The CRISPR/Cas9 system, known for its efficient and easy gene-editing capabilities, is widely preferred and extensively used in various fields, attracting considerable attention from researchers both domestically and internationally. Extracellular vesicles, as natural nanoscale carriers with abundant sources, are proving to be a highly attractive delivery vehicle for the CRISPR/Cas9 system. Compared to conventional viral or non-viral vectors, extracellular vesicles offer significant advantages in terms of safety, capacity, penetrability, targeting specificity, and transformative potential. They are poised to become the optimal carriers for delivery of the CRISPR/Cas9 system. This article summarizes common delivery strategies and loading methods for the CRISPR/Cas9 system, contrasts extracellular vesicles with other carriers, and provides a comprehensive review of the advantages, domestic and international research progress, and applications of extracellular vesicles in delivering the CRISPR/Cas9 system. The goal is to contribute to the advancement of the field of gene-editing delivery.
Extracellular vesicles (EVs) are cell-derived nanovesicles that can deliver bioactive cargoes among cells or organs, including proteins, nucleic acids and metabolic molecules. Recently, mitochondrial contents-containing EVs (mitoEVs) were identified as a new subtype of EVs, containing mitochondrial DNA, proteins, and damaged or active mitochondria. Secretion of mitoEVs contributes to maintaining homeostasis of donor cells, rescuing the metabolic status of recipient cells and participating in the regulation of immune microenvironment. The biological information carried by mitoEVs could serve as crucial targets for disease diagnosis, and the active mitochondrial components had proved potential therapeutic effects in various diseases. In conclusion, summarizing the formation mechanism, function, isolation and characterization of mitoEVs, and prospecting its future clinical application prospects, can provide potential targets and insights for the development and treatment of disease diagnostic markers.
Exosomes are nanoscale vesicular structures with a diameter of 40-150 nm that are shed from the cell membrane or secreted by cells. They consist of a bilayer membrane and encapsulate various substances such as DNA, mRNA, microRNA, circRNA, and proteins. Almost all living cells, including tumor cells, can secrete exosomes, which play a critical role in cell communication, signal transduction, cargo delivery, tumor therapy, and diagnosis. The unique biological properties, preferential homing to tumors, and targeted drug delivery capabilities make exosomes attractive biomarkers and effective tools for cancer diagnosis and treatment. Therefore, comprehensive research on exosomes as nanomaterials will undoubtedly accelerate the progress of their application in cancer treatment. This article summarizes the targeted delivery function of exosomes and discusses their role in cancer diagnosis, immunotherapy, tumor microenvironment, and drug resistance. It is expected to provide insights into the development and evaluation of exosome-based tumor therapy.
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive dysfunction and behavioral impairment, accounting for approximately 70% of all caese of dementia. Currently, no cure has been identified, and the predominant treatment approach remains symptomatic relief. Astrocyte-derived extracellular vesicles (ADEVs), which contain diverse components such as proteins, nucleic acids, and lipids, serve as a critical mode of intercellular communication. Under physiological conditions, ADEVs contribute to the amelioration of AD by maintaining neuronal activity, promoting neurogenesis, and enhancing synaptic plasticity. Conversely, under pathological conditions, they participate in exacerbating AD pathology by promoting neuroinflammation and inhibiting neuronal dendritic growth. This paper provides a comprehensive review of the multiple effects of ADEVs on the development of AD, and aims to provide a theoretical basis for the potential use of ADEVs in the treatment of AD.
Liver fibrosis is a major problem in the treatment of liver disease. Mesenchymal stem cells can be used to treat liver fibrosis, but are limited by their potential carcinogenicity, and the large number of cells required for transplantation. In recent years, mesenchymal stem cell exosomes have become a research hotspot due to their smaller size, lower immunogenicity and non-carcinogenicity. However, the clinical application of mesenchymal stem cell exosomes is limited for the following reasons. First, there is a lack of standard and uniform methods for extracting and identifying exosomes from different types of mesenchymal stem cells. Second, the mechanism of mesenchymal stem cell exosomes to treat liver fibrosis is not clear. Finally, mesenchymal stem cell exosome therapy has some problems such as weak ability to target aHSC, low exosome production, low drug loading ability and low delivery efficiency. In view of the above reasons, the factors causing the difference in exosome extraction effect of common mesenchymal stem cells, the mechanism of mesenchymal stem cell exosomes ameliorating liver fibrosis and its optimization strategy were reviewed, providing new understanding and new ideas for the treatment of liver fibrosis by mesenchymal stem cell exosomes.
Reducing lactic acid and ammonia accumulation has long been a goal of the mammalian cell biotechnology industry, and process control is a more convenient and efficient way to achieve this. That is, the accumulation of lactic acid and ammonia is reduced by controlling lower concentrations of glucose and glutamine during culture. Based on online Raman monitoring technology and proportional-integral-derivative (PID) automatic feedback control technology, this research realized the control of low glucose concentration (about 0.5 g/L) and low glutamine concentration (about 1.5 mmol/L) during the production of BHK-21 cell PRV vaccine. Lactic acid and ammonia accumulation were reduced by 26.0% and 30.3%, respectively. The pH at virus inoculation was maintained above 6.75, and the optimal feeding, inoculation, and harvest time of the virus were determined by online live cell detection technology. After the feeding operation, the maximum specific cell growth rate was delayed for 12 h and the high cell activity state was maintained for a longer time (24-48 h). While maintaining the virus-producing ability of single cells, the cell density at virus inoculation was effectively increased. The maximum virus titer was 10 times that of the high-glucose control group and 1.4 times that of the manual feeding group. Instead of the traditional manual feeding operation, the intelligent production process of PRV vaccine controlled by BHK-21 cell reactor PAT based on low glucose and glutamine concentration as well as online Raman and online live cell detection was successfully established, which laid the foundation for the intelligent production of biological drugs.
Objective: To explore the role of Kupffer cells in APAP-induced liver injury by using the Cre/iDTR system to specifically deplete Kupffer cells. Methods: Clec4fCre/iDTR mice were injected intraperitoneally with 1 μg DT (Diphtheria Ttoxin, diphtheria toxin), and the depletion efficiency of Kupffer cells in the liver was detected by flow cytometry. Then, Clec4fCre/iDTR mice were divided into four groups, namely control group, DT group, APAP group, DT and APAP group. 300 mg/kg APAP was injected intraperitoneally after 18 h of starvation to establish an acute liver injury model. ELISA and CBA analysis were used to detect the release of ALT and AST and the secretion of inflammatory factors in serum. H&E staining and TUNEL staining were used to detect the necrosis of liver tissue and the apoptosis of hepatocytes. Immunohistochemistry was used to detect inflammatory cell infiltration in the liver and immunoblotting to detect activation of the JNK signaling pathway. qPCR was used to detect the transcriptional expression of inflammatory factors in the liver. Results: Hepatic Kupffer cells in Clec4fCre/iDTR mice were almost completely depleted at 12 and 24 h after injection of 1 μg DT. Depletion of Kupffer cells potently ameliorated APAP-induced liver injury, as manifested by decreased serum ALT and AST levels, less hepatic necrotic area and hepatocyte apoptosis, reduced inflammatory cell infiltration and production of inflammatory cytokines, and abrogated activation of the JNK pathway. Conclusion: This study demonstrated that removal of Kupffer cells significantly attenuated APAP-induced liver injury, suggesting an essential contribution of Kupffer cells to APAP hepatotoxicity.
