Electrospinning is a straightforward and versatile method to prepare 1D SNFs with programmable structures, manageable dimensions, and modifiable properties, which hold great potential in many cutting-edge applications including aerospace, nanodevice, and energy. In this review, substantial advances in the structural design, controllable synthesis, and multifunctional applications of electrospun SNFs are highlighted. We begin with a brief introduction to the fundamental principles, available raw materials, and typical apparatus of electrospun SNFs. We then discuss the strategies for preparing SNFs with diverse structures in detail, especially stressing the newly emerging three-dimensional SiO2 nanofibrous aerogels. We continue with focus on major breakthroughs about brittleness-to-flexibility transition of SNFs and the means to achieve their mechanical reinforcement. In addition, we showcase recent applications enabled by electrospun SNFs, with particular emphasis on physical protection, health care and water treatment. In the end, we summarize this review and provide some perspectives on the future development direction of electrospun SNFs. One-dimensional (1D) SiO The use of SiO2 is increasingly important in today’s world. It is no exaggeration to say that SiO2 is an essential part of the modern industrial foundation, employed in many industrial areas from glass making to oil extraction: a full list of usages would take up many pages. The significance of SiO2 is indisputable, and it is hard to imagine a world in which significant restrictions are imposed on its use . . 4@PVB nkes Fig. i. Furthe2-TiO2 nanofibrous membranes by electrospinning, on which MnO2 nanoparticles were in situ deposited [2 nanoparticles increased gradually with the increase in the number of synthesis cycles. Because the MnO2 nanoparticles were fixed stably on the surface of the nanofibers, it has excellent catalytic oxidation effect on formaldehyde gas. The formaldehyde removal efficiency of the prepared sample was nearly 100% within 20 min, and the sample still showed a formaldehyde removal efficiency of 91.57% after 5 cycles of use. Moreover, Zhan and colleagues designed SiO2-doped mesoporous TiO2 nanofibers and verified their excellent photocatalytic performance through degrading gaseous toluene under ultraviolet light [2-TiO2 composite nanofibers showed a toluene degradation efficiency of 90.6% under the appropriate SiO2 doping, which was far superior to pure TiO2 nanofibers (69.6%) and commercial Degussa P25 (70.5%). The strong coupling effect between the doped SiO2 and the TiO2 may reduce the photoexcitation level and reduce the difficulty of toluene degradation removal. At the same time, these catalysts were used for the removal of organic pollutants in the form of electrospun nanofibrous membranes, which effectively avoided the drawbacks of conventional powder catalysts such as easy agglomeration and difficult recovery.In addition to chemical warfare agents in war, there are also volatile organic compounds in daily life, such as formaldehyde, toluene, and xylene, and prolonged exposure to these compounds can be extremely harmful to the human body. Cui et al. fabricated soft SiOeposited . It was et light . The resSome progress has been made in the degradation of toxic substances by electrospun SNFs, especially when they were compounded with other functional nanomaterials, showed the robust synergistic effects that greatly improve the overall properties. However, in the face of increasingly complex living environment, all kinds of harmful substances to human health need to be considered, so it is particularly necessary to improve the simultaneous degradation performance of materials to a variety of toxic substances. In addition, for toxics in different states, such as gas, liquid, and aerosol, the structure of materials needs to be elaborately designed to meet the use requirements of different scenarios.2, CaO, and P2O5 [2 precursor sol to prepare composite biomaterials for bone tissue regeneration by electrospinning technology [2, took full advantage of these two materials to successfully create effective biomaterials for bone tissue engineering.The natural bone extracellular matrix (ECM) is composed of 60–70 wt% inorganic components and 10–30 wt% organic components and presents a network structure of nanofibers . Electroand P2O5 . The terchnology . It was 2-TiO2 hybrid nanofibers with different SiO2 content and evaluated their osteogenic potential [2-TiO2 nanofibrous membrane enhanced the osteogenic differentiation of mesenchymal stem cells (MSC), especially when the average diameter of the nanofibers was larger and the crystallinity of the nanofibrous membrane was higher.The abovementioned works did not involve calcination of the as-spun nanofibers, and it is known that the conventional preparation of ceramic nanofibers inevitably required calcination to remove organic components. Therefore, different calcination conditions had a great impact on whether the obtained nanofibers were suitable for bone tissue engineering. Sakai and colleagues found in their study that apatite particles with a diameter of 10 μm were formed on the SNFs without calcination after soaking in SBF solution for 1 week . Furtherotential . The resThe electrospun SNFs prepared above were generally 2D nanofibrous membranes with pore sizes of several microns, which were adverse to the transport of nutrients and metabolites and difficult to provide enough space for cell growth. Wang et al. developed a 3D ceramic nanofibrous scaffold assembled by electrospun SNFs with CTS as the bonding site via freeze-drying technology . The res2 showed limited biomineralization activity and was difficult to accelerate bone regeneration in osteoporosis. Wang and co-workers further prepared bioactive glass (SiO2-CaO) nanofibers and combined them with CTS as cross-linking agent to synthesize 3D nanofibrous scaffolds [2-CaO nanofibers and CTS had superior mechanical properties. In addition, the scaffolds possessed continuous nanofiber-assembled cell walls, which were structurally highly consistent with the natural ECM  and dynamic (1.8 × 103 mg g−1) protein adsorption capacity, with processing flux, was up to 1.5 × 104 L h−1 m−2 only driven by the buffer’s own gravity (~ 1 kPa).Although the introduction of these electrospun SNFs based on 2D nanofibrous membrane promoted the actual performance of protein separation, their dense structure led to problems such as high resistance of protein molecule adsorption and mass transfer and slow liquid penetration velocity, which limited the full play of structural advantages of nanofibers. Fu and colleagues developed a highly carboxylated nanofibrous aerogels consisted of flexible electrospun SNFs and a functional polymer cladding layer . The as-−1 Fig. e. When t−1 Fig. f. MoreovIt can be found that most of these materials were organic/inorganic hybrid nanofibrous materials, in which the organic components will inevitably swell in the long-term practical application. This will seriously affect the morphology, pore structure, and mechanical stability of the material, which will significantly reduce the adsorption and separation performance of the material for protein. In addition, the electrospun SNFs applied in the field of protein separation in current studies basically relied on surface modification or in situ blending methods and pay little attention to the importance of nanofibrous intrinsic structure. Therefore, continuous efforts should be made in the relationship between composition controlled nanofibrous microstructure and their performance.E. coli and Gram-positive bacteria S. aureus due to the presence of Ag nanoparticles. Furthermore, Liu and co-workers developed a superhydrophilic N-halamine/SiO2 nanofibrous membranes through the combination of electrospinning technique and followed chlorination [8 CFU mL−1E. coli and S. aureus within 3 min. In addition to grafting modification on the surface of nanofibrous membrane, Shan et al. designed and prepared a new type of C/SiO2 nanofibrous antibacterial material supported by Co nanoparticles [2 nanofibrous membranes could effectively activate peroxymonosulfate and rapidly produce a large amount of reactive oxygen species, which could inactivate E. coli and S. aureus with a 7 log reduction within 3 min. The membranes also showed excellent dynamic bactericidal performance and could achieve high bactericidal efficiency of 99.99999% with flux up to 3.4 × 104 L m−2 h−1 only under gravity drive. It is worth mentioning that a novel spray sterilizer based on nanofibrous membranes was designed, which could sterilize the solid surfaces conveniently and efficiently.Because of their excellent biocompatibility, good structural tunability, and easy surface modification, electrospun SNFs have impressive antibacterial performance. Wan et al. prepared composite nanofibrous membranes by combining electrospinning and surface modification technology . The Ag rination . The obtarticles . The metE. coli and S. aureus) and could effectively sterilize the bacteria-containing sewage with ultra-high throughput (5.76 × 104 L m−2 h−1). In addition, Wang and colleagues also developed a superflexible nanofibrous aerogel with antibacterial and antiviral functions targeting harmful microorganisms such as bacteria and viruses that may exist in the public health environment [2 framework  and antiviral activity (6 log reduction against bacteriophage within 5 min). These positive results indicate that the prepared aerogel can be used as a scalable antibacterial and antiviral air filter, which means that it has great potential for health care.Although these membranes showed good antibacterial effects, their antibacterial function gradually decreased with the depletion of antibacterial ingredients. At the same time, the narrow and unconnected pore structure of the nanofibrous membrane makes it easy for bacteria to accumulate in the pore, resulting in a sharp decrease in the treatment flux. Wang et al. prepared a superelastic nanofibrous aerogel with rechargeable bactericidal properties by electrospun SNFs and functional Si–O-Si bonding networks . The excironment . The aerAt present, most of the researches on electrospun SNFs in antibacterial field focus on inactivating bacteria existing in liquid environment, and the killing of pathogens and microorganisms in air environment is relatively rare. The continuing spread of the COVID-19 in the world since 2020 has alarmed us and highlighted the importance of developing integrated nanofibrous filters with broad-spectrum biocidal activity as well as high efficiency fine particulate filtration