Date: 28th May 2020
Coronary heart disease (CHD) is the leading cause of deaths worldwide, killing over 7 million people each year. The primary cause of CHD is atherosclerosis, a condition where the arteries become narrowed and hardened due to an accumulation of plaque around the artery wall. Now scientists report a biomimetic drug delivery system derived from macrophage membrane coated ROS (reactive oxygen species)-responsive nanoparticles (NPs) that can treat the disease.
The current treatment of atherosclerosis by pharmacotherapy has limited therapeutic efficacy, however, biomimetic drug delivery systems, especially cell membrane coated nanoparticles (NPs), have been attracting increasing attention in an effort to improve the repertoire of tools available for treating it. Macrophages in particular show promise and their membranes, for example, have previously been employed as vehicles for targeted delivery of drugs to the lungs. However, research using intact macrophages versus macrophage membrane components in vivo remains limited.
Now a team of scientists from the Institute of Chinese Medical Sciences, led by Ruibing Wang have used macrophage-biomimetic nanoparticles to deliver targeted pharmacotherapