loss and various parameters (i.e., protein type, protein concentration, throughput, membrane morphology, and protein removal mechanism) was also investigated. This study provides important insights into the nature of protein loss during membrane processes as well as a methodology for quantifying protein yield loss in bioprocesses.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891440","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Self-assembly of biological elements into biomimetic cargo carriers for targeting and delivery is a promising approach. However, it still holds practical challenges. We developed a functionalization approach of DNA origami (DO) nanostructures with neuronal growth factor (NGF) for manipulating neuronal systems. NGF bioactivity and its interactions with the neuronal system were demonstrated in vitro and in vivo models. The DO elements fabricated by molecular self-assembly have manipulated the surrounding environment through static spatially and temporally controlled presentation of ligands to the cell surface receptors. Our data showed effective bioactivity in differentiating PC12 cells in vitro. Furthermore, the DNA origami NGF (DON) affected the growth directionality and spatial capabilities of dorsal root ganglion neurons in culture by introducing a chemotaxis effect along a gradient of functionalized DO structures. Finally, we showed that these elements provide enhanced axonal regeneration in a rat sciatic nerve injury model in vivo. This study is a proof of principle for the functionality of DO in neuronal manipulation and regeneration. The approach proposed here, of an engineered platform formed out of programmable nanoscale elements constructed of DO, could be extended beyond the nervous system and revolutionize the fields of regenerative medicine, tissue engineering, and cell biology.
将生物元素自组装成生物仿生货物载体以进行靶向和输送是一种很有前景的方法。然而,它仍然面临着实际挑战。我们开发了一种用神经元生长因子(NGF)操纵神经元系统的 DNA 折纸(DO)纳米结构功能化方法。NGF 的生物活性及其与神经元系统的相互作用已在体外和体内模型中得到证实。通过分子自组装制造的 DO 元件通过向细胞表面受体静态呈现配体的空间和时间控制来操纵周围环境。我们的数据显示,它在体外分化 PC12 细胞方面具有有效的生物活性。此外,DNA 折纸 NGF (DON) 通过沿功能化 DO 结构梯度引入趋化效应,影响了培养中背根神经节神经元的生长方向性和空间能力。最后,我们发现这些元素在大鼠坐骨神经损伤模型中增强了轴突再生能力。这项研究证明了 DO 在神经元操作和再生中的功能。这里提出的方法,即由 DO 构建的可编程纳米级元件形成的工程平台,可以扩展到神经系统之外,并为再生医学、组织工程和细胞生物学领域带来革命性的变化。
{"title":"DNA origami scaffold promoting nerve guidance and regeneration","authors":"Jonathan Giron, Merav Antman-Passig, Neta Zilony, Hadas Schori, Ido Bachelet, Orit Shefi","doi":"10.1002/biot.202300734","DOIUrl":"10.1002/biot.202300734","url":null,"abstract":"<p>Self-assembly of biological elements into biomimetic cargo carriers for targeting and delivery is a promising approach. However, it still holds practical challenges. We developed a functionalization approach of DNA origami (DO) nanostructures with neuronal growth factor (NGF) for manipulating neuronal systems. NGF bioactivity and its interactions with the neuronal system were demonstrated in vitro and in vivo models. The DO elements fabricated by molecular self-assembly have manipulated the surrounding environment through static spatially and temporally controlled presentation of ligands to the cell surface receptors. Our data showed effective bioactivity in differentiating PC12 cells in vitro. Furthermore, the DNA origami NGF (DON) affected the growth directionality and spatial capabilities of dorsal root ganglion neurons in culture by introducing a chemotaxis effect along a gradient of functionalized DO structures. Finally, we showed that these elements provide enhanced axonal regeneration in a rat sciatic nerve injury model in vivo. This study is a proof of principle for the functionality of DO in neuronal manipulation and regeneration. The approach proposed here, of an engineered platform formed out of programmable nanoscale elements constructed of DO, could be extended beyond the nervous system and revolutionize the fields of regenerative medicine, tissue engineering, and cell biology.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300734","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891342","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ju Young Lee, Hyunbin D. Huh, Dong Ki Lee, So Yeon Park, Ji Eun Shin, Heon Yung Gee, Hyun Woo Park
As the biopharmaceutical industry continues to mature in its cost-effectiveness and productivity, many companies have begun employing larger-scale biomanufacturing and bioprocessing protocols. While many of these protocols require cells with anchorage-independent growth, it remains challenging to induce the necessary suspension adaptations in many different cell types. In addition, although transfection efficiency is an important consideration for all cells, especially for therapeutic protein production, cells in suspension are generally more difficult to transfect than adherent cells. Thus, much of the biomanufacturing industry is focused on the development of new human cell lines with properties that can support more efficient biopharmaceutical production. With this in mind, we identified a set of “Adherent-to-Suspension Transition” (AST) factors, IKZF1, BTG2 and KLF1, the expression of which induces adherent cells to acquire anchorage-independent growth. Working from the HEK293A cell line, we established 293-AST cells and 293-AST-TetR cells for inducible and reversible reprogramming of anchorage dependency. Surprisingly, we found that the AST-TetR system induces the necessary suspension adaptations with an accompanying increase in transfection efficiency and protein expression rate. Our AST-TetR system therefore represents a novel technological platform for the development of cell lines used for generating therapeutic proteins.