performance. In addition, special attention should be paid to the recycling properties of these antibacterial materials in order to reduce the pressure on resources and the environment. Therefore, the future research of such materials should consider more convenient and easily scalable preparation methods combined with energy-saving high-performance regeneration means.2 g−1, the pore volume increased to 0.317 cm3 g−1, and the mesopore size concentrated at 10 nm  and high separation efficiency . Notably, the BiOBr/PANI heterojunction structure and 3D PANI conductive networks endowed the nanofibrous membrane visible light induced self-cleaning properties. In addition, Zhang et al. also prepared another intriguing nanofibrous membrane by constructing periodic knots on electrospun SNFs and further encapsulating the shell of PANI [Electrospun SNFs have unique advantages in the treatment of oily wastewater due to their high chemical stability, interconnected pore channel, high porosity, and regulable surface properties. Li et al. synthesized the hierarchical nanofibrous membranes composed of SiOspraying . Thanks bstrates . Due to  of PANI . The mem2 nanoparticles were deposited on the nanofiber surface to further enhance the nanoscale roughness of the hierarchical aerogel  deposited on deposited on porous electrospun SNFs [Solar evaporator is an effective method to treat highly saline water with clean and renewable energy and realize seawater desalination. Electrospun SNFs have good chemical durability and high porosity, which facilitates evaporation of water vapor from the pores between the nanofibers. Huang and colleagues fabricated core-sheath structure and amphiphobicity SNFs via coaxial electrospinning technology . The SiOpun SNFs . The eng2 nanofibrous aerogel which was then deposited on the nanofibrous framework by simple CNTs impregnation coating [−2 h−1 within 15 min, which was in the same level with most materials at present. However, it should be emphasized that the evaporation rate of the aerogel prepared has little change in the treatment of water with high salinity, while the evaporation rate of the conventional SiO2 aerogel as the control group decreased significantly, proving that the well-designed aerogel could effectively transport salt and prevent salt crystallization , health care , and water treatment .In this review, we have summarized the advances in electrospun SNFs, covering the rational structure design from solid to core-sheath, hollow, porous, hierarchical, aligned, and 3D-assembled structure, and relevant synthetic strategies including coaxial electrospinning method, sacrificial template method, 2 precursor sol and polymer solution spinning and then calcination. Therefore, the distinctions of spinning dope properties and the influences of calcination process should be concerned in the preparation of SNFs with 2D nanostructured networks.At present, most reports focus on core-sheath, porous, hierarchical structure of electrospun SNFs, and more attractive nanostructures are yet to be developed. For example, the widely reported spider-web-like 2D nanonets in electrospun polymer materials. These nanonets possess a highly porous structure with ultrafine diameters (10 ~ 40 nm) and small pore sizes, which hold great potential in energy, filtration, and protection applications . Differe2 nanofibrous aerogel has been widely studied in recent years. As we have seen, several methods  have been developed to prepare electrospun SiO2 nanofibrous aerogel, and its application in many areas has been explored. However, it should be acknowledged that the current method of preparing SiO2 nanofibrous aerogel is generally cumbersome, time-consuming, and energy intensive, especially involving the acquisition of staple slurry from electrospun SNFs and subsequent freeze drying. Recently, a novel approach to fabricate nanofibrous aerogel using 3D reaction electrospinning has opened a new avenue for us [As a promising new material, electrospun SiOe for us . The key2 nanofibrous aerogels prepared by freeze-drying method is generally less than 20 kPa. These materials are still difficult to meet the requirements of mechanical properties for some specific applications. It is an urgent problem to further improve the mechanical properties of SNFs. The key to solve this problem lies in the fabrication of strong and tough single SiO2 nanofiber. On this basis, building strong adhesion structure between nanofibers or improving the orientation of nanofibers might be effective strategies to enhance the mechanical properties of SNFs. In addition, it is of great significance to establish theoretical models of the mechanical relationship between single nanofiber and nanofibrous assemblies in the future work, which could guide researchers to design SNFs with higher strength and better toughness.The current tensile strength of flexible SNFs membranes is normally less than 5 MPa, while the compressive stress of SiO2 material itself, and more creativity can be generated from the aspects of structure, interface and multi-scale assembly. For example, the construction of ordered hierarchical pores in SNFs will bring a significant driver for hydrogen storage, molecular separation, and gas capture. In addition, combining the fertile surfaces of SNFs with other specific materials promises infinite possibilities in the fields of sensing, electromagnetic shielding, and flexible energy. In short, more substantial and effective applications of electrospun SiO2 nanofibrous materials are worthy of further study, and the road to the application library will never be limited.In the future, advanced applications of electrospun SNFs will go far beyond the scope of our discussion, and the existing wealth of results will continue to inspire us to create new materials. There is no need to elaborate on the characteristic advantages of SiO2 precursor sol. Therefore, needleless electrospinning is equipped with the advantages of simple construction, uniform electric field, and high efficiency, which is expected to become the main force of mass production of electrospun SNFs in the future.The large-scale manufacture of electrospun SNFs is the cornerstone to realize their commercial value. Currently, most of the synthesis methods of functional SNFs  are limited to laboratory research and are not suitable for large-scale production. Although some polymer nanofibers could be manufactured on an industrial scale, the impact of different spinning solution systems on mass production is significant, especially for spinneret devices. Some auxiliary means can be well utilized, such as high-speed airflow and rotary centrifugation, which are expected to significantly improve manufacturing efficiency. In addition, the continuity, uniformity, and efficiency of conventional multi-needle electrospinning are seriously affected because of the electric field disturbances between multiple needles and the faster gelation rate of SiOAlthough the great progress has been made, there are still many challenges in the design, preparation, and application of electrospun SNFs, and more work needs to be continued in the future. Some viewpoints are put forward here, which are expected to play a constructive role in promoting the rapid development of this field.Challenge goes together with chance, and difficulty coexists with hope. Even though there are many troubles to be faced in the future, there is no doubt that electrospinning technology is still the most powerful weapon to prepare advanced SNFs. In the future, the rapid development of SNFs will be realized through the cross-fusion of multidisciplinary knowledge of materials science, material mechanics, and mechanical engineering. We firmly believe that the design, manufacture, and application of electrospun SNFs will have an exciting and bright future with continuously push.


Early detection and prevention of precancerous lesions can significantly reduce the morbidity and mortality of colorectal cancer (CRC). Here, we developed new candidate CpG site biomarkers for CRC and evaluated the diagnostic value of their expression in blood and stool samples of CRC and precancerous lesions.We analyzed 76 pairs of CRC and adjacent normal tissue samples, 348 stool samples, and 136 blood samples. Candidate biomarkers for CRC were screened using a bioinformatics database and identified using a quantitative methylation-specific PCR method. The methylation levels of the candidate biomarkers were validated using blood and stool samples. The divided stool samples were used to construct and validate a combined diagnostic model and to analyze the independent or combined diagnostic value of candidate biomarkers in stool samples of CRC and precancerous lesions.Two candidate CpG site biomarkers for CRC, cg13096260 and cg12993163, were identified. Although both biomarkers demonstrated diagnostic performance to a certain extent when using blood samples, they showed better diagnostic value for different stages of CRC and AA with stool samples.cg13096260 and cg12993163 detection in stool samples could be a promising approach for screening and early diagnosis of CRC and precancerous lesions.The online version contains supplementary material available at 10.1186/s13148-023-01443-7. Colorectal cancer (CRC) is an important public health problem, accounting for approximately 10% of global cancer cases and deaths. It is the third most commonly diagnosed cancer and the second leading cause of cancer-related deaths globally . In contMost cases of CRC originate from neoplastic precursor lesions over a period of 10–15 years . UnfortuCurrently, the main screening methods for CRC include colonoscopy, flexible sigmoidoscopy, guaiac-based fecal occult blood test (gFOBT), fecal immunochemical test (FIT), stool DNA test, blood-based tests, and computed tomography colonography (CTC) . The effSince CRC cells are excreted in feces; abnormal DNA changes during CRC can be detected using fecal samples. Multitarget stool DNA (mt-sDNA) testing is a combination of FIT and aberrant DNA methylation tests. It has a sensitivity of 92.3% for CRC and advanced adenoma (AA) and a specificity of 86.6% . MeanwhiCurrently, many candidate DNA methylation biomarkers for CRC have been reported in various studies –19. In oTherefore, this study aimed to explore novel and valuable DNA methylation biomarkers for CRC and precancerous lesions. Candidate biomarkers were identified by bioinformatics analysis. Moreover, we analyzed these candidate biomarkers in clinical tissue, stool, and blood samples to evaluate their clinical performance for the early detection of CRC and precancerous lesions.SDC2 promoter