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Recently, researchers from Columbia University's Aaron Diamond AIDS Research Center published a paper in the journal Vaccines, with an impact factor of 7.8, on the results of a research study on the development of a SARS-CoV-2 vaccine based on the NanoGeneratorTM Flex System, which demonstrated a major breakthrough in the application of the LNP technology to the development of a COVID vaccine. LNP technology has made a major breakthrough in the application of LNP technology, which can be successfully applied to the development of COVID vaccines.
Over the past few years, novel coronaviruses have brought us a global epidemic of infectiousness that has had a dramatic impact on health-care systems around the world. While several coronavirus vaccines have been previously approved for use and have proven effective in reducing the severity and transmission of infection. However, there are also drawbacks due to the rapid evolution of the virus and the toxicity of the vectors that have been used for LNP delivery, which has led to the development of a new generation of SARS-CoV-2 vaccines on the agenda to address the threat of this and potential future pandemics.
Subunit vaccines offer a higher safety profile compared to inactivated vaccines and are an alternative to several nucleic acid vaccines such as Pfizer and Moderna. However, these vaccines are typically less immunogenic and often require the addition of adjuvants or rely on delivery platforms to increase the biological half-life of the antigenic material to achieve an improved immunomodulatory cytokine response. In addition, preliminary clinical trial investigations have shown that most of the vaccine candidates under development use the structural s protein (SP) of SARS-CoV-2 as a target, as SP is considered to be the most suitable antigen for inducing neutralizing antibodies. However, COVID-19 vaccine candidates should not be limited to the SP; studying the entire proteome of SARS-CoV-2 may reveal other nonstructural proteins (NSPs) or open reading frame (ORF) auxiliary proteins, which may also be critical for viral virulence, viral adhesion, replication, and host invasion. Therefore, more optimal next-generation vaccines or vaccine platforms must be designed to induce broadly neutralizing antibody and cellular responses for comprehensive and durable protection.
Much attention has been paid to the development of a platform for layer-by-layer (LbL) delivery of multiple antigens by nanoparticles dependent on trimethylated chitosan (TMC) based on a microfluidic synthesis system using a scalable microfluidic system synthesis technology route. This layered (LbL) synthesis platform allows for the co-delivery of SARS-CoV-2 s-protein/peptide (SP) and non-structural or accessory protein/ t-cell epitope peptides, while also providing for the addition of adjuvants to the delivery complexes. Chitosan, a product of chitin deacetylation, is a specific cationic polysaccharide. It has the advantages of non-toxicity, biocompatibility, biodegradability, adhesion and cost-effectiveness in vaccine and drug delivery. This delivery platform based on LbL trimethylated chitosan can enhance antigen stability, prolong the duration of action, control drug release, optimize the solubilization of insoluble peptides, and increase the cell membrane permeability of hydrophobic antigens such as peptides. However, most of the reported methods for synthesizing chitosan nanoparticles are not suitable for large-scale synthesis, and scale-up production to meet the requirements for potential vaccine rollout is considered a major challenge for chitosan-based nanoparticle platforms.
Based on the NanoGeneratorTM Flex System , researchers at the Aaron Diamond AIDS Research Center have developed a platform for the layer-by-layer (LbL) delivery of trimethylated chitosan (TMC) dependent nanoparticles for the large-scale production of LbL trimethylated chitosan (TMC)-cov19, an NP-based vaccine candidate designed to allow "plug-and-play" delivery of different antigens from viral variants or t-cell epitope peptides. NP-based vaccine candidate (LbL- cov19), which is designed to allow "plug-and-play" modification of different antigens derived from viral variants or t-cell epitope peptides to enhance preparedness for future pandemic disease outbreaks.
During the course of the new vaccine research, the team carried out the synthesis of trimethylated chitosan (TMC) nanoparticles and LbL-CoV19 formulations successively using the nanogenerator-TM Flex microfluidic nanoparticle synthesis system.The nanogenerator-TM Flex system integrates the UI Advanced Mode flow control software, where the flow of liquid in each line is controlled by a flow sensor and thus automated. Liquid flow is controlled by flow sensors in each line for automation. A micro-mixing chip (CHP-MIX-3) is then used to achieve fast, efficient and controlled mixing of different precursor solutions or liquid phases.
Synthesis of trimethylated chitosan (TMC) nanoparticles
In order to produce TMC nanoparticles faster using the nanogenerator-TM Flex microfluidic nanoparticle synthesis system, the team first used formulations of the same concentration as in the traditional method to allow for direct comparisons between methodologies. A preset pressure from the PG-MFC pressure controller was applied to each sample tube. Each precursor solution was pushed through the tubes into the two inlets of the micro-mixer chip and mixed within the channels of the microfluidic chip. The TMC solution was added to sample pathway #1 at a concentration of 1 to 5 mg /mL. TPP solution at a concentration of 1 mg /mL was added to sample pathway #2. When the ratio of TMC to TPP was 5:1, 1 mL of TMC solution was consumed, 0.2 mL of TPP solution was consumed, and approximately 1.2 mL of NP solution was obtained, with a total reaction time for this volume of 12 s. Different ratios of TMC and TPP were tested according to the developed ratios of precursor feeds for NP synthesis (10:1-3:1) to determine the optimal synthesis conditions. Our method obtained better reproducibility (3 replicates), narrower size range of NPs (180-210 nm) and smaller polydispersity index (PDI) of NPs. The average size (by DLS) and zeta potential values of the NPs did not change significantly after 24 h of refrigeration, which further confirmed the successful production of NPs and their stability under refrigeration (Table 2).
Table 2. 3 replicates of empty TMC NP size and zeta potential data were synthesized using a microfluidic device and aged after 24 h. The data are shown in Table 2.
Meanwhile, another advantage of using the NanogeneratorTM Flex microfluidic synthesis system is that a larger amount of product can be obtained in a shorter period of time, with a yield of 10 mL in less than 10 minutes. With the traditional method, we could only obtain 1 mL of product in less than 1 hour. Additionally, we can use a larger yield synthesis device, the NanoGenerator Max Microfluidic Synthesis System, to obtain a 1 L yield.
Synthesis of LbL-CoV19 formulation
To encapsulate antigenic subunit proteins or peptides in TMC nanoparticles, the team premixed the proteins/peptides with TMC or TPP based on the isoelectric point (pI) value of the protein or peptide, and then loaded the solution into the reactor reservoir. If the pI value of the antigen is higher than pH 7 (the pH of the reaction solution), the antigen must be premixed with TMC; otherwise, we premixed it with TPP solution to allow attachment between the peptide/protein and the precursor. From conventional methods to microfluidic synthesis, we found that the DLS sizes of the obtained TMC-antigen-TPP NPs varied.TMC was dissolved in ultrapure DI water at a concentration of 1.5 mg/mL, and TPP was dissolved in ultrapure DI water at a concentration of 2 mg/mL. Spike protein or peptide (antigen) solution in a volume of 50 μL at a concentration of 40 mg/mL was added to the TMC solution; the antigen concentration was 0.3 mg/mL, and the ratio of TMC to antigen was 5:1. After mixing, the TMC/antigen solution was placed in a 15 ml FALCON tube, which could be inserted into the Bit 2 connecting line to serve as precursor solution 1 in the microfluidic system. The TPP solution was placed into the position 1 connection line as precursor solution 2. The flow rate of TMC was 5 mL/min, the flow rate of TPP was 1 mL/min, and the total flow rate was 6 mL/min. 1 ml of TMC solution and 0.2 mL of TPP solution were consumed for each reaction. the ratio of TMC to TPP was 5:1, and the total amount of the TMC- spike -TPP solution obtained was about was 1.2 mL and the reaction time was 0.2 min (12 s). The final mass ratios of the TMC:TPP: Spike or TMC:TPP:peptide: Spike components of the monolayer or bilayer formulations were 5:1:1 or 5:1:0.5:1, respectively.The sizes of the NPs using the microfluidic production system (200-300 nm) were smaller than those generated by the original synthesis method (300-400 nm). Moreover, the standard deviation of synthesized TMC - antigen - tpp (LbL-CoV19) nanoparticles was also reduced from 163 nm to 32 nm, and that of TMC nanoparticles from 63 nm to 19 nm.This production method is feasible for future GMP production. In the SEM results (Fig. 2B), we observed that the nanoparticles of LbL-CoV19 formulation had a diameter of 94.6 ± 27.7 nm and were generated from 60 nanoparticles.
Figure 2. scanning electron microscope images of TMC nanoparticles (A) and LbL-CoV-1 formulation nanoparticles (B). The images inserted in the lower right corner are high magnification images of TMC nanoparticles and LbL-CoV-1 formulation nanoparticles.
Through the systematic study in this article, the team developed a method for synthesizing TMC nanoparticles and lbl vaccine formulations based on the Nanogenerator-TM Flex microfluidic synthesis system. Common bottlenecks in traditional methods were overcome by synthesizing nanoparticle formulations within the microchannels of a microfluidic device that utilizes continuous flow to obtain the final product. A rapid increase in yield to 1 L was also achieved using another model of the device (NanoGenerator Max.) This method reduces reaction time while providing better control of the final NP composition, narrowing the particle size distribution, and improving the reproducibility of the synthesis, which has the potential for large-scale vaccine production.
The NanoGeneratorTM Flex system has been published over 80 articles and cited nearly 1000 times by many famous research universities around the world. Representative customers include Tsinghua University, Shanghai Jiao Tong University, Shanghai University of Science and Technology, Shenzhen Advanced Research Institute of the University of Hong Kong, Soochow University, Harvard University, Yale University, Stanford University, University of California, Berkeley, Broad institute, MD Anderson Cancer center, MIT, NASA, Livermore National Laboratory, Nanyang Technological University, Gateway University, and many others. National Laboratory, Nanyang University of Technology, Mendeleev University, University of Salerno, Italy, etc.
Original: https://doi.org/10.3390/vaccines12030339