{"title":"Reprogramming anchorage dependency to develop cell lines for recombinant protein expression","authors":"Ju Young Lee, Hyunbin D. Huh, Dong Ki Lee, So Yeon Park, Ji Eun Shin, Heon Yung Gee, Hyun Woo Park","doi":"10.1002/biot.202400104","DOIUrl":"https://doi.org/10.1002/biot.202400104","url":null,"abstract":"<p>As the biopharmaceutical industry continues to mature in its cost-effectiveness and productivity, many companies have begun employing larger-scale biomanufacturing and bioprocessing protocols. While many of these protocols require cells with anchorage-independent growth, it remains challenging to induce the necessary suspension adaptations in many different cell types. In addition, although transfection efficiency is an important consideration for all cells, especially for therapeutic protein production, cells in suspension are generally more difficult to transfect than adherent cells. Thus, much of the biomanufacturing industry is focused on the development of new human cell lines with properties that can support more efficient biopharmaceutical production. With this in mind, we identified a set of “Adherent-to-Suspension Transition” (AST) factors, IKZF1, BTG2 and KLF1, the expression of which induces adherent cells to acquire anchorage-independent growth. Working from the HEK293A cell line, we established 293-AST cells and 293-AST-TetR cells for inducible and reversible reprogramming of anchorage dependency. Surprisingly, we found that the AST-TetR system induces the necessary suspension adaptations with an accompanying increase in transfection efficiency and protein expression rate. Our AST-TetR system therefore represents a novel technological platform for the development of cell lines used for generating therapeutic proteins.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400104","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140826183","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Justin S. Antony, Anabel Migenda Herranz, Tahereh Mohammadian Gol, Susanne Mailand, Paul Monnier, Jennifer Rottenberger, Alicia Roig-Merino, Bettina Keller, Claudia Gowin, Maria Milla, Tobias A. Beyer, Markus Mezger
Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.
中国仓鼠卵巢(CHO)细胞是生产重组蛋白(包括单克隆抗体)的常用哺乳动物宿主系统。然而,CHO 生产的单克隆抗体上显示出的不利的非人类糖型对产品质量、药代动力学和治疗效率产生了负面影响。糖工程(如通过基因消除 CHO 细胞中参与糖基化途径的基因)是一种可行的解决方案,但由于时间较长、工作流程繁琐而受到限制。在这项概念验证(PoC)研究中,我们提出了一种被称为 CellEDIT 的新方法,利用 FluidFM 技术将 CRISPR 成分通过核内递送到单细胞,从而对 CHO 细胞进行工程化改造。将靶向 BAX、DHFR 和 FUT8 的 CRISPR 系统直接注入单个细胞的细胞核,使我们能够在短时间内生成三重基因敲除的 CHO-K1 细胞系。所提出的技术确保了单克隆性的起源,而无需进行极限稀释、细胞分选或阳性选择。此外,该方法还能在 CHO 细胞中开发单基因和多基因敲除克隆(FUT8、BAX 和 DHFR)。对单个和多个基因敲除克隆的进一步分析证实了目标基因破坏和蛋白质表达的改变。基因敲除 CHO-K1 克隆在随后的传代过程中显示出基因编辑的持续性,与无血清化学定义培养基兼容,并显示出与亲本克隆相同的转基因表达。
{"title":"Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9","authors":"Justin S. Antony, Anabel Migenda Herranz, Tahereh Mohammadian Gol, Susanne Mailand, Paul Monnier, Jennifer Rottenberger, Alicia Roig-Merino, Bettina Keller, Claudia Gowin, Maria Milla, Tobias A. Beyer, Markus Mezger","doi":"10.1002/biot.202300505","DOIUrl":"https://doi.org/10.1002/biot.202300505","url":null,"abstract":"<p>Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (<i>FUT8</i>, <i>BAX</i>, and <i>DHFR</i>) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300505","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140633740","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Martin Bereta, Michal Teplan, Tomáš Zakar, Hoang Vuviet, Michal Cifra, Djamel Eddine Chafai
The application of pulsed electric fields (PEFs) is becoming a promising tool for application in biotechnology, and the food industry. However, real-time monitoring of the efficiency of PEF treatment conditions is challenging, especially at the industrial scale and in continuous production conditions. To overcome this challenge, we have developed a straightforward setup capable of real-time detection of yeast biological autoluminescence (BAL) during pulsing. Saccharomyces cerevisiae culture was exposed to 8 pulses of 100 µs width with electric field strength magnitude 2–7 kV cm−1. To assess the sensitivity of our method in detecting yeast electroporation, we conducted a comparison with established methods including impedance measurements, propidium iodide uptake, cell growth assay, and fluorescence microscopy. Our results demonstrate that yeast electroporation can be instantaneously monitored during pulsing, making it highly suitable for industrial applications. Furthermore, the simplicity of our setup facilitates its integration into continuous liquid flow systems. Additionally, we have established quantitative indicators based on a thorough statistical analysis of the data that can be implemented through a dedicated machine interface, providing efficiency indicators for analysis.
{"title":"Biological autoluminescence enables effective monitoring of yeast cell electroporation","authors":"Martin Bereta, Michal Teplan, Tomáš Zakar, Hoang Vuviet, Michal Cifra, Djamel Eddine Chafai","doi":"10.1002/biot.202300475","DOIUrl":"https://doi.org/10.1002/biot.202300475","url":null,"abstract":"<p>The application of pulsed electric fields (PEFs) is becoming a promising tool for application in biotechnology, and the food industry. However, real-time monitoring of the efficiency of PEF treatment conditions is challenging, especially at the industrial scale and in continuous production conditions. To overcome this challenge, we have developed a straightforward setup capable of real-time detection of yeast biological autoluminescence (BAL) during pulsing. <i>Saccharomyces cerevisiae</i> culture was exposed to 8 pulses of 100 µs width with electric field strength magnitude 2–7 kV cm<sup>−1</sup>. To assess the sensitivity of our method in detecting yeast electroporation, we conducted a comparison with established methods including impedance measurements, propidium iodide uptake, cell growth assay, and fluorescence microscopy. Our results demonstrate that yeast electroporation can be instantaneously monitored during pulsing, making it highly suitable for industrial applications. Furthermore, the simplicity of our setup facilitates its integration into continuous liquid flow systems. Additionally, we have established quantitative indicators based on a thorough statistical analysis of the data that can be implemented through a dedicated machine interface, providing efficiency indicators for analysis.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140633741","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ruixia Zhao, Sinan Cheng, Xue Bai, Danying Zhang, Hongming Fang, Wanlin Che, Wenxuan Zhang, Yujuan Zhou, Wei Duan, Qiumin Liang, Li Xiao, Guochao Nie, Yingchun Hou
Hepatocellular carcinoma (HCC) is a digestive tract cancer with high mortality and poor prognosis, especially in China. Current chemotherapeutic drugs lead to poor prognosis, low efficacy, and high side effects due to weak targeting specificity and rapidly formed multidrug resistance (MDR). Based on the previous studies on the doxorubicin (DOX) formulation for cancer targeting therapy, we developed a novel DOX delivery formulation for the targeting chemotherapy of HCC and DOX resistant HCC. HCSP4 was previously screened and casein kinase 2α (CK2α) was predicted as its specific target on HCC cells in our lab. In the study, miR125a-5p was firstly predicted as an MDR inhibiting miRNA, and then CK2α was validated as the target of HCSP4 and miR125a-5p using CK2α-/-HepG2 cells. Based on the above, an HCC targeting and MDR inhibiting DOX delivery liposomal formulation, HCSP4/Lipo-DOX/miR125a-5p was synthesized and tested for its HCC therapeutic efficacy in vitro. The results showed that the liposomal DOX delivery formulation targeted to HCC cells specifically and sensitively, and presented the satisfied therapeutic efficacy for HCC, particularly for DOX resistant HCC. The potential therapeutic mechanism of the DOX delivery formulation was explored, and the formulation inhibited the expression of MDR-relevant genes including ATP-binding cassette subfamily B member 1 (ABCB1, also known as P-glycoprotein), ATP-binding cassette subfamily C member 5 (ABCC5), enhancer of zeste homolog 2 (EZH2), and ATPase Na+/K+ transporting subunit beta 1 (ATP1B1). Our study presents a novel targeting chemotherapeutic drug formulation for the therapy of HCC, especially for drug resistant HCC, although it is primarily and needs further study in vivo, but provided a new strategy for the development of novel anticancer drugs.