region, and cg12993163 was from the of SHOX2 gene body region. The methylation β values of cg13096260 and cg12993163 were analyzed using TCGA database .Based on the screening process of CRC-related candidate CpG sites Fig. , and to P < 0.001), while cg12993163 was not significantly correlated with the transcriptional expression of SHOX2 (P > 0.05). There was a significant positive correlation between the methylation levels of the two CpG sites in CRC tissues (P < 0.001) , and SHOX2 expression was significantly higher in tumor tissues (P < 0.01)   for the two candidate CpG sites and the reference gene, we diluted the synthetic plasmids with candidate CpG site sequences into samples of different concentrations for three replicate assays. Both the candidate CpG sites and the reference gene were detected in samples with a minimum of five copies and a maximum Ct value of 25 . The methylation level of cg13096260 was higher in 93.4% (71/76) of tumor tissues than in paired normal tissues, and cg12993163 was higher in 90.8% (69/76) of tumor tissues than in paired normal tissues. This is consistent with the results obtained by analyzing the database.To validate the results screened through the database and the methylation levels of candidate CpG sites in CRC tissues, we used qMSP to detect the methylation of cg13096260 and cg12993163 in cancerous and normal precancerous tissues of 76 CRC patients. The ΔCt value was used to indicate the site methylation level, with lower ΔCt values indicating higher methylation levels , whereas the rest were not significantly different , and the AA group had significantly lower methylation levels than the CRC group (P < 0.001) . The methylation level of cg12993163 was significantly lower in the NED group than in the AA and CRC groups (P < 0.001), whereas there was no significant difference in methylation levels between the AA and CRC groups (P > 0.05).The methylation levels of cg13096260 and cg12993163 were detected in 162 CRC, 46 AA, and 120 no evidence of disease (NED) stool samples. Cg13096260 had significantly lower methylation levels in the NED group than in the AA and CRC groups , 31 AA cases, and 80 NED cases. The validation set consisted of stool samples from 54 CRC cases , 15 AA cases, and 40 NED cases. The training set was used to evaluate the predictive ability of two candidate CpG sites for the disease and construct a combined diagnostic model. The validation set was used to establish the cut-off values, determine the clinical significance, and verify the diagnostic efficacy of the two sites.Based on the Ct values of the two candidate CpG sites in the training set and their corresponding sample types, ROC curves of the two sites for different grouped samples were obtained, and area under the curve (AUC) values were calculated. The detection results were used to construct a combined logistic diagnostic model (score = 15.7174 − 0.3146 × cg13096260 − 0.2224 × cg12993163)  and 100% (9/9), respectively, with an overall negative rate of 90%. The negative rates of cg12993163 in other digestive tract tumors and colitis were 72.73% (8/11) and 88.89% (8/9), respectively, with an overall negative rate of 80%. The negative rates of combined diagnostic models for other digestive tract tumors and colitis were 81.81% (9/11) and 100% (9/9), respectively, and the overall negative rate was 90%. The overall negative rate of interfering diseases and the assay specificity of three assays were very similar.P < 0.05), while the rest were not significantly different , stage I and II CRC (P < 0.01), and stage III and IV CRC (P < 0.001) groups, while there was no significant difference between the AA and CRC group (P > 0.05) , whereas the methylation levels were not significantly different between the AA and CRC groups (P > 0.05).Methylation of two candidate CpG sites was detected in 128 blood samples using qMSP, including 64 CRC, 17 AA, and 47 NED samples. cg13096260 had significantly lower methylation levels in the NED group than in the AA group  and 81.25% (CI 69.15–89.53%), respectively; for early CRC was 67.57% (CI 50.11–81.44%) and 81.08% (CI 64.29–91.44%), respectively; and for AA was 70.59% (CI 44.05–88.62%) and 76.47% (CI 49.76–92.18%), respectively. The specificities were 82.98% (CI 68.65–91.86%) and 74.47% (CI 78.01–91.29%), respectively.To further investigate the specificity of DNA methylation detection in blood samples, blood samples from eight patients with other interfering diseases but without colorectal tumors were included in this study and tested for methylation levels of candidate CpG sites. The negative rate of cg13096260 in both gastric malignancy and colitis was 75% (3/4), and the overall negative rate was 75%. The negative rates of cg12993163 in gastric malignancy and colitis were 50% (2/4) and 75% (3/4), respectively, with an overall negative rate of 62.5%. The overall negative rate of interfering diseases and the assay specificity of two assays were different.P < 0.05). cg12993163 methylation levels differed between male and female participants (P < 0.05). The remaining parameters did not differ significantly between the groups  DNA methylation datasets and current research advances, we screened two candidate CpG sites for CRC, cg13096260 and cg12993163. We found that both sites had significantly higher methylation levels in tumor tissues than in normal tissues adjacent to cancer. Cg13096260 is located in the SDC2 promoter region and cg12993163 is located in the SHOX2 gene body region. Both genes are methylation biomarkers for CRC but show limited ability to identify early lesions , 30, 31.The common methods currently used to detect DNA methylation include genomic bisulfite sequencing and qMSP , 33. AltIn this study, we first detected cg13096260 and cg12993163 methylation levels in CRC, AA, and NED stool samples and subsequently constructed and validated a combined diagnostic model using the training and validation sets. The sensitivities of two CpG sites for CRC detection were very similar, whereas cg12993163 was more sensitive for AA detection, and cg13096260 had higher specificity. The combined diagnostic model achieved both high detection specificity and a certain sensitivity for AA. Other interfering diseases had no significant effects on any of the three assays. The clinical diagnostic performance of such a single- or dual-site methylation assay is robust. Moreover, the diagnostic sensitivity of our stool DNA methylation test was not affected by most clinicopathological characteristics, ensuring the same diagnostic sensitivity for different populations and tumor types, which is a required feature for CRC screening and diagnosis.Stool DNA testing has been used for CRC screening and diagnosis for several years. Cologuard, a stool DNA multi-targeting test kit approved by the US FDA in 2014, has an overall sensitivity of 92.3% for CRC, 42.4% for AA, and a specificity of 86.7% . HoweverBased on the DNA methylation results of blood samples, we found that the diagnostic sensitivity and specificity of cg13096260 and cg12993163 for colorectal tumors were approximately 70–80%, and other interfering diseases may have some influence on the detection of blood samples. Owing to the limited sample size, we did not design the training and validation sets, nor did we evaluate the diagnostic performance after combining the two sites. Although DNA methylation detection in blood samples demonstrated diagnostic performance for colorectal tumors to a certain extent, the diagnostic performance was relatively weak when compared to stool samples. Studies have shown that ctDNA produced by tumor cells in the blood is limited and diluted by whole-body blood, whereas DNA in stool is directly derived from tumor tissue in the intestine. Therefore, tumor DNA in stool is more concentrated and tissue-specific, making CpG sites detection using stool samples more sensitive and specific. This explains our results, which are consistent with those in similar studies , 36. EpiThis study has several advantages. First, it is innovative in screening candidate CpG site biomarkers for CRC and validating the CpG sites proposed for the first time in tissue samples. Second, a diverse variety of clinical samples were collected, and rich clinicopathological characteristics were evaluated, ensuring more comprehensive and reliable results. Third, this study provides more objective data to assess the diagnostic value of stool methylation detection through the design of training and validation sets of stool samples. Fourth, the sensitivity of the assays involved in the study was greater than 60% for early stage lesions, and cg12993163 had a sensitivity of 82.6% for AA in stool samples, which provides a significant advantage in both traditional and novel CRC screening and early diagnosis methods.Nevertheless, this study still has some limitations. First, the study had a limited sample size, especially for AA and blood samples, and some samples were not collected with corresponding clinicopathological characteristics. Second, the excellent detection performance at a single center needs to be validated in a large multicenter population. Third, although other interfering diseases were analyzed, the types of diseases analyzed and number of tests utilized remained very limited. Further expansion of the sample is required for statistical analysis. Fourth, all subjects in this study belonged to a Chinese population with a similar genetic background, and ethnic differences should also be taken into account. Further, the levels of some ctDNAs in blood may follow circadian rhythms . TherefoWe developed two candidate CpG site biomarkers for CRC and precancerous lesions: cg13096260 and cg12993163. The two sites were validated to have excellent diagnostic value in stool samples, either independently or in combination, for the detection of CRC at different stages and AA. Thus, detecting cg13096260 and cg12993163 in stool samples may serve as a promising approach for the screening and early diagnosis of CRC and precancerous lesions.This study explored and validated novel colorectal tumor biomarkers through the analysis of the biological databases and detection of clinical samples using qMSP. This study focused on the alteration of DNA CpG sites in the stool and blood of patients with colorectal tumor(s) and controls. The clinical trial was registered with the China Clinical Trials Registry, which is a part of the WHO International Clinical Trials Registry, under the trial registration number Chi-CTR-2100048569. The main evaluation indexes include sensitivity and specificity.The study enrolled patients who were hospitalized in the Department of Gastrointestinal Surgery of Renji Hospital, Shanghai Jiaotong University School of Medicine between October 2020 and October 2021 and had been pathologically diagnosed with CRC or AA , as welClinical samples collected for this study included cancerous and normal paracancerous tissue samples from patients with CRC, and blood and stool samples from patients with CRC, AA, interfering disease, and NED. Tissue samples were intraoperatively excised from isolated tumor specimens of cancerous tissue and paired with paracancerous normal tissue from the bowel wall tissue. Samples were then snap-frozen in liquid nitrogen and stored at a low temperature of − 80 °C. Stool samples were collected preoperatively from five random locations without bowel preparation, weighing approximately 5 g, and placed in 50 ml centrifuge tubes containing 25 ml of preservation fluid. Long-term storage was performed at − 80 °C. Blood samples were collected preoperatively from approximately 10 ml of whole blood per subject using Streck tubes, separated from the plasma, and stored at − 80 °C for extended periods.Clinical data collected from the participants included age, sex, tumor size, selected tumor indicators , tumor location, histological type, lymphatic invasion, distant metastasis, pathological staging according to the AJCC 8th edition TNM tumor staging system for CRC, pathological staging, vascular invasion, nerve invasion, microsatellite status, and selected gene mutations .A total of 515 participants were enrolled in this study, and 422 were included in the final analysis. 