Inhalable Charge-Assisted Stabilized LNPs for Mucosal Immunization

The success of mRNA vaccines has opened up new prospects for the development of mRNA drugs in other disease areas, especially in the treatment of lung diseases and respiratory immune activation, where inhaled mRNA drugs show great potential. However, due to the complex physiological structure and powerful defense mechanisms of the respiratory system, the development of inhaled mRNA drugs faces many challenges. To address these issues, the researchers explored a variety of delivery vectors, including polymers, exosomes, and lipid nanoparticles (LNPs), aimed at improving the delivery efficiency of inhaled mRNA drugs. Although LNP has achieved some success in clinical application, its performance in inhalation administration still needs to be improved.

During inhalation preparation, the LNP solution needs to be converted into micron droplets via a nebulizer to ensure effective penetration into the deep tissues of the lungs. However, the high shear force generated during atomization may lead to the aggregation and disintegration of LNP particles, and even mRNA leakage, thus weakening the transfection efficiency of LNP in the lung. Therefore, how to maintain the stability of LNP during atomization has become a key challenge in the development of inhalable mRNA-LNP preparations. Although the stability of LNP can be improved to a certain extent by increasing the content of PEG lipid, it will also reduce the mRNA encapsulation efficiency, the efficiency of uptake by cells and the efficiency of endosomal escape, and ultimately adversely affect the mRNA expression level. In view of this, there is an urgent need to develop more effective LNP formulations to improve the overall effectiveness of inhaled mRNA delivery. Lu et al. developed a charge-assisted stabilized LNP (CAS-LNP) inhalation preparation. By introducing negatively charged peptide-lipid couplings, this technique significantly enhanced the atomization stability of LNP and improved the delivery efficiency of mRNA after inhalation.

Preparation of charge-assisted stabilized LNP

Amino acids can effectively regulate charge and hydrophilicity, and it has good biocompatibility and easy preparation characteristics, so the researchers designed aspart-serine-serine-cysteine peptide (DSSC), in which cysteine can be used for covalency coupling with lipids, aspartic acid provides negative charge, and two serines improve the hydrophilicity of peptide-lipid coupling compounds. It can be stably distributed on the LNP surface. The DSSC-DOPE conjugate was integrated into SM102-LNP to form CAS-LNP. The surface charge of CAS-LNP can be adjusted by adjusting the incorporation of DSSC-DOPE (0.6%, 2.5%, and 10%). The experimental results showed that 2.5% CAS-LNP exhibited good colloidal stability in PBS, and the surface potential of CAS-LNP decreased gradually with the increase of DSSC-DOPE content, confirming the successful integration of DSSC-DOPE. Meanwhile, the assembly, mRNA encapsulation efficiency and morphology of CAS-LNP were similar to those of SM102-LNP. While successfully introducing negatively charged DSSC-DOPE, LNP can be given a negative surface charge without affecting its basic physicochemical properties, laying the foundation for further development of mRNA delivery systems for inhalation drug delivery.

Synthesis process of CAS-LNP. (Liu, S., 2024)

Stability and mRNA delivery efficiency of CAS-LNP

To evaluate the colloidal stability of CAS-LNP during atomization, the authors atomized with a vibrating screen nebulizer and quantified the percentage of intact LNP by measuring the mRNA encapsulation rate before and after atomization. Only about 17% of SM102-LNP particles remain intact after atomization. In contrast, all CAS-LNPs demonstrated greater stability, particularly at formulations with a DSSC-DOPE concentration of 2.5% (about 40% of the particles remained intact at 2.5% CAS-LNP). The authors then further evaluated whether the enhanced stability of CAS-LNP during atomization could improve the lung delivery efficiency of mRNA. The experimental results showed that the mRNA expression level in the lungs of mice was 6.9 times that of SM102-LNP after aerosol inhalation of 2.5% CAS-LNP. However, in vitro uptake experiments of dendritic cells, the authors found that the cellular uptake rate of 2.5% CAS-LNP was 2.4 times lower than that of SM102-LNP, which may be due to the more negative charge of CAS-LNP. Therefore, the effective inhalation delivery of mRNA by CAS-LNP benefits from the improved colloid stability, thus making up for the lack of cellular uptake.

Related services at BOC Sciences

CAS-LNP efficient delivery efficiency mechanism

The presence of salt can shield the electrostatic repulsion between LNPS, thus damaging the surface charge and enhancing the stability. Therefore, low PBS concentration can make CAS-LNPs more stable after atomization. The researchers tried to replace DSSC-DOPE with different negatively charged molecules and found that the PKa and hydrophilicity of these molecules were critical to the stability of LNP during atomization. To further validate the role of CAS, the researchers designed three peptides - SSSC, DDSC, and DDDC - carrying one, three, and four carboxylic acid groups, respectively, to be coupled to DOPE, replacing DSSC-DOPE. The results show that these LNPs have high stability and enhanced mRNA delivery ability, and higher negative surface charge can provide better stability, but when the surface charge reaches a certain threshold, the increase of negative charge will not continue to improve the efficacy of LNPs. In subsequent studies, it was found that the carboxyl group on the peptide is of great importance to the negative surface charge of CAS-LNP, which is far more effective than the phosphate group on DOPE. The excellent stability of CAS-LNP after atomization is driven by the electrostatic repulsion between all LNPS.

Inhaled CAS-LNP induces mucosal immune responses

CAS-LNP can efficiently deliver mRNA in mice and large animals such as pigs and dogs. At the same time, it can penetrate the mucus barrier to reach lung cells and transfect mRNA into immune cells. To evaluate the potential of CAS-LNP for inhaled mRNA vaccine application, mRNA encoding SARS-CoV-2 Omicron mutant spike protein was encapsulated into CAS-LNP and inoculated into mice by aerosol inhalation. Experimental results showed that CAS-LNP could induce higher levels of IgG and IgA than traditional SM102-LNP, which fully demonstrated the effectiveness of CAS-LNP in inducing systemic humoral and mucosal immune responses. In addition, CAS-LNP significantly enhanced the cellular immune response in the lungs, which was manifested by a significant increase in the number of IFN-γ-secreting cells and tissue-resident memory T cells (TRM). In general, CAS-LNP, as a respiratory preventive vaccine, has demonstrated a strong ability to induce systemic and mucosal immune responses, providing an important reference basis for future vaccine design in enhancing respiratory mucosal immunity.

CAS-LNP for mucosal vaccination

Finally, in order to evaluate the potential application value of CAS-LNP as an mRNA cancer vaccine, the authors packaged mRNA encoding OVA and GP70 into CAS-LNP, respectively, to prepare preventive and therapeutic cancer vaccines, and carried out anti-tumor immunity experiments in mice. The experimental results showed that CAS-LNP, as a preventive cancer vaccine, could effectively induce the production of OVA-specific CD8+ T cells and significantly reduce the number of metastases. As a therapeutic cancer vaccine, CAS-LNP significantly reduced the number of lung metastases, extended the survival time of experimental animals, and activated M1-type macrophages with anti-tumor activity. These experimental results fully demonstrate the great potential of CAS-LNP as an inhaled mRNA vaccine in the treatment of lung cancer, and provide important theoretical support for future clinical application.

Prospect for charge-assisted stabilized LNP

To sum up, the core of the charge-assisted stabilization strategy proposed by the researchers is to introduce negatively charged DSSC-DOPE couplet into the traditional LNP components to form electrostatic repulsion on the LNP surface, effectively avoiding the possible aggregation and decomposition of LNP during the atomization stage, and thus significantly improving the atomization stability of LNP. This strategy is different from traditional cationic lipid screening strategies or excessive addition of PEG lipids, and has a wider application prospect, and can effectively improve the mRNA inhalation delivery efficiency of multiple LNP formulations. In the process of experimental verification, CAS-LNP showed excellent mucus penetration, mRNA expression efficiency and targeting ability against dendritic cells, and was able to induce strong systemic and mucosal immune responses, fully demonstrating the huge development potential of CAS-LNP as a platform for inhaled mRNA vaccine.