ABSTRACT
Recent advancements in nanotechnology have resulted in improved medicine delivery to the target site. Nanosponges are three-dimensional drug delivery systems that are nanoscale in size and created by cross-linking polymers. The introduction of Nanosponges has been a significant step toward overcoming issues such as drug toxicity, low bioavailability, and predictable medication release. Using a new way of nanotechnology, nanosponges, which are porous with small sponges (below one microm) flowing throughout the body, have demonstrated excellent results in delivering drugs. As a result, they reach the target place, attach to the skin's surface, and slowly release the medicine. Nanosponges can be used to encapsulate a wide range of medicines, including both hydrophilic and lipophilic pharmaceuticals. The medication delivery method using nanosponges is one of the most promising fields in pharmacy. It can be used as a biocatalyst carrier for vaccines, antibodies, enzymes, and proteins to be released. The existing study enlightens on the preparation method, evaluation, and prospective application in a medication delivery system and also focuses on patents filed in the field of nanosponges.Copyright © 2023 The Authors.
ABSTRACT
The 6XS6 is the structure of the SARS-CoV-2 spike protein. The physiological role of the spike protein is relative to the respiratory syndrome coronavirus and has a stronger infect on the human body than the ancestor virus. The purification of the 6XS6 is in the homo sapiens cell by the affinity chromatography, PBS supplemented and Size Exclusion chromatography. At last, using the Cryo-Electron Microscopy to see the structure. This paper is using the D614G mutation to illustrate the structure of the 6XS6. The N-terminal domain and C-terminal domain of the 6XS6 protein are ALA27 and VAL1137. Furthermore, the mutation doesn't have the hydrogen bond because the Asp614 is substituted by the Gly614, and the molecule that interacts with the Ala 647 may occur. While the 6XS6 structure has lots of non-covalent and disulfide bonds. Comparing the structure of the 6XS6 and 6VXX, both are glycoproteins, have three monomers, have two subunits, and have the same category of expression and classification. The different conformations of the two structures can affect the binding ability with the ACE2. This paper can help the researchers to further understand the structure and function of the 6XS6 which can be used in future experiments. © 2023 SPIE.
ABSTRACT
At the Center of Molecular Immunology (Havana, Cuba), the fusion protein SARS-CoV-2 S protein (RBD)-hFc was synthesized linking the receptor-binding domain (RBD) of the SARS-CoV-2 virus and the crystallizable fragment of a human immunoglobulin. This fusion protein was used in the construction of a diagnostic device for COVID-19 called UMELISA SARS-CoV-2-IgG. Given the relevance of this protein, the characterization of three batches (A1, A2 and A3) was carried out. The molecular weight of the protein was determined to be 120 kDa, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its isoelectric point was estimated between 8.3 and 9 by isoelectric focusing. The molecular integrity was evaluated by size exclusion liquid chromatography and SDS-PAGE after one year of the production of the protein;the presence of aggregates and fragments was detected. Batches A1 and A2 have a purity percentage higher than 95% and they can be used for the construction of new diagnostic devices. © 2022 Walter de Gruyter GmbH, Berlin/Boston.
ABSTRACT
The separation of highly pure single-chirality single-walled carbon nanotubes (SWCNTs) is challenging and also in demand due to their intrinsic physical, optical, and electronic properties. The use of single-chirality and their performance characteristics makes them a selective candidate for multifunctional applications and opens a new front in nanotube development. It has previously been reported that SWCNTs can be separated in various ways by employing direct control and post-synthesis approaches. Herein, we review the separation of single-chiralities of SWCNTs on account of simplicity and time/cost effectiveness by using gel chromatography. The most recent progress in the controlled synthesis of SWCNTs is comprehensively reviewed in terms of selective-diameter, single-chirality, and specific geometric shape. The method to achieve the single-chirality of SWCNTs is also highlighted. Besides addressing COVID-19 characteristics, epidemiology, and pathology, we also review the most recent developments in nano-biosensors for the rapid and early detection of COVID-19. Furthermore, the photothermal/bioimaging response of single-chirality is reviewed in order to enhance the cytotoxicity of drugs against cancer cells over simple carbon nanotubes (CNTs). The single-chirality allows for precise imaging (due to efficient absorption and emission) of tumors/blood vessels up to ~10-fold higher by injecting a low dose. We hope this review stimulates further study on single-chirality controlled SWCNTs for practical applications. Copyright © 2023 The Royal Society of Chemistry.