12 subjects were excluded due to incomplete pathological information, 25 due to unsuccessful collection of tissue, stool, or blood samples, and 32 due to insufficient reference genes. An additional 24 patients with interfering diseases were tested for DNA methylation in stool or blood samples . Among the 422 participants included in the analysis, 222 had CRC, 46 had AA, and 154 had NED. The flow of sample collection is shown in Additional file This study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Ethics Committee of Renji Hospital, Shanghai Jiaotong University School of Medicine . All participants signed an informed consent form.The public databases involved in this study were TCGA and GEO. Data from TCGA consisted mainly of Infinium Human Methylation450 BeadChip DNA methylation data , including tumor tissue from 408 CRC patients and 45 normal tissues. In addition, the GSE40279 and GSE41169 peripheral blood DNA methylation datasets from the GEO were downloaded for 751 cases of peripheral blood DNA methylation in non-tumor populations. A stepwise screening approach was used to identify and select CRC-associated DNA methylation biomarker candidates . DNA from the blood samples was extracted using a MagicPure® Cell-Free DNA Kit . All kits were used according to the manufacturers’ instructions, and all collected supernatants were stored at − 20 °C.Tissue samples were extracted using a DNeasy Blood & Tissue Kit . Stool samples were extracted by grinding the sample with glass beads until completely homogenized and then centrifuged at 4000×EZ DNA Methylation-Gold Kit  was used to transform the extracted DNA samples with sulfite. The assay was performed according to the manufacturer’s instructions. Concentration of the collected DNA was quantified using a spectrophotometer, and the samples were stored at − 20 °C.Probe Ex Taq (Probe qPCR)  kit was used to perform qMSP on DNA from sulfite-transformed samples, and PCR was performed according to the manufacturer’s instructions. Primers and probes were designed according to the specific sequences of CpG sites, with ACTB as the reference gene, refer to Additional file Detection limits of the methylation biomarker assay were determined using plasmids with candidate CpG site sequences constructed by Nanjing Kingsray Biotechnology Co. Specific sequences are listed in Additional file The methylation levels of candidate CpG sites in tissue samples were determined using the ΔCt value which is the difference between the Ct values of the target and reference gene (ACTB) normalized to the amount of DNA in the tissue samples. DNA in the stool and blood samples was mostly fragmented. Therefore, the average Ct value of the three replicate wells obtained using qMSP indicated the detection of the reference gene and the methylation level of the candidate CpG sites. The Ct value of the ACTB reference gene was used to verify the sample quality. If the Ct value of ACTB was greater than 33 in 2/3 of the wells, the sample was considered invalid. If the mean Ct value of cg13096260 in the stool sample was less than 31, it was considered positive for methylation. If the mean Ct value of cg12993163 was less than 32, it was considered positive for methylation. If the mean Ct value of cg13096260 or cg12993163 in the blood samples was less than 25, the samples were considered positive for methylation. If the sample quality was acceptable, but the candidate CpG sites were not detected in 2/3 replicate wells, a Ct value of 33 was assigned to the target CpG sites for subsequent statistical analysis.t test and chi-square test were performed: otherwise, the rank-sum test was performed. The chi-squared test or Fisher's exact probability method was used to compare and correlate groups of clinicopathological data. CpG sites were screened using the rank-sum test and random forest algorithm. The AUC and 95% confidence interval were calculated using the subject operating characteristic curve (ROC curve) to assess the diagnostic performance of candidate CpG sites and guide the cut-off value. A logistic regression model for combined diagnosis was constructed using the glm function. Specificity, sensitivity, and 95% confidence interval were used to assess the clinical value of candidate sites and diagnostic models. All statistical analyses were performed using SPSS 23.0 and R 3.6.3 software. The criteria for determining statistical significance were *P < 0.05, **P < 0.01, ***P < 0.0001, and ns for no statistical significance.When the sample data followed a normal distribution, the Additional file 1: Table S1. Limit of detection of methylation markers. Table S2. Relationship between the sensitivity of methylation biomarker assays in stool samples of CRC and clinicopathological characteristics. Table S3. Relationship between sensitivities of methylation biomarkers assay in blood samples of CRC and clinicopathological characteristics. Table S4. Characteristics of the study subjects. Table S5. Primer and probe sequences for CpG sites and reference gene. Table S6. Artificially synthesized plasmid sequences. Figure S1. Relationship between CpG site methylation levels and gene expression according to data from TCGA. Correlation between cg13096260 methylation levels and SDC2 gene transcript expression (a). Correlation between cg12993163 methylation levels and SHOX2 gene transcript expression (b). Correlation between cg13096260 methylation levels and cg12993163 methylation levels (c). Differences in the expression of SDC2 and SHOX2 genes between tumor and normal tissues (d). Survival analysis of cg13096260 methylation levels in colorectal cancer (e). Survival analysis of cg13096260 methylation levels in colorectal cancer (f). Figure S2. Relationship between methylation levels of cg13096260 and cg12993163, and clinicopathological characteristics of tissue samples. a–i show age, sex, tumor size, tumor location, histological type, lymphatic invasion, distant metastasis, PIK3CA mutation, and KRAS mutation. Figure S3. Flowchart of sample collection.


As Mongolia is considered one of the most resource extraction-dependent countries globally, significant mining-related environmental and human health risks are expected. The aim of this study was to (I) assess the impacts of mining on soil pollution with metals in Mongolia's key coal mining towns  and (II) review the current knowledge on soil pollution with metal(loid)s and related health risks in Mongolia. The results showed predominantly low soil contents of Cd, Cu, Pb and Zn and a related absence of severe pollution and potential health risk in the coal mining towns. Urban design, rather than the presence of mines, controlled the pollution distribution. Despite the methodological shortcomings of several studies on soil pollution in Mongolia, their results suggest a similarly low threat in the three largest cities  and several mining areas. While the generally highlighted risk of As seems like an artificially escalated issue, the content of Cr in urban soil may be a neglected threat. Further pollution research in Mongolia should focus on street dust and drinking water pollution. The mining industry's origin is dated to 1924 when the first coal mine was opened in Nalaikh2. Its rapid development associated with the intensive release of mining licensing began in the 1990s after the transition to an open economy and is ongoing5. Simultaneously, the expansion of major official mining operations has been accompanied by artisanal and small-scale mining7. There are approximately 3,000 deposits and 50 different minerals explored and researched in Mongolia1, with the most economically important and mined commodities being black and brown coal, fluorite, Au, Cu, Ag, Mo, Pb and Zn9.Mongolia is considered one of the most resource extraction-dependent countries globally10. According to UNDP5, 77% of Mongolian land is classified as degraded or desertified. While mining-related environmental pollution in Mongolia has been intensively studied in recent years7, this is still insufficient because of the country's size and the number of mining areas. In the semi-arid to arid conditions of Mongolia11 with limited water resources12, water pollution poses a critical risk16. Insufficient scientific attention is paid to soil pollution, although mining of most minerals can be associated with soil contamination with metal(loid)s. Furthermore, there is no effective management system to assess the environmental health of mining sites9.However, mining development and related economic growth have accelerated land degradation12. The concentration of people in these areas may be risky due to daily exposure to potentially hazardous substances in the environment released from mining operations and stored mining waste materials. Lkhasuren et al.2 stated that the high levels of dust associated with coal and gold mining are behind the high and growing number of lung diseases in Mongolia. Suvd et al.6 found typical chronic Hg intoxication symptoms in artisanal miners from gold mining areas in two sums. Recently, Surenbaatar et al.9 identified different patterns of mining's