Immune cell function is regulated by co-inhibitory and costimulatory receptors. Although immune checkpoint inhibitors are widely utilized in tumor therapy, their efficacy is hampered by the immunosuppressive tumor microenvironment. Immunoagonistic drugs, such as agonistic antibodies or their corresponding ligands targeting costimulatory receptors, have the potential to enhance the immune system’s anti-tumor response and are emerging as a new generation of cancer treatment strategies. This review aims to introduce various types of immunoagonistic drugs developed in recent years and to evaluate the research progress of antibody-targeted immunoagonistic drugs in the field of tumor immunotherapy, in order to provide new insights for the development of the next generation of immunoagonistic drugs.
As a revolutionary technology, messenger RNA (mRNA) vaccines have shown immense potential in the prevention and treatment of infectious diseases. Compared to novel DNA vaccines, mRNA vaccines avoid the risk of genomic integration, and they significantly accelerate development speed and increase production efficiency compared to widely used protein-based vaccines. mRNA vaccines can encode multiple antigens, adapt to different pathogen variants, and have dual functions as antigen encoders and adjuvants, effectively eliciting robust immune responses. However, further optimization of mRNA stability, immunogenicity, and safety, as well as the efficiency enhancement of delivery systems, especially lipid nanoparticles (LNPs), remain key challenges in technology development. Globally, the successful use of mRNA vaccines in diseases such as COVID-19 has opened up a new direction in vaccine research, indicating their potential for broader application in disease prevention and treatment. On this basis, the latest advances in mRNA vaccine sequence optimization strategies, delivery systems, and their application in infectious diseases are reviewed with the aim of providing a comprehensive reference for future research and applications.
Rapid and accurate molecular diagnostics play an important role in food safety, disease diagnosis, environmental monitoring and other life science fields. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has become an effective molecular diagnostic tool due to its advantages such as sensitivity, specificity, convenience and speed. However, most current detection methods based on CRISPR/Cas still rely on the pre-amplification of nucleic acid, which can produce non-specific amplicons, prolong the detection time, and increase the risk of carryover contamination. Therefore, more amplification-free strategies have been developed to maximize the sensitivity while achieving target detection in a single step. In this review, the importance of amplification-free CRISPR systems, influencing factors and related strategies to optimize the system are summarized, followed by the three main strategies to amplify the signal of target amplification-free CRISPR/Cas-based technology, including the digital CRISPR detection platforms, surface-enhanced Raman spectroscopy, and electrochemical (EC)/ECL biosensors. Finally, the future of the CRISPR/Cas system is envisioned to provide theoretical guidance for the application and development of amplification-free CRISPR/Cas detection systems.
Astaxanthin is a crimson ketone carotene, which is mainly produced by algae, bacteria and phytoplankton. As a natural pigment and antioxidant, it is widely used in daily chemical products, pharmaceutical industry, food health industry, aquatic animal husbandry. Compared with natural extraction and chemical synthesis, the heterologous synthesis of astaxanthin by heterologous microorganisms is a green, economical and efficient method. The biosynthetic pathway of astaxanthin was introduced, the research status of microbial synthesis of astaxanthin and the engineering strategy of astaxanthin synthesis by yeast were summarized, and the future research prospects were discussed.
L-Alanyl-L-Glutamine (Ala-Gln) is an important dipeptide that can be decomposed as L-glutamine (L-Gln) in the body, which plays an irreplaceable physiological and biochemical roles. The traditional chemical synthesis method suffers from the disadvantages of high pollution, low conversion, and low purity, and is accompanied by the generation of toxic by-products, which limits its large-scale application. With the rapid development of biology, researchers have analyzed the synthetic methods of Ala-Gln and directed the modification of strains, which opens up a new era for the green and efficient production of Ala-Gln. This paper reviews the application of Ala-Gln, the metabolic pathways of Ala-Gln synthesized by traditional chemical synthesis and microbial synthesis, and the progress of microbial production of Ala-Gln, with the aim of exploring the use of synthetic biology to optimize the metabolic pathway of Ala-Gln, providing a theoretical basis for the efficient production of Ala-Gln, and promoting the industrial production of Ala-Gln.