ABSTRACT
Introduction: Due to Covid-19 restrictions on collecting and processing sputum samples in real time in clinic, we designed a novel sputum home collection method with immediate freezing and delayed processing (“home”). A validation study was carried out to compare key sputum endpoints using the “home” vs “real time (RT)” collection and processing methods. Sputum soluble phase proteomics, mucins and RNA/DNA endpoints were measured and compared between the 2 methods to assess the validity of the “home” method. Methods: Spontaneous sputum samples were collected from N=10 healthy adult volunteers. Each sample was split evenly by weight and processed, half by the “home” method and half by the RT method. Home method samples were first aliquoted into 3 collection tubes (T) as follows: T1: 100-250mg for mucin analysis (refractive index, gel chromatography, and CsCl gradients);T2 and T3: equal weights each, T2 for proteomic analysis (MesoScale Discovery) and T3 for RNA/DNA analysis (Isohelix collection kit). Each was immediately frozen at -20 deg C (24-48hr), then at -80 deg C (2-4 weeks) without any processing. Thawed home T1 and T2 samples were processed by treating with 8M Urea (1:1) to deactivate SARS-CoV-2 if present. T1 was then stored at 2-4 deg C, and T2 was processed with 7x DPBS, centrifuged and recovered supernatants stored at -80 deg C. In contrast, the RT sputum was first treated with 8M Urea (1:1) soon after collection, and then processed for mucins and proteomics per the “home” method above. The remaining cell pellet from the RT processed sample was stored in Zymo research RNA/DNA shield (0.5ml) and, along with home T3 samples, extracted and analyzed for qualitative and quantitative yield, as well as for genes of interest. Paired T-Test analysis compared all sputum endpoints between the home and RT method. Results: There were no statistically significant differences (p<0.05) between the home and RT method for any mucin (MUC5B, MUC5AC, MUC5AC:MUC5B ratio, total mucin) or proteomic endpoint (IL-1a, IL-6, IL-8, TNFalpha, TIMP1, TIMP2, MMP-9, CRP, MPO). In addition, except for CRP and MUC5AC, correlation between sample pairings was strong (correlation coefficient R, range = 0.5-0.9) and statistically significant (p<0.05) for all sputum endpoints. RNA/DNA results are still pending. Conclusion: The sputum “home collection method with immediate freezing and delayed processing” does not result in significantly different proteomic and mucin measurements when compared to the same samples being processed in real time in an identical manner.
ABSTRACT
Various types of COVID-19 vaccines, including adenovirus, mRNA, and inactivated ones, have been developed and approved for clinical use worldwide. Inactivated vaccines are produced using a proven technology that is widely used for the production of vaccines for the prevention and control of infectious diseases, including influenza and poliomyelitis. The development of inactivated whole-virion vaccines commonly includes several stages: the production of cellular and viral biomass in cell culture; inactivation of the virus; filtration and ultrafiltration; chromatographic purification of the viral antigen; and formulation with stabilizers and adjuvants. In this study, the suitability of four resins for Size-Exclusion Chromatography was investigated for the purification of a viral antigen for the human COVID-19 vaccine.