impact on children's health in areas of southern Mongolia. However, the results generally indicated the local effect of mining on the content of Pb in blood, As in urine and Hg in blood and hair.In recent decades, mining areas in Mongolia have been urbanised by originally nomadic inhabitants20, other cities21 and environments22. Other typical and frequently mentioned sources of contamination in Mongolia include transport and various industries23. However, the studies carried out so far on urban soil pollution with metal(loid)s in Mongolia strictly focus on the three largest cities , of which only Erdenet is directly linked to mining.The most resonant issue in urban soil pollution studies in Mongolia is coal combustion. Coal combustion can lead to a significant release of metal(loid)s into the environment and is ranked among the primary sources of contamination in the capital of Ulaanbaatar24, metal(loid)s can be deposited over considerable distances. For example, mining activities in Mongolia are associated with the contamination of Lake Baikal in the Russian Federation14. Another problem may be traditional livestock grazing11. The accumulation of metal(loid)s in plants growing on polluted soils that livestock graze can endanger the vitality of the animals and the quality of food produced, leading to another human health risk25.The pollution-related health risk may not only concern the immediate vicinity of mines. Due to aeolian dispersion and water erosion, the most common means of pollutants transport in arid and semi-arid environments26, it can be assumed that mining also poses serious environmental and health risks in Mongolia. The absence of relevant studies on this issue points to the need for extensive research. The aims of this study are to: (1) assess the pollution of urban soils with metals in the key coal mining towns of Baganuur, Nalaikh and Sharyn Gol; (2) summarise and evaluate studies on soil pollution with metal(loid)s in Mongolia; and (3) assess the human health risks posed by soil pollution in Mongolia.Given that the impacts of mining on soil pollution are serious and pose high health risks to the public in neighbouring, similarly highly mining-dependent China27. Strong winds that can easily enhance dust particles dispersion from mining areas significantly influence Mongolian steppe and desert areas. Wind erosion affects up to 90% of the country to some extent. The major soil types are Kastanozem soils, Cambisols and Chernozems28.Mongolia is characterised by extreme semi-arid to arid climate conditions, with an average annual rainfall reaching about 400 mm. Average annual temperatures range from approximately − 50 °C to 40 °C30. While official mining in Nalaikh has already ended22, small-scale illegal mining continues7. The open-pit  or underground  coal mines are to the south of the towns. The quality of mined coal varies between lignite and sub-bituminous32.For this study, three key coal mining towns were sampled: Baganuur, Nalaikh and Sharyn Gol  Fig. . These tFurthermore, the available studies (up to 2021) were reviewed to evaluate the status of metal(loid) soil pollution in Mongolia.2 were taken in each town according to a regular hexagonal sampling network. Forty-four samples were taken from Sharyn Gol, 50 from Nalaikh and 48 from Baganuur: 142 samples in total. The samples were air-dried at room temperature and sieved through a 2.0 mm nylon sieve following the standard ČSN ISO 11464.Topsoil (0–10 cm) samples composited from three sub-samples from approximately 1 mThe decomposition of the samples was performed in two replications according to ČSN EN 16174 by microwave extraction with aqua regia at the microwave extractor ETHOS ONE . Half gram of soil sample was weighed to Teflon tubes, and 2 ml of Suprapur nitric acid (65%) and 6 ml of hydrochloric acid (37%) in ACS quality (Sigma Aldrich) were added. Extraction was performed at 175 °C for 20 min. Total metal  contents in soil samples were determined by flame atomic absorption spectrometry using Varian SpectrAA-30 . Air-acetylene flame atomisation (gas flow 13.5 L/min and 2.0 L/min) and an ultrasensitive hollow cathode lamp for Cd, Cu, Pb and Zn  were used. Cd, Cu, Pb and Zn were measured at 228.8 nm, 324.7 nm, 217.0 nm and 213.9 nm, respectively. For calibration, standard solutions of Cd, Cu, Pb and Zn (1 g/L) (Merck) and MilliQ water  were used. Certified reference material METRANAL 31  was used for quality control.33, they were not analysed in the remaining samples with the expectation of a low probability of pollution risk at the town scale.Based on the analysis of these basic metal pollutants, additional As, Cr, Hg and Ni content measurements were performed in several samples to verify the possibility of severe town-scale pollution with these metal(loid)s. The test samples were selected with regard to their increased content of Cd, Cu, Pb or Zn and diverse spatial distribution; up to fifteen samples were analysed in total. Contents of As, Cr and Ni in the soil samples were analysed using inductively coupled plasma mass spectrometer Agilent 7900  with the SPS 4 autosampler  after microwave extraction with aqua regia at the microwave extractor ETHOS ONE . Half gram of soil sample was weighed to Teflon tubes, and 3 ml of nitric acid (67–69%) and 9 ml of hydrochloric acid (36%)  were added. Extraction was performed at 200 °C for 15 min. For total Hg content analysis, a single-purpose atomic absorption spectrophotometer AMA-254  was used without the necessity of further sample pre-treatment or pre-concentration. The quality control was the same as in the previous measurements. The median values of As, Cr, Hg and Ni in test samples were 10.9, 31.3, 0.03 and 17.4 mg/kg, respectively. Due to their prevailing contents being below the limits of the soil pollution standard34 and the internationally recognised target and intervention values of the Dutch Soil Guidelines (DSG)33. Contamination/pollution indicators (indices) calculate the level of environmental contamination/pollution based mainly on local background values. However, the level of pollution determined in this way only weakly indicates the severity of the threat. Therefore, the adapted Integrated Nemerow Pollution Index (IPIN)35 with the Mongolian standard values  considered was chosen:i is the pollution index for a metal(loid), Ci is the content of the metal(loid), Ti is the permissible content of the soil pollutant given by MNS 5850:201934, PIavg is the mean value of all the PIi of the metal(loid)s, and PImax is the maximum PIi value of the metal(loid)s. IPIN classes are as follows: ≤ 0.7: safe; 0.7–1: precaution; 1–2: slight pollution; 2–3: moderate pollution; ≥ 3: heavy pollution.The content of the metal(loid)s in soils was assessed in compliance with the permissible value of the Mongolian standard on soil quality (MNS 5850:2019)soil) arises through ingestion (ADDing), dermal contact (ADDderm) and inhalation. However, because ingestion and dermal contact pose the greatest health risks35, attention was paid only to these two pathways. An explanation of the input parameters and the values used for the calculations are summarised in Tables 36:The Hazard Index (HI) and Carcinogenic Risk (CR) were calculated to assess the potential health risk posed by metal(loid)s in Mongolian soils. The HI and CR calculations are based on average daily dose (ADD), reference dose (RfD), and slope factor (SF) values of metal(loid)s. Human exposure to metal(loid)s in soils 35:–6 indicate virtual safety, whereas values ≥ 10–4 indicate an unacceptable risk. The acceptable risk range for regulatory purposes is 10–6 to 10–4.The carcinogenic risk was calculated for considered carcinogenic metals (Cd and Pb) as followsP > 0.01). One-way ANOVA was used for normal series; if at least one of the series did not pass the normality test, a nonparametric Kruskal–Wallis ANOVA was used. Spatial data of the pre-processing and geostatistical analysis were conducted in SW ESRI ArcGIS Desktop 10.8 using the Geostatistical Analyst extension. All output rasters of the interpolated values were calculated with a spatial resolution of 10 m. The geostatistical kriging method was selected for spatial data interpolation, specifically, Empirical Bayesian Kriging where the mean prediction errors are more accurate for small data sets compared to other kriging methods. Spatial data transformation (with any base function) was not applied. The thin Plate Spline semivariogram model was chosen by testing several different models of semivariogram. Setting the semivariogram has always been simulated for 100 models, and the results for the map outputs were checked by cross-validation. A smooth circular interpolation option with a radius of 500 m and a smoothing factor of 0.5 was selected in search neighbourhood parameters to control the output.Statistica 12® was used for statistical analyses of the dataset. Differences in the soil contents of metals between the towns were tested. Data normality was investigated using the Shapiro–Wilk normality test s in Mongolia have focused strictly on the three largest cities Table . In addi35; however, its contents were atypically low (≤ 0.25 mg/kg) in most studies or have not been studied  exceeded the former Mongolian limit , which was usually used in the studies concerned. However, the value given by that standard can generally be considered extremely low, even compared to background values. In the context of DSG target value and current Mongolian standard34, the urban soils content of As is rather low. In addition, Nottebaum et al.7, partly studying urban soils of Nalaikh, stated that As does not pose a ubiquitous risk there. Extreme Cr contents  higher local emissions of Zn. However, we can relatively reject hypotheses I and II, given the possible background values  compared to other cities and standards Table  was founts Table  commonlyPb Table  are alsoPb Table  may be r50 mg/kg 37, reporting the predominant absence of contamination in the three largest Mongolian cities. However, there is no discussion of other studies' results concerning these cities or a better explanation of the results, reducing the credibility of the conclusions.Due to the different calculation methods, the pollution level mentioned in the summarised studies is not comparable. Despite the oft-cited moderate or high contamination or pollution (the ambiguity is mainly based on incorrectly used terminology) by at least one element Table , compari21, Chonokhuu et al.37 and Bilguun et al.20. Using local background values is less common, and the values for the same cities vary considerably in different studies , soil metal(loid) contents