{"title":"Development of an efficient liposomal DOX delivery formulation for HCC therapy by targeting CK2α","authors":"Ruixia Zhao, Sinan Cheng, Xue Bai, Danying Zhang, Hongming Fang, Wanlin Che, Wenxuan Zhang, Yujuan Zhou, Wei Duan, Qiumin Liang, Li Xiao, Guochao Nie, Yingchun Hou","doi":"10.1002/biot.202400050","DOIUrl":"https://doi.org/10.1002/biot.202400050","url":null,"abstract":"<p>Hepatocellular carcinoma (HCC) is a digestive tract cancer with high mortality and poor prognosis, especially in China. Current chemotherapeutic drugs lead to poor prognosis, low efficacy, and high side effects due to weak targeting specificity and rapidly formed multidrug resistance (MDR). Based on the previous studies on the doxorubicin (DOX) formulation for cancer targeting therapy, we developed a novel DOX delivery formulation for the targeting chemotherapy of HCC and DOX resistant HCC. HCSP4 was previously screened and casein kinase 2α (CK2α) was predicted as its specific target on HCC cells in our lab. In the study, miR125a-5p was firstly predicted as an MDR inhibiting miRNA, and then CK2α was validated as the target of HCSP4 and miR125a-5p using CK2α-/-HepG2 cells. Based on the above, an HCC targeting and MDR inhibiting DOX delivery liposomal formulation, HCSP4/Lipo-DOX/miR125a-5p was synthesized and tested for its HCC therapeutic efficacy in vitro. The results showed that the liposomal DOX delivery formulation targeted to HCC cells specifically and sensitively, and presented the satisfied therapeutic efficacy for HCC, particularly for DOX resistant HCC. The potential therapeutic mechanism of the DOX delivery formulation was explored, and the formulation inhibited the expression of MDR-relevant genes including ATP-binding cassette subfamily B member 1 (ABCB1, also known as P-glycoprotein), ATP-binding cassette subfamily C member 5 (ABCC5), enhancer of zeste homolog 2 (EZH2), and ATPase Na<sup>+</sup>/K<sup>+</sup> transporting subunit beta 1 (ATP1B1). Our study presents a novel targeting chemotherapeutic drug formulation for the therapy of HCC, especially for drug resistant HCC, although it is primarily and needs further study in vivo, but provided a new strategy for the development of novel anticancer drugs.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140633739","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The use of a combination of several antibacterial agents for therapy holds great promise in reducing the dosage and side effects of these agents, improving their efficiency, and inducing potential synergistic therapeutic effects. Herein, this study provides an innovative antibacterial treatment strategy by synergistically combining R12-AgNPs with H2O2 therapy. R12-AgNPs were simply produced with the supernatant of an ionizing radiation-tolerant bacterium Deinococcus wulumuqiensis R12 by one-step under room temperature. In comparison with chemically synthesized AgNPs, the biosynthesized AgNPs presented fascinating antibacterial activity and peroxidase-like properties, which endowed it with the capability to catalyze the decomposition of H2O2 to generate hydroxyl radical. After the combination of R12-AgNPs and H2O2, an excellent synergistic bacteriostatic activity was observed for both Escherichia coli and Staphylococcus aureus, especially at low concentrations. In addition, in vitro cytotoxicity tests showed R12-AgNPs had good biocompatibility. Thus, this work presents a novel antibacterial agent that exhibits favorable synergistic antibacterial activity and low toxicity, without the use of antibiotics or a complicated synthesis process.