ABSTRACT
At the Center of Molecular Immunology (Havana, Cuba), the fusion protein SARS-CoV-2 S protein (RBD)-hFc was synthesized linking the receptor-binding domain (RBD) of the SARS-CoV-2 virus and the crystallizable fragment of a human immunoglobulin. This fusion protein was used in the construction of a diagnostic device for COVID-19 called UMELISA SARS-CoV-2-IgG. Given the relevance of this protein, the characterization of three batches (A1, A2 and A3) was carried out. The molecular weight of the protein was determined to be 120 kDa, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its isoelectric point was estimated between 8.3 and 9 by isoelectric focusing. The molecular integrity was evaluated by size exclusion liquid chromatography and SDS-PAGE after one year of the production of the protein;the presence of aggregates and fragments was detected. Batches A1 and A2 have a purity percentage higher than 95% and they can be used for the construction of new diagnostic devices.
ABSTRACT
Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil-derived extracellular vesicles (NDEVs) involved in immune and thrombo-inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID-19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta.
Subject(s)
COVID-19 , Extracellular Vesicles , Extracellular Vesicles/chemistry , Female , Flow Cytometry/methods , Humans , Neutrophils , Pregnancy , Protein TransportABSTRACT
Purpose: Research has shown that ACE2 is one of the important targets of SARS - CoV - 2 virus infection. And receptor binding domain (receptorbindingdomain, RBD), a recombinant spike protein extracted from SARS - CoV - 2 virus, can bind to the angiotensin converting enzyme 2 (ACE2) in human body. This study aims to observe the biodistribution of [124I]I-RBD in mammals, evaluate the ability of [124I]I-RBD to detect ACE2-positive lesions, and explore the potential of RBD against COVID-19. Method: The binding ability of RBD to human ACE2 receptor was studied by surface plasmon resonance (SPR). RBD was labeled with 124I by N-Bromosuccinimide (NBS) mediated method, and high quality radiopharmaceutics [124I]I-RBD were obtained after purification. The binding potency of [124I]I-RBD to human ACE2 was detected by a modified enzyme linked immunosorbent assay (ELISA) method. The biodistribution of [124I]I-RBD in normal BALC/c mice was observed at 0.5 h, 2 h, 24 h and 60 h after tail vein injection. The human radiation dosimetry was estimated based on the animal studies. Results: The labeled [124I] I-RBD was purified by size-exclusion chromatography (PD-10), yielding a radio-chemical purity over 99%, as tested by radio-TLC. Specific activity of the product [124I]I-RBD was 28.9 GBq/nmol, and the radio chemistry purity (RCP) was over in saline for seven days. RBD binds to ACE2 with K<INFD= 14.08 nM, while the binding potency of [124I]I-RBD to ACE2 was calculated as 75.7 nM. Biodistribution data in normal balc/c mice showed that, RBD had a moderate metabolic rate, and its 24-hours-p.i. distribution was basically consistent with the high expression distribution of ACE2 in human body. Human radiation dosimetry estimates an indicated effective dose of 6.42 x 10-2 mSv/MBq. Conclusion: These experimental results confirmed the penitential of 124I-RBD as a novel molecular targeting probe for COVID-19. (Figure Presented) The probing of ACE2 expression via radiolabeled RBD may not only be used for non-invasive ACE2 mapping in mammals, but also have the prospect of radiotherapy to suppress the pandemic of COVID-19 by simply changing the iodine isotopes.
ABSTRACT
The potential of lipid nanoparticles (LNPs) as nucleic acid delivery vehicles has been demonstrated in recent years, culminating in the emergency use approval of LNP-based mRNA SARS-CoV-2 vaccines in late 2020. The determination of RNA content relative to LNP size can be important to the understanding of efficacy and adverse effects. This work presents the first description of a facile and rapid analytical method for online, size-dependent RNA payload distribution measurement using data from multi-angle light scattering, ultraviolet and refractive index detectors following separation of the LNPs by size-exclusion chromatography. The analysis was validated by size-based fractionation of the LNPs with subsequent offline analysis of the fractions. Four LNPs formulated with different PEG-lipids and different lipid compositions were tested. Good agreement was observed between the online and offline size-based RNA distributions among all four LNPs, demonstrating the utility of the online method for LNP-encapsulated RNA in general, and suggesting a means for simplified biophysical quantitation of a dosing-related critical quality attribute.
