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In order to overcome the current LNP-mRNA delivery system's weakness of poor stability and rapid degradation by nuclease, a novel chol-CGYKK molecule and then the new phospholipid liposome were designed and prepared. A solid phase approach synthesized CGYKK and connected it to cholesterol via a disulfide linker to form the desired chol-CGYKK. Four formulated samples with different proportions of excipients were prepared by freeze-drying cationic liposomes and packaged S-mRNA. The stability test shows that after six months at 4 °C, the encapsulation rate of this novel phospholipid liposome was still approximately 90%, which would significantly improve the storage and transportation requirement. Transmission electron microscopy, atomic force microscopy, and scanning electron microscopy indicated that the liposomes were spherical and uniformly dispersed. On comparing the levels of mRNA protein expression of the four formulated samples, the S protein vaccine expression of formulated sample 1 was the highest. Uptake by vector cells for formulated sample 1 showed that compared to Lipo2000, and the transfection efficiency was 66.7%. Furthermore, the safety evaluation of the CGYKK and mRNA vaccine liposomes revealed no toxic effects. The in vivo study demonstrated that this novel mRNA vaccine had an immune response. However, it was still not as good as the LNP group right now, but its excellent physicochemical properties, stability, in vitro biological activity, and in vivo efficacy against SARS-CoV-2 provided new strategies for developing the next generation of mRNA delivery system.
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Péptidos de Penetración Celular , Liposomas , Liposomas/química , Esteroles , Transfección , FosfolípidosRESUMEN
Some studies have shown that lyophilization significantly improves the stability of mRNA-LNPs and enables long-term storage at 2-8 °C. However, there is little research on the lyophilization process of mRNA-lipid nanoparticles (LNPs). Most previous studies have used empirical lyophilization with only a single lyoprotectant, resulting in low lyophilization efficiency, often requiring 40-100 h. In the present study, an efficient lyophilization method suitable for mRNA-LNPs was designed and optimized, shortening the total length of the lyophilization process to 8-18 h, which significantly reduced energy consumption and production costs. When the mixed lyoprotectant composed of sucrose, trehalose, and mannitol was added to mRNA-LNPs, the eutectic point and collapse temperature of the system were increased. The lyophilized product had a ginger root-shaped rigid structure with large porosity, which tolerated rapid temperature increases and efficiently removed water. In addition, the lyophilized mRNA-LNPs rapidly rehydrated and had good particle size distribution, encapsulation rate, and mRNA integrity. The lyophilized mRNA-LNPs were stable at 2-8 °C, and they did not reduce immunogenicity in vivo or in vitro. Molecular dynamics simulation was used to compare the phospholipid molecular layer with the lyoprotectant in aqueous and anhydrous environments to elucidate the mechanism of lyophilization to improve the stability of mRNA-LNPs. This efficient lyophilization platform significantly improves the accessibility of mRNA-LNPs.
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In order to overcome the pandemic of COVID-19, messenger RNA (mRNA)-based vaccine has been extensively researched as a rapid and versatile strategy. Herein, we described the immunogenicity of mRNA-based vaccines for Beta and the most recent Omicron variants. The homologous mRNA-Beta and mRNA-Omicron and heterologous Ad5-nCoV plus mRNA vaccine exhibited high-level cross-reactive neutralization for Beta, original, Delta, and Omicron variants. It indicated that the COVID-19 mRNA vaccines have great potential in the clinical use against different SARS-CoV-2 variants.
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COVID-19 , SARS-CoV-2 , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacuna BNT162 , COVID-19/prevención & control , Humanos , ARN Mensajero/genética , SARS-CoV-2/genética , Vacunas Sintéticas , Vacunas de ARNmRESUMEN
The current COVID-19 epidemic has greatly accelerated the application of mRNA technology to our real world, and during this battle mRNA has proven it's unique advantages compared to traditional biopharmaceutical and vaccine technology. In order to overcome mRNA instability in human physiological environments, mRNA chemical modifications and nano delivery systems are two key factors for their in vivo applications. In this review, we would like to summarize the challenges for clinical translation of mRNA-based therapeutics, with an emphasis on recent advances in innovative materials and delivery strategies. The nano delivery systems include lipid delivery systems (lipid nanoparticles and liposomes), polymer complexes, micelles, cationic peptides and so on. The similarities and differences of lipid nanoparticles and liposomes are also discussed. In addition, this review also present the applications of mRNA to other areas than COVID-19 vaccine, such as infectious diseases, tumors, and cardiovascular disease, for which a variety of candidate vaccines or drugs have entered clinical trials. Furthermore, mRNA was found that it might be used to treat some genetic disease, overcome the immaturity of the immune system due to the small fetal size in utero, treat some neurological diseases that are difficult to be treated surgically, even be used in advancing the translation of iPSC technology et al. In short, mRNA has a wide range of applications, and its era has just begun.
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Sistema de Administración de Fármacos con Nanopartículas/química , ARN Mensajero/química , COVID-19/prevención & control , COVID-19/virología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/química , Humanos , Liposomas/química , Micelas , Nanopartículas/química , Péptidos/química , ARN Mensajero/metabolismo , SARS-CoV-2/aislamiento & purificaciónRESUMEN
PURPOSE: We sought to develop diagnostic models incorporating mpMRI examination to identify PCa (Gleason score≥3+3) and CSPCa (Gleason score≥3+4) to reduce overdiagnosis and overtreatment. METHODS: We retrospectively identified 784 patients according to inclusion criteria between 2016 and 2020. The cohort was split into a training cohort of 548 (70%) patients and a validation cohort of 236 (30%) patients. Age, PSA derivatives, prostate volume, and mpMRI parameters were assessed as predictors for PCa and CSPCa. The multivariable models based on clinical parameters were evaluated using area under the curve (AUC), calibration plots, and decision curve analysis (DCA). RESULTS: Univariate analysis showed that age, tPSA, PSAD, prostate volume, MRI-PCa, MRI-seminal vesicle invasion, and MRI-lymph node invasion were significant predictors for both PCa and CSPCa (each p≤0.001). PSAD has the highest diagnostic accuracy in predicting PCa (AUC=0.79) and CSPCa (AUC=0.79). The multivariable models for PCa (AUC=0.92, 95% CI: 0.88-0.96) and CSPCa (AUC=0.95, 95% CI: 0.92-0.97) were significantly higher than the combination of derivatives for PSA (p=0.041 and 0.009 for PCa and CSPCa, respectively) or mpMRI (each p<0.001) in diagnostic accuracy. And the multivariable models for PCa and CSPCa illustrated better calibration and substantial improvement in DCA at threshold above 10%, compared with PSA or mpMRI derivatives. The PCa model with a 30% cutoff or CSPCa model with a 20% cutoff could spare the number of biopsies by 53%, and avoid the number of benign biopsies over 80%, while keeping a 95% sensitivity for detecting CSPCa. CONCLUSION: Our multivariable models could reduce unnecessary biopsy without comprising the ability to diagnose CSPCa. Further prospective validation is required.
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Background: Aberrant transcript alternative splicing is an important regulatory process closely connected with oncogenesis. Purpose: The objective of this study was to determine the phenotype and function of a novel long noncoding RNA (lncRNA) LINC00477 in gastric cancer. Patients and methods: The gastric cancer samples of 140 from Oncomine database and 17 from our own hospital, as well as three gastric cancer cell lines MKN-45, AGS and KATO III were used in this study. The expression of the spliced isoforms of LINC00477 were tested. The tumor effects of LINC00477 on gastric cancer were investigated in vitro and in vivo. The mechanism of LINC00477 interacted with aconitase 1 (ACO1) was further examined by RIP and pull down assay. Results: The overall expression of LINC00477 was reduced in gastric cancers compared to normal gastric tissues. The isoform 1 of LINC00477 was down-regulated while the isoform 2 was up-regulated in gastric cancer cells. The opposite role of isoforms 1 and 2 in the proliferation and migration of cancer cells in vitro and in vivo was observed. Furthermore, isoform 1 of LINC00477 was determined to interact with ACO1 and suppress the conversion ability from citrate to isocitrate by ACO1. Conclusion: we presented the important roles of the spliced isoforms of long noncoding RNA, LINC00477 in gastric carcinogenesis.