are within the acceptable levels of the Mongolian standard34 in the vicinity of the Boroo gold mine. However, the authors pointed out that the waste material may be a source of future contamination. Later, Oyuntsetseg et al.55 found Cu, As and Pb enrichment in soils at the Boroo small-scale mining area that exceeded the standard in As (167 mg/kg) and Cu (109 mg/kg) in the gold washing location. In Zaamar Goldfield, Jarsjö et al.56 found elevated soil contents of As (13 mg/kg), Ni (28 mg/kg), Cu (25 mg/kg) and Cr (69 mg/kg) compared to non-local backgrounds; however, no metal(loid) exceeded the current standard. Thus, only As can pose a potential threat in limited and extreme cases of gold mining operations.Cu Most attention has been focused on Cu-Mo mining in Erdenet. Battogtokh et al.57 stated that the soils in the Erdenet mining area are highly contaminated with Cu. While Timofeev et al.51 designated the studied soils as urban, we assigned the overall study conclusions to this chapter due to the predominance of out-of-city sampling. The authors found contamination and potential environmental risk related to Cu and As exceeding the former Mongolian standard. Yondonjamts et al.58 achieved the same conclusions. Dust production by grinding and transportation associated with the mine operation and wind erosion of technogenic sands of the tailings were highlighted as important factors for contamination of the mining area59.Coal Park et al.60 expect the risk of As poisoning due to above-standard (former) As contents (5.57–14.2 mg/kg) in the soil dust from the Baganuur mine; however, they draw conclusions only from five samples and still low As contents in the context of other standards  accompanying soil assessment in the study of water contamination and II) assessment of mining areas partly including urban units. The studies concern three basic mineral resources: Au  Cu (Erdenet) and coal .Although many of the mentioned studies highlighted the issue of soil pollution with As in particular, its content is predominantly low by other international standards Table . Assumed37 confirmed this assumption, not finding serious health threats based on potential health risk assessments in Ulaanbaatar, Darkhan or Erdenet. Conversely, Battsengel et al.45 emphasised possible health risks in Ulaanbaatar. However, the description of the results in both studies suggests a partial misunderstanding of the calculation, as the authors present the generally applicable facts arising from the calculation methodology as unique results. In addition, errors can be found in both cases. The conclusions of Battsengel et al.45 can, therefore, be considered incorrect.Given that soil contents of metal(loid)s in Mongolia are predominantly low s into the environment within such a short time. Furthermore, limited local ore processing followed by direct export could also contribute to the current favourable situation. The last important factor is probably the wind erosion and wind transport of dust particles far from the deposits. However, the positives of this phenomenon can be ambiguous as particle accumulation must occur somewhere, and the creation of pollution hotspots can be expected.Low metal contents in Mongolian mining areas . The risk of livestock intoxication and unsuccessful phytostabilisation must be reduced by fencing and permanent monitoring.The results showed that the contents of Cd, Cu, Pb and Zn are low without significant pollution and health risk in the coal mining towns of Baganuur, Nalaikh and Sharyn Gol. Based on the available studies, the same conclusions can be drawn for other Mongolian cities. Only the Cr content associated with the leather industry can likely pose a significant threat and, therefore, warrants increased attention. The generally highlighted risk of As contamination, on the contrary, acts more like a virtual problem. Many soil contamination studies focused on Mongolia are based on the inappropriate methodology or an insufficient number of samples, reducing their results' quality and conclusions' relevance. Due to the potential for the long-term growth of the Mongolian mining sector, a precise metal(loid)s contamination assessment is highly needed. Given the results, future research on pollution in Mongolia should focus more on drinking water and street and mining dust pollution.Supplementary Information.


Chemically modified mRNA (modRNA) has proven to be a versatile tool for the treatment of various cancers and infectious diseases due to recent technological advancements. However, a safe and effective delivery system to overcome the complex extracellular and intracellular barriers is required in order to achieve higher therapeutic efficacy and broaden clinical applications. Here, we explored All-Fect and Leu-Fect C as novel transfection reagents derived from lipopolymers, which demonstrated excellent biocompatibility, efficient delivery capabilities, and a robust ability to escape the lysosomes. These properties directly increase mRNA stability by preventing mRNA degradation by nucleases and simultaneously promote efficient gene translation in vitro and in vivo. The modRNA delivered with lipopolymer vectors sustained effective transfection in mouse hearts following direct intramyocardial injection, as well as in major organs (liver and spleen) after systemic administration. No observable immune reactions or systemic toxicity were detected following the systemic administration of lipopolymer-mRNA complexes to additional solid organs. This study identified commercial reagents for the effective delivery of modRNA and may help facilitate the advancement of gene-based interventions involving the safe and effective delivery of nucleic acid drug substances. Messenger RNA (mRNA) vaccines have rapidly emerged as a novel platform for prophylactic use and in the treatment of certain cancers ,3,4,5,6.In recent years, viral and non-viral vectors have been explored to ferry various expression systems to the target cells ,30,31,32Polyethyleneimine (PEI) has emerged as the most widely studied cationic polymer as a non-viral delivery system owing to its spectrum of functional amines, the facile chemistry for further functionalization, its cost-effectiveness and safety profiles ,45,47,48The aim of this study was to identify and develop appropriate modRNA vectors that exhibit excellent biocompatibility, efficient cellular uptake and have the ability to easily escape the endosomes. A series of in vitro and in vivo investigations was conducted to screen and identify top-performing commercial polymeric vectors from modified PEIs. Our results demonstrated that the chosen vectors not only protected the modRNA from degradation and facilitated cellular uptake, but also promoted release from endosomal compartments to the cytoplasm for rapid protein expression. The toxicity of the modRNA formulations with polymeric vectors was minimal in animal models and they were well tolerated when delivered via either local or systemic administration. These results show polymeric mRNA complexes as a feasible, efficient, stable, and safe biomolecule delivery system with potential clinical applications.TM MessengerMAX transfection reagent (MAX) was purchased from ThermoFisher Scientific . Cell culture-related reagents such as 0.25% Trypsin-EDTA (1×), Dulbecco’s Phosphate Buffered Saline (DPBS), Dulbecco’s Modified Eagles Medium (DMEM), Penicillin-Streptomycin antibiotic, and Fetal Bovine Serum (FBS) were obtained from Life Technologies . Opti-MEM reduced serum medium (1×) was purchased from Gibco . The cell counting kit-8 (CCK8) was purchased from Dojindo . EdU Cell Proliferation Kit with Alexa Fluor 555 was purchased from Beyotime . Plasmid DNA used in this project was purchased from Sanggon Biotech . DEPC-Treated nuclease-free water was purchased from Ambion . MEGAscript T7 Transcription kit and transcription cleanup kit was purchased from Ambion (USA). Lyso-Tracker Probes were purchased from Life Technologies (USA). siRNA tracker Cy3 kit purchased from Mirus . The primary antibodies used in this study were CD3  and CD68 . A Donkey Anti-Mouse IgG H&L  and a Donkey Anti-Rabbit IgG H&L  were used as secondary antibodies. Cell lines of HeLa , 3T3 , and MCF-7  used in the projects were purchased from the Cell Bank of the Chinese Academy of Sciences . The human induced pluripotent stem cells (hiPSCs) were donated by Yanxin Li’s laboratory at Shanghai Children’s Medical Center’s Institute of Pediatric Translational Research.Lipopolymer transfection reagents used in this experiment were purchased from RJH Bioscience . LipofectamineAll modRNA used in this study was produced using in vitro transcription techniques, as previously described ,26,51. BThe efficiency of gene transfection and cytotoxicity are significantly influenced by the polyplexes’ particle size and zeta potential. To determine the mean diameter size and the polydispersity index (PDI) of each sample, modRNA and different carriers were mixed at a 1:2 ratio, diluted in 200 μL distilled water, and loaded into a quartz cuvette. The mean diameter size of the polyplexes was measured at an angle of 173° at room temperature (25 °C) by dynamic light scattering using a Zetasizer Nano series . Each experiment was repeated three times and the results were averaged.The zeta potential of polyplexes was measured using the Zetasizer Nano series , as previously described ,40,46. BTo prove the structure of the modRNA wrapped by the polymeric vectors remain unchanged, the conformation of naked modRNA and the modRNA-polymer complexes were tested by a MOS-500 Circular Dichromatic  spectrometer in the range of 220~320 nm.To assess the nuclease resistance capability of modRNA encapsulated in the vector, agarose electrophoresis was used to evaluate the integrity of modRNA as previously reported . Either To test the morphology and size of the polyplexes, we used a 200 kV JEOL 2100 transmission electron microscope for analysis as previously reported ,40,46. F2 atmosphere. The medium was replaced every 2 days and cell passaging was executed when the monolayer of adherent cells reached ~90% confluence.HeLa cells and 3T3 cells were cultured in DMEM cell medium supplemented with 10% FBS and 1% penicillin-streptomycin antibiotic at 37 °C under a 5% CO5 cells were seeded in a 6-well plate and cultured at 37 °C for 24 h. Then, modRNA (2 μg) and vectors  were separately diluted in serum-free Opti-MEM medium and incubated for 5 min. To generate modRNA-vector complexes, the separate mixes were gently pooled together and left to incubate for 15 min at room temperature (RT). Finally, the transfection complex was added directly to