{"title":"Deinococcus wulumuqiensis R12 synthesized silver nanoparticles with peroxidase-like activity for synergistic antibacterial application","authors":"Jingjia Liu, Nan Zhang, Bowen Shen, Liling Zhang, Zhidong Zhang, Liying Zhu, Ling Jiang","doi":"10.1002/biot.202300584","DOIUrl":"https://doi.org/10.1002/biot.202300584","url":null,"abstract":"<p>The use of a combination of several antibacterial agents for therapy holds great promise in reducing the dosage and side effects of these agents, improving their efficiency, and inducing potential synergistic therapeutic effects. Herein, this study provides an innovative antibacterial treatment strategy by synergistically combining R12-AgNPs with H<sub>2</sub>O<sub>2</sub> therapy. R12-AgNPs were simply produced with the supernatant of an ionizing radiation-tolerant bacterium <i>Deinococcus wulumuqiensis</i> R12 by one-step under room temperature. In comparison with chemically synthesized AgNPs, the biosynthesized AgNPs presented fascinating antibacterial activity and peroxidase-like properties, which endowed it with the capability to catalyze the decomposition of H<sub>2</sub>O<sub>2</sub> to generate hydroxyl radical. After the combination of R12-AgNPs and H<sub>2</sub>O<sub>2</sub>, an excellent synergistic bacteriostatic activity was observed for both <i>Escherichia coli</i> and <i>Staphylococcus aureus</i>, especially at low concentrations. In addition, in vitro cytotoxicity tests showed R12-AgNPs had good biocompatibility. Thus, this work presents a novel antibacterial agent that exhibits favorable synergistic antibacterial activity and low toxicity, without the use of antibiotics or a complicated synthesis process.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140633793","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
It was previously demonstrated that polypod-like nanostructured DNA (polypodna) comprising three or more oligodeoxynucleotides (ODNs) were useful for the delivery of ODNs containing cytosine–phosphate–guanine (CpG) motifs, or CpG ODNs, to immune cells. Although the immunostimulatory activity of single-stranded CpG ODNs is highly dependent on CpG motif sequence and position, little is known about how the position of the motif affects the immunostimulatory activity of CpG motif-containing nanostructured DNAs. In the present study, four series of polypodna were designed, each comprising a CpG ODN with one potent CpG motif at varying positions and 2–5 CpG-free ODNs, and investigated their immunostimulatory activity using Toll-like receptor-9 (TLR9)-positive murine macrophage-like RAW264.7 cells. Polypodnas with the CpG motif in the 5′-overhang induced more tumor necrosis factor-α release than those with the motif in the double-stranded region, even though their cellular uptake were similar. Importantly, the rank order of the immunostimulatory activity of single-stranded CpG ODNs changed after their incorporation into polypodna. These results indicate that the CpG ODN sequence as well as the motif location in nanostructured DNAs should be considered for designing the CpG motif-containing nanostructured DNAs for immune stimulation.
{"title":"Tuning CpG motif position in nanostructured DNA for efficient immune stimulation","authors":"Mengmeng Tan, Natsuki Makiguchi, Kosuke Kusamori, Shoko Itakura, Yuki Takahashi, Yoshinobu Takakura, Makiya Nishikawa","doi":"10.1002/biot.202300308","DOIUrl":"https://doi.org/10.1002/biot.202300308","url":null,"abstract":"<p>It was previously demonstrated that polypod-like nanostructured DNA (polypodna) comprising three or more oligodeoxynucleotides (ODNs) were useful for the delivery of ODNs containing cytosine–phosphate–guanine (CpG) motifs, or CpG ODNs, to immune cells. Although the immunostimulatory activity of single-stranded CpG ODNs is highly dependent on CpG motif sequence and position, little is known about how the position of the motif affects the immunostimulatory activity of CpG motif-containing nanostructured DNAs. In the present study, four series of polypodna were designed, each comprising a CpG ODN with one potent CpG motif at varying positions and 2–5 CpG-free ODNs, and investigated their immunostimulatory activity using Toll-like receptor-9 (TLR9)-positive murine macrophage-like RAW264.7 cells. Polypodnas with the CpG motif in the 5′-overhang induced more tumor necrosis factor-α release than those with the motif in the double-stranded region, even though their cellular uptake were similar. Importantly, the rank order of the immunostimulatory activity of single-stranded CpG ODNs changed after their incorporation into polypodna. These results indicate that the CpG ODN sequence as well as the motif location in nanostructured DNAs should be considered for designing the CpG motif-containing nanostructured DNAs for immune stimulation.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300308","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140633742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}