Subject(s)
COVID-19 Vaccines/chemistry , Chromatography, Gel/methods , Drug Carriers/chemistry , Nanoparticles/chemistry , RNA, Messenger/chemistry , RNA, Viral/chemistry , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Vaccines/immunology , Drug Delivery Systems , Humans , Lipids/chemistry , Particle Size , RNA, Messenger/immunology , RNA, Viral/immunology , SARS-CoV-2/chemistry , SARS-CoV-2/immunologyABSTRACT
The novel coronavirus disease 2019 (COVID-19) outbreak has brought to light unprecedented challenges to global public health security. Researchers have devoted their efforts to in-depth research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bring the epidemic under control as rapidly as possible. Among the many areas of burgeoning SARS-CoV-2 related research, various analytical technologies have been applied to the advancement of virus detection, and development of vaccines and innovative therapies. Separation technologies with the merits of simple operation, high separation efficiency, and high selectivity, have become widely used and are key to progress in life science, medicine, pharmaceutical discovery and development, and other fields. Separation technologies have played an irreplaceable role in the isolation, detection, diagnosis, treatment, and prevention of this novel coronavirus. In this review, an overview of the relevant literature is presented from ISI Web of Science spanning Jan. 1st, 2020-Dec. 31, 2020, using "SARS-CoV-2" or "COVID-19" as keywords. The top 20 research directions are summarized, based on papers published in high impact international journals (e. g. Nature, Science, and Cell). Incorporating the impact of published papers, this review summarizes the primary separation technologies applied in these coronavirus studies, and discusses contributions of the following six technologies: affinity chromatography and size exclusion chromatography, liquid chromatography, magnetic bead separation technology, centrifugal technology, micro/nano-separation technology, and electrophoresis. First, affinity chromatography and size exclusion chromatography are discussed, which are the most frequently used protein purification techniques in Nature, Science, and Cell. The SARS-CoV-2 related proteins purified by affinity chromatography and size exclusion chromatography are summarized, and their applications in coronavirus transmission, infection mechanisms, and drug screening are introduced. Subsequently, high performance liquid chromatography (HPLC) is introduced, which is mainly employed for assessing the purity of candidate drugs. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) incorporates the strengths of HPLC and MS, offering both high separation efficiency and structural analysis capabilities with extended applications. LC-MS/MS has been applied to characterization of the binding of SARS-CoV-2 related proteins to potential inhibitors, and to metabolic analyses of candidate drugs. In SARS-CoV-2 nucleic acid tests, magnetic bead separation technology plays a crucial role in the separation of novel coronaviruses. In combination with other analytical techniques, magnetic bead separation technology can be applied to cytological analyses and immunological detection by functionalization of bead surfaces. Centrifugal technology is undoubtedly the most basic separation technology. It has been employed in almost all SARS-CoV-2 related researches. By controlling centrifugation speed, centrifugal technology can rapidly isolate virus particles or cultured cells from complex samples. Micro-nano separation technologies, such as microfluidics, offer advantages including small size, low sample consumption, rapid diffusion, and large surface area. In general, microfluidic technologies are often used in combination with other technologies to realize highly sensitive detection of SARS-CoV-2 related proteins. Finally, the applications of electrophoresis are introduced, which commonly engages in the analysis of polymerase chain reaction (PCR) products. In novel coronavirus studies, the application of electrophoresis has been relatively limited but has potential with further development to contribute significantly to future research. In conclusion, this review summarizes the contributions of six primary separation technologies to novel coronavirus studies, including epidemic detection and prevention, analyzes the main problems facing coronavirus detection efforts, and discusses the role of separation technologies in addressing these problems, with the aim of providing references for broader application of separation technologies.