the cells in a complete culture medium and incubated for 24 h for testing. To transfect modRNA in vivo, 20 μg RNA and 40 μL transfection reagent were diluted separately in 200 μL Opti-MEM medium, incubated for 5 min at room temperature, then mixed and incubated for another 20 min at RT before tail vein injection [For in vitro transfection, 2 × 10njection .To quantify the extent of modRNA expression, the transfection efficiency and fluorescence intensity of modRNA were measured by flow cytometry 24 h after transfection, as previously described . Cells w4 cells in a 24-well plate 24 h before transfection. After transfection for 24 h, EdU was added to the medium according to the reagent instructions to make a final concentration of 10 μM. Then, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min. To detect EdU, the configured click reaction mixes were added to each well, incubated for 30 min at room temperature, and protected from light. Lastly, to detect the proportion of cell proliferation, nuclei were stained using Hoechst 33,342 and imaged with a Leica inverted fluorescence microscope .To test the cell proliferation, Click-iT EdU Imaging Kits  was used according to the manufacturer’s instructions, as previously studied . BrieflyIn order to observe the cellular uptake and escape from the lysosome of modRNA complexes, GFP modRNA and luciferase modRNA were labeled with the fluorophore Cy3, as in previously published studies ,40. Init4 cells per well and cultured overnight. Then, the cells were treated with GFP modRNA complexed with MessengerMAX, ALL-Fect, or Leu-Fect C at 37 °C, and 5% CO2 for different times, respectively. For control groups, cells were either exposed to naked modRNA or left untreated. The vector-to-modRNA mixing ratio was 3:1 and all groups were performed in five parallel wells. After different time treatments , cells were washed with DPBS and incubated with 10 μL of CCK-8 and 90 μL of DMEM cell medium at 37 °C for 2 h. Two hours later, the absorbance of the plates was measured at wavelengths of 450 nm by a multifunctional microplate reader .The in vitro cytotoxicity of the vectors was assessed by the Cell Counting Kit-8 . BrieflyIn vivo biocompatibility of the vectors was assessed by histological evaluation and hematological examination, as previously described ,26,40,46For the systemic toxicity study, C57BL/6 male mice were administered 500 μL mixed solution of reagents (50 μL) and DPBS (450 μL) by tail vein injection, once every two days for 7 injections. The mice’s body weights were also measured. On day 14, mice were sacrificed, blood was drawn, and the serum was isolated. The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and counts of white blood cells (WBC), lymphocytes (Lymph cell), monocyte (Mon cell), and neutrophils were measured using individual assay kits. Meanwhile, organs  were gathered for histological analysis. The organs were fixed in 4% paraformaldehyde overnight, embedded in paraffin followed by sectioning (5 μm), and stained with hematoxylin and eosin. The slides were pathologically evaluated using a Leica DM6000 B system.To study in vivo transfection efficiency of modRNA, the complexes were delivered via direct intramyocardial injection or systemic tail vein infusion ,40,43. RAdditionally, the biodistribution of mRNA was also evaluated using tail vein injection. The injection was prepared by adding 20 μg of Cre modRNA and 40 µL of vector to 200 µL of the reaction mixture. Then, the solution was injected into the Rosa26mTmG mice at three different sites on their tails. Frozen sections of the liver, spleen, heart, and kidney were taken for staining to survey the effect of modRNA expression after three days.The isolated organs were washed clean of residual blood with sterile DPBS and fixed in 4% paraformaldehyde for 24 h at 4 °C. The following day, the organs were washed three times with DPBS and soaked in a 30% sucrose solution to dehydrate until they precipitated to the bottom. Then, they were embedded in OCT compound and rapidly frozen at −80 °C. Tissue sections were performed using a cryostat  with a section thickness of 5 μm and stored at −80 °C. Before staining, sections were defrosted and dried at room temperature for 30 min, then washed three times in DPBS, and permeabilized with 0.5% Triton-X 100 solution for 30 min. Then, the slides were rinsed three times every 5 min with DPBS and blocked with 5% normal goat serum for 2 h at room temperature. The sections were then probed with the appropriate concentration of primary antibody at 4 °C overnight. Following three washes, the tissue sections were incubated for two hours at room temperature and shielded from light with the appropriate fluorescently labeled secondary antibodies. Finally, imaging was carried out using a Leica TSC SP8 laser confocal microscopy system.To count the number of cells, the pictures were first processed with the Image J software version 1.8.0, as previously described . The imat-test, values of ** p < 0.01 and *** p < 0.001 were regarded as statistically significant.The results are presented as the mean ± standard deviation (SD). Statistical analyses were performed by Modified mRNA holds significant promise for gene and recombinant protein therapies but selecting the appropriate RNA delivery vehicle remains a challenge for unlocking the potential of modRNA therapies. Ideal carriers should have the ability to deliver modRNA to specific cell types and permit significant transgene expression for a specified duration in order to attain a satisfactory therapeutic effect, while avoiding activation from host immune responses, and/or by exhibiting ‘stealth’ features for optimal safety. A common delivery agent of choice for many RNA studies has been lipid nanoparticles , which effectively express exogenous genes with high efficiency ,53,54,55We explored the capacity at which four lipopolymer carriers could safely and efficiently deliver modRNA in both in vitro and in vivo model systems. MessengerMAX was chosen as a reference carrier since its transfection efficiency was reported to be as high as ~80% for in vitro cells of human fibroblasts . We firsThe nature of the modRNA complexes was further analyzed with TEM and the results of analysis from a representative carrier (ALL-Fect) are shown in To investigate the transfection efficiency of modRNA delivered by the chosen carriers, we initially transfected HeLa cells with a GFP modRNA at different mixing ratios (1:1 to 1:3), where different amounts of carriers were mixed with a fixed amount of modRNA (2 μg). The cells were incubated with the complexes for 24 h and analyzed by fluorescence microscopy A,B and fTo further explore the performance of ALL-Fect and Leu-Fect C, we conducted similar experiments in 3T3 and human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) in addition to HeLa cells using GFP expressing modRNAs (eGFP and nGFP), as well as a modRNA expressing mCherry. As can be seen from To verify the biocompatibility of the chosen carriers, we first tested their cytotoxicity through a series of cell culture experiments. The cytotoxic effects of the carriers were evaluated by GFP modRNA transfection in HeLa cells, using the CCK-8 assay which provides a measure of the total metabolic activity of the cells . Under oNext, we conducted an EdU proliferation assay to further assess the biocompatibility of the carriers. The proliferation of HeLa cells was assessed by EdU staining after 1, 2, and 3 days post-seeding and transfection C–E. A siFollowing intracellular uptake, subsequent endosomal/lysosomal escape of the complexes is necessary for successful gene expression. To track intracellular uptake and localization of modRNA complexes in cells, Cy3 fluorophore was tagged to GFP modRNA so we could follow the intracellular localization of modRNA complexes. In The escape of the modRNA complexes from the endosomes/lysosomes was explored next. Luciferase modRNA was labeled with Cy3 and LysoTracker DND-26 (green) was used to inspect the co-localization of modRNA and lysosomal bodies . The redEncouraged by the suitable biocompatibility and effective transfection results in vitro, we next investigated PEI-modRNA delivery in a mouse model. Previous studies have shown that modRNA therapy prevents cardiomyocyte death and induces neovascularization with minimal side effects . HoweverIntravenous (IV) injection is considered to be an efficient way to induce target protein expression in the liver and spleen, since most nano-sized complexes are typically deposited into the liver and spleen from the systemic circulation . To test+ and CD68+ lymphocytes and monocytes, indicators of local immune responses  into the pancreas, a major secretory organ of the body A. At thresponses C,D. Inde control E,F.Next, we further explored the biosafety of the delivery systems by repeat administration into the tail vein once every 2 days over the course of 2 weeks, for a total of 7 times A. Body wOur in vitro study results highlighted excellent transfection efficiencies when transfecting modRNA in cells using ALL-Fect and Leu-Fect C polymer carriers. Transfection efficiencies reached >80% at the optimal ratio of 3:1, yielding higher delivery efficiency as compared to the other polymeric carriers. Further investigation showed that these two novel reagents can mediate endosomal escape and release of modRNA cargo, which may be due to the small particle size of the resultant complexes. Importantly, ALL-Fect and Leu-Fect C demonstrated potent delivery capability in vivo at low dose administration. We also observed ideal biocompatibility and tolerability in mouse models with no adverse effects under the study conditions. The toxicity studies and histopathology analysis revealed that repeated administration is well tolerated even at higher doses, where no evidence of tissue necrosis or organ failure was documented. Of interest were our suggestive findings that alluded to lipopolymer tissue tropism in spleen and liver. More experiments are needed in order to decipher the biological mechanisms underpinning this translocation. Furthermore, our findings may suggest that these lipopolymer carriers could be more potent for modRNA delivery compared to native polymers or other commercial transfection reagents. This is the first report on the use of these new lipopolymer carriers to effectively deliver modRNA in animal models and these carriers are expected to play an important role in the future delivery of modRNA for gene therapy. Therefore, this study may open up new avenues for modRNA delivery in vivo and provide great hope to generate a new avenue for the targeted treatment of human diseases.