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Cervical cancer remains the second-most prevalent female malignancy around the world, leading to a great majority of cancer-related mortality that occurs mainly in developing countries. Developing an effective and low-cost vaccine against human papillomavirus (HPV) infection, especially in medically underfunded areas, is urgent. Compared with vaccines based on HPV L1 viruslike particles (VLPs) in the market, recombinant HPV L1 pentamer expressed in Escherichia coli represents a promising and potentially cost-effective vaccine for preventing HPV infection. Hybrid particles comprising a polymer core and lipid shell have shown great potential compared to conventional aluminum salts adjuvant and is urgently needed for HPV L1 pentamer vaccines. It is well-reported that particle sizes are crucial in regulating immune responses. Nevertheless, reports on the relationship between the particulate size and the resultant immune response have been in conflict, and there is no answer to how the size of particles regulates specific immune response for HPV L1 pentamer-based candidate vaccines. Here, we fabricated HPV 16 L1 pentamer-loaded poly(d,l-lactide- co-glycolide) (PLGA)/lecithin hybrid particles with uniform sizes (0.3, 1, and 3 µm) and investigated the particle size effects on antigen release, activation of lymphocytes, dendritic cells (DCs) activation and maturation, follicular helper CD4+ T (TFH) cells differentiation, and release of pro-inflammatory cytokines and chemokines. Compared with the other particle sizes, 1 µm particles induced more powerful antibody protection and yielded more persistent antibody responses, as well as more heightened anamnestic responses upon repeat vaccination. The superior immune responses might be attributed to sustainable antigen release and robust antigen uptake and transport and then further promoted a series of cascade reactions, including enhanced DCs maturation, increased lymphocytes activation, and augmented TFH cells differentiation in draining lymph nodes (DLNs). Here, a powerful and economical platform for HPV vaccine and a comprehensive understanding of particle size effect on immune responses for HPV L1 pentamer-based candidate vaccines are provided.
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Proteínas de la Cápside , Papillomavirus Humano 16/inmunología , Inmunidad Celular , Nanopartículas/química , Proteínas Oncogénicas Virales , Vacunas contra Papillomavirus , Vacunación , Animales , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/farmacología , Células Dendríticas/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/inmunología , Proteínas Oncogénicas Virales/farmacología , Vacunas contra Papillomavirus/química , Vacunas contra Papillomavirus/inmunología , Vacunas contra Papillomavirus/farmacología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/farmacologíaRESUMEN
Previous studies have demonstrated that microRNAs (miRNAs) are associated with tumor development and progression. miRNA-524-5p (miR-524-5p) has been reported to be involved in the development and progression of several types of cancer, but its role in gastric cancer has not been fully elucidated to date. Therefore, the aim of the present study was to investigate the expression levels and function of miR-524-5p in human gastric cancer. The expression levels of miR-524-5p were assessed in gastric cancer specimens and cell lines, including MKN-45, SGC-7901 and MGC-803 cell lines and gastric epithelial mucosa GES-1 cells, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell proliferation and cell apoptosis assays and invasion analysis in gastric cancer cell lines were performed to evaluate the effects of miR-524-5p on gastric cancer cells in vitro. The expression levels of matrix metallopeptidase (MMP)-2 and MMP-9 were determined by RT-qPCR and western blot analysis. The expression of miR-524-5p was significantly decreased in gastric cancer tissues and cell lines. Additionally, the results of the in vitro experiments demonstrated that overexpression of miR-524-5p inhibited cell proliferation and invasion, and promoted cell apoptosis in gastric cancer cells. Human gastric cancer SGC-7901 and MGC-803 cell lines transfected with miR-524-5p exhibited reduced expression levels of MMP-2 and MMP-9. Taken together, the results of the present study indicated that miR-524-5p may function as a novel tumor suppressor gene in gastric cancer, and may serve as a biomarker and therapeutic target for the treatment of gastric cancer.
