In an article published in the Journal of Controlled Release, researchers used three kinds of porous nanoparticles (NPs), including porous crosslinked cyclodextrin carriers (CD-NP), mesoporous magnesium-phosphate carrier (MPC-NP), and a mesoporous silica (MSN) to target necrosulfonamide (NSA), a novel gasdermin D (GSDMD) inhibitor to both mouse and human macrophages. All three NPs displayed high-loading abilities for this effective hydrophobic drug.
Study: Inhibition of IL-1β release from macrophages targeted with necrosulfonamide-loaded porous nanoparticles. Image Credit: urfin/Shutterstock.com
Time-lapse observations using high-throughput, live-cell fluorescence microscopy were used to monitor intracellular NSA delivery and cellular uptake. The findings showed quick nanoparticle absorption and efficient NSA-targeted delivery to phagocytic cells. Notably, a potent cytostatic impact was perceived when macrophage cell lines were exposed to free NSA.
Functional assays revealed that CD-NP, followed by MSN-NP, had the most significant suppressive influence on the human macrophages when used for NSA administration. Contrarily, cell growth was less impacted when NSA was administered using nanoparticle carriers. Significant concentration-dependent inhibition of IL-1β cytokine secretion from newly differentiated human macrophages and primary murine was seen when NSA-loaded nanoparticles were used.
Additionally, when loaded with NSA, MPC-NP inhibited macrophage metabolic activities. Thus, in this work, the authors illustrated the efficiency of the hydrophobic drug delivery to macrophages for inhibiting inflammatory responses using porous nanoparticles.
Understanding the Feasibility of NP-Aided NSA Targeting
An organism's physiological response to infections is inflammation, which is crucial for pathogen defense. Chronic inflammation, however, can be harmful to various diseases, including autoimmune and cardiovascular conditions and certain cancers.
The cytokines interleukin (IL)-18 and IL-1β, secreted from immune cells like macrophages when activated by endogenous stress signals or danger signals from pathogens, are two primary mediators of inflammation. These cytokines can initiate and maintain inflammatory processes across the body.
Immune cells release IL-1β and IL-18 as a result of a sequential process. The activation of the caspase-1 by proteolytic cleavage begins with a cascade of intracellular processes caused by the identification of viral or bacterial components by sensor proteins, which serve as danger signals. Then the proforms of IL-18 and IL-1β, as well as the pore-forming protein GSDMD are cleaved by the activated caspase-1.
Ultimately, pore formation caused by GSDMD leads to the production of proinflammatory mediators like IL-18, IL-1β, adenosine triphosphate (ATP), and high mobility group box 1 (HMGB1), which causes pyroptosis, a type of inflammatory cell death.
In this study, the authors investigated the viability of porous NP targeting NSA to macrophages. In addition to their capacity to cause inflammation, macrophages can ingest small particles, like dead cells or bacteria, through phagocytosis.
The authors took advantage of this function by employing NP as a medium to deliver NSA precisely to macrophages. The findings demonstrated that these NSA-loaded carriers allowed for a solvent-free NSA administration that prevented the release of proinflammatory IL-1β by exposing human macrophages and murine.
Three internally generated nanocarriers with various compositions and structures were assessed. MSN, freshly created CD-NP, and MPC-NP made up the nanocarriers. The efficiency of NSA administration, NP cellular uptake, and toxicity were investigated using macrophage cell lines and primary mouse splenocytes. Also, the efficiency of cytokine inhibition following NSA administration was then assessed using human monocyte-derived macrophages (MDM) and murine bone marrow-derived macrophages (BMDM).
The efficiency of NSA delivery to immune cells from three distinct materials was tested. Every porous nanocarrier utilized in this study was biocompatible, and they were selected for their advantageous traits in solvent-free, sustained drug delivery. A potential "off-the-shelf" application was possible by the excellent NP stability, which enabled years of storage in ethanolic solutions.
The first category of materials utilized, MSN, was continually over the years and was applied in numerous biomedical applications due to its outstanding flexibility regarding pore and particle size, NP constitution, and molecular functionalization. The second category of materials consisted of new NPs made of cyclodextrin (CD-NP). The third material was magnesium phosphate citrate NP (MPC-NP), which was entirely biogenic.
Since macrophages specialized in the uptake of particulate material, their use as a delivery mechanism enabled the specific targeting of these cells. The findings demonstrated that the hydrophobic NSA could be loaded into three distinct nanocarriers with high loading, enabling NSA distribution without needing solvents. Thus, the NSA cargo carried by MSN and CD particles was quickly ingested by macrophages and delivered to the cells.
A large percentage of dendritic cells and macrophages were positive for CD particles after 24 hours of exposure to the mixed population of immune cells. Interestingly, after 24 hours, a sizable portion of B lymphocyte cells was also CD particle positive. Contrarily, a few non-phagocytic T cells were CD particle positive. Thus, these findings showed that targeting macrophages and dendritic cells, the critical inflammatory initiators, by porous NP delivery of NSA might be a successful strategy.
MSN and CD NP were more tolerable to macrophages when loaded with NSA than the free NSA compounds, which aggregated in an aqueous medium. In contrast, even at lower NSA concentrations, NSA-loaded MPC particles prevented macrophage metabolic activity entirely. Small amounts of surfactant, which could be harmful to these cells, were needed by MPC particles to load NSA. Therefore, distribution by porous NP might improve the compatibility of NSA compounds with macrophages, except for MPC particles.
NSA-Loaded Porous NPs and the Future of Human Inflammatory Responses
The authors used macrophage cell lines and newly differentiated primary macrophages from human donors and mice in various in vitro examinations. They also showed excellent compatibility of the unloaded carriers with the employed cell lines, even in long-term incubation investigations stretching over 45 hours.
Two excellent options for NSA delivery were identified through the proposed particle screening in the form of CD-CDI and MSN NPs. These particles could deliver significant concentrations of the hydrophobic molecule to phagocytic cells without adding a capping layer, thereby suppressing the IL-1β cytokine. Also, these NPs were simple to synthesize, storable, scalable, and biodegradable.
The findings showed that MSN NP was quickly taken up in huge numbers by macrophages. However, CD-NP was much more efficient in intracellular NSA release, contributing to the most significant suppression of the IL-1β cytokine.
Here, the authors concentrated on the characterization of several NSA-NP forms and their impact on macrophage cell lines and other immune cells when tested in vitro. The findings demonstrated the potential of porous biocompatible NPs for the efficient, targeted delivery of possibly lethal, hydrophobic drugs to control inflammatory responses in human immune cells and primary murine. Also, these NP formulations offered further advantages in vivo.
Boersma, B et al. (2022). Inhibition of IL-1β release from macrophages targeted with necrosulfonamide-loaded porous nanoparticles. Journal of Controlled Release. https://www.sciencedirect.com/science/article/pii/S0168365922006666?via%3Dihub