There are concerns about microorganisms present on cannabis materials used in clinical settings by individuals whose health status is already compromised and are likely more susceptible to opportunistic infections from microbial populations present on the materials. Most concerning is administration by inhalation where cannabis plant material is heated in a vaporizer, aerosolized, and inhaled to receive the bioactive ingredients. Heating to high temperatures is known to kill microorganisms including bacteria and fungi; however, microbial death is dependent upon exposure time and temperature. It is unknown whether the heating of cannabis at temperatures and times designated by a commercial vaporizer utilized in clinical settings will significantly decrease the microbial loads in cannabis plant material.Escherichia coli were performed to optimize culture and recovery parameters. Subsequent studies were carried out using the cannabis placebo, low delta-9 tetrahydrocannabinol (THC) potency and high THC potency cannabis materials exposed to either no heat or heating for 30 or 70 seconds at 190°C. Phosphate-buffered saline was added to the samples and the samples agitated to suspend the microorganism. Microbial growth after no heat or heating was evaluated by plating on growth media and determining the total aerobic microbial counts and total yeast and mold counts.To assess this question, bulk cannabis plant material supplied by National Institute on Drug Abuse (NIDA) was used to assess the impact of heating by a commercial vaporizer. Initial method development studies using a cannabis placebo spiked with Enterobacteriaceae, Staphylococcus, Pseudomonas, and Aspergillus.Overall, while there were trends of reductions in microbial counts with heating, these reductions were not statistically significant, indicating that heating using standard vaporization parameters of 70 seconds at 190°C may not eliminate the existing microbial bioburden, including any opportunistic pathogens. When cultured organisms were identified by DNA sequence analyses, several fungal and bacterial taxa were detected in the different products that have been associated with opportunistic infections or allergic reactions including  Cannabis sativa L.) is a plant of the Cannabaceae family and contains more than eighty biologically active chemical compounds (Cannabis sativa L. plant have been controlled under the Controlled Substances Act (CSA) since 1970 under the drug class “Marihuana” (commonly referred to as “marijuana”) [21 U.S.C. 802(16)] (Cannabis (ompounds . Parts o802(16)] . The Agr802(16)] . An esti802(16)] . In addi802(16)] ; however802(16)] . There h802(16)] . Even as802(16)] .Aspergillus, Mucor, and Penicillium and bacteria including Clostridium, Streptococcus, Salmonella, and certain Escherichia  applications proposing to utilize cannabis for therapeutic indications in patients, the FDA must consider the safety of the product that is being proposed . When usherichia .Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests” and <62> “Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms”  and were grown at the University of Mississippi. Three types of cannabis material were utilized in the studies: placebo without measurable THC, low potency containing 1.90% delta-9 THC, and high potency containing 6.50% delta-9 THC Table 1.Escherichia coli BL-21 (Invitrogen) was used for cannabis spiking experiments. The culture was removed from -80°C storage, streaked onto a tryptic soy agar (TSA) with 5% sheep’s blood and incubated at 37°C overnight. Colonies were used to inoculate Luria-Bertani (LB) broth, which was incubated at 37°C overnight. The next morning, the optical density was measured spectrophotometrically at 600nm  to estimate the cell numbers and the cell concentrations adjusted to the desired level with DPBS and placed at 4°C for spiking experiments.Sample preparation for microbial testing was based on methods recommended in USP Chapter <61> . InitialE. coli suspension of approximately 5 x 108 CFU/ml (OD600 of ~1.0) in DPBS or 20 µl of sterile DPBS and aseptically mixed and stored on ice for 2 hours. Additionally, 20 µl samples of the E. coli suspension were added to tubes and processed to serve as a reference to determine the efficiency of the microbial recovery techniques. After incubation, 4 ml of DPBS was added to each sample and the tubes were rotated for 15 min at room temperature. Serial dilutions were prepared and 50 µl was plated in triplicate onto TSA plates. The plates were incubated at 37°C overnight and counted. To calculate the efficiency of recovery, the number of E. coli cultured from the cannabis materials was divided by the number of E. coli that grew from the reference suspensions. The ratio was then multiplied by 100 to determine the percent recovery from each sample. Each set of samples was prepared in triplicate and the experiments were done three times .Humidified placebo (70 mg each) was spiked with either 20 µl of prepared E. coli suspension (5 x 108 CFU/ml) as described above. This initial spiking volume was found to be too large for the 70 mg samples when added to the sample holder in the vaporizer, so spike volumes were reduced to 5 µl with an ~8 x 108 CFU/ml (OD600 of ~1.025) suspension. The spiked cannabis material was placed in the vaporizer sample holder and subjected to 1 of 3 conditions: no heating, heating for a 30-second preheating cycle (recommended time prior to attachment of the vapor collection bag) or heating for 70-seconds . Each set of samples contained three replicates that were plated in triplicate and the experiments were done a total of four times . To assess the impact of heating on the spiked samples, the numbers of E. coli isolated from the heated cannabis samples were compared to those isolated from the no heat samples to determine the relative reductions in recoveries.A commercial vaporizer, Volcano Digit (Storz & Bickel GmbH & Company) was used to assess the impact of heating on microbial levels. The vaporizer was turned on to heat up for 5 minutes prior to sample introduction. Based on manufacturer use recommendations for volatilization of the bioactive compounds in plant materials, a temperature of 190°C was used throughout the study. To optimize testing conditions for the impact of heating, initial testing was conducted on the cannabis placebo spiked with 20 µl of the Placebo, low and high potency cannabis materials were exposed to no heat, 30 seconds, or 70 seconds of heating in the Volcano vaporizer. To each sample, 4 ml of DPBS was added and they samples were processed and plated on TSA as described above to determine aerobic microbial levels. Additionally, the suspensions were plated onto Sabouraud dextrose agar (SDA) and potato dextrose agar (PDA) plates to assess fungal growth. The SDA and PDA plates were incubated at 25°C for up to 5 days and the resulting colonies were counted to determine the impact of heating on fungal populations. Plating was done in triplicate and the experiments repeated a total of three times (experiments 1-3).To further characterize the microorganisms in the sample, metagenomics experiments were conducted on the samples utilized in experiment 3. These efforts consisted of two sets of sequencing experiments. The first examined the bacterial and fungal populations that were present in the microbial suspensions from the cannabis materials prior to plating . The second set of sequencing focused on the identification of microorganisms that grew following plating. To prepare the DNA to conduct the sequencing for the first set of experiments, the undiluted microbial suspensions in DPBS from six representative subsamples each of placebo, low potency and high potency were added to the lysis tubes from the ZR Fungal/Bacterial DNA MiniPrep™ kit (Zymo Research) for template DNA extraction. For the second set of experiments, growth from six representative tryptic soy agar (TSA) and six Sabouraud dextrose agar (SDA) plates from each cannabis material type were collected by scraping the surface of the plates with an inoculating loop and suspending the growth in the dilution buffer from the ZR Fungal/Bacterial DNA MiniPrep™ kit. The DNA was then extracted from each sample according to manufacturer’s instructions (Zymo Research). The isolated DNA served as template to amplify the 16s rRNA V3/V4 or internal transcribed spacer (ITS) variable regions. The 16s rRNA sequencing was conducted following the methods described by 6 CFU/g were recovered from the low potency and 1.76 X 106 CFU/g were recovered from the high potency samples  of the spiked amount, indicating that the recovery technique was efficient for extracting microorganisms for the cannabis materials.Heating and subsequent vaporization of bioactive cannabis compounds followed by inhalation by individuals is a predominant method of cannabis use . Thus, t samples Table 1.handling . The comE. coli, the experiments focused on investigating the impact of heating on microbial populations. E. coli from spiked placebo that was heated for 30 seconds and 70 seconds. The average reduction after heating for 30 seconds (preheating cycle) was 50.4% and after heating for 70 seconds  was 46.1%. There was some experiment-to-experiment variability, especially in the second experiment where the first replicate appeared to have more E. coli following treatment and the other two replicates showing significant reductions  were not cultured from the placebo; however, Acinetobacter species which was cultured from the samples accounted for about 2.5% of sequencing reads (i.e. sequencing output) from the placebo. Likewise, Bacillus species and members of the Enterobacteriaceae family which were also cultured also were detected in the sequencing results of the placebo samples, although they demonstrated low abundance with less than 0.1% of sequencing reads . For low potency samples, there was variability in results across the experiments. On the SDA media, the most common result was a slight increase in fungal numbers with 30-second heating, followed by a reduction in 70-second samples to levels close to no-heat levels  did not lead to significant reductions in microorganisms present in the cannabis materials and in some cases, the heating seemed to enhance the numbers of organisms recovered. This latter observation may be due to the heat volatilizing some residual compounds that may have facilitated “sticking” of microorganisms to the plant material. Finally, it is unknown if the vaporizer airflow could facilitate the transfer of microorganisms from the cannabis material into the vapor collection bag that the end-user inhales. However, the findings demonstrate that heating with a vaporizer may not be considered an effective means to limit microbial hazards associated with inhaled cannabis use. Therefore, other microbial reduction methodologies, such as gamma irradiation, ultraviolet germicidal irradiation or pasteurization may be needed to limit microbial hazards in cannabis material .https://www.ebi.ac.uk/ena/browser/view/PRJEB58799).The sequence data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB58799 (CT, BO, MS-R and SF initiated the studies and provided leadership and overview for the studies. CT secured the funding for the research from the FDA Center for Drug Evaluation and Research. DS, JH, MV, BE, QW and SF conducted the studies. SF, CT, MS-R, DS, and BE conducted data analyses and interpretation. SF, CT, MS-R, and BE drafted the manuscript and all listed authors contributed to final editing and revisions of manuscript.