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Nonmuscle myosin IIA (NMIIA) has been found to be overexpressed in gastric cancer tissues. However, the roles of NMIIA in the migration and invasion of gastric cancer cells have not yet been elucidated. The aim of the present study was to assess the effects of NMIIA knockdown on the migratory and invasive capacities of gastric cancer cells and to investigate the potential underlying mechanisms in vitro. First, the expression of NMIIA was assessed in gastric cancer tissues and noncancerous tissues using western blot analysis. The expression levels of NMIIA protein in 51 out of 63 gastric cancer tissue specimens were higher compared to those in their matched nontumoric gastric tissue specimens, and differences between the two groups were statistically significant (P<0.001). After downregulation of NMIIA using RNA interference, the migratory and invasive abilities of the SGC7901 and MGC803 gastric cancer cell lines were observed using a woundhealing assay and a Transwell assay, respectively. Knockdown of NMIIA significantly decreased the amount of wound closure as well as the number of cells which transgressed through the Matrigel (P<0.01). Finally, the levels of cJun Nterminal kinase (JNK), phosphorylated (p)JNK, cJun and pcJun were detected using western blot analysis in order to explore the association between NMIIA and the JNK signaling pathway. Knockdown of NMIIA decreased the levels of pJNK and pcJun in the two gastric cancer cell lines (P<0.05). In conclusion, the present study indicated that knockdown of NMIIA inhibited the migration and invasion of gastric cancer cells, which may be, at least in part, mediated via the JNK signaling pathway.
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Movimiento Celular/genética , Proteínas Quinasas JNK Activadas por Mitógenos/biosíntesis , Miosina Tipo IIA no Muscular/biosíntesis , Neoplasias Gástricas/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Invasividad Neoplásica/genética , Miosina Tipo IIA no Muscular/genética , Transducción de Señal/genética , Neoplasias Gástricas/patologíaRESUMEN
This study was designed to investigate the proliferation inhibition and apoptosis-promoting effect under hyperthermia and chemotherapy treatment, at cellular level. Human gastric cancer cell line SGC-7901 was cultivated with 5-fluorouracil at different temperatures. Cell proliferation and apoptosis were determined, and expression of Bcl-2 and HSP70 was measured at different treatments. Cell survival rates and inhibition rates in chemotherapy group, thermotherapy group, and thermo-chemotherapy group were drastically lower than the control group (P<0.05). For tumor cells in the thermo-chemotherapy group, survival rates and inhibition rates at three different temperatures were all significantly lower than those in chemotherapy group and thermotherapy group (P<0.05). 5-Fluorouracil induced apoptosis of SGC-7901 cells with a strong temperature dependence, which increased gradually with increase in temperature. At 37°C and 43°C there were significant differences between the thermotherapy group and chemotherapy group and between the thermo-chemotherapy group and thermotherapy group (P<0.01). The expression of Bcl-2 was downregulated and HSP70 was upregulated, with increase in temperature in all groups. Cell apoptosis was not significant at 46°C (P>0.05), which was probably due to thermotolerance caused by HSP70 accumulation. These results suggested that hyperthermia combined with 5-fluorouracil had a synergistic effect in promoting apoptosis and enhancing thermotolerance in gastric cancer cell line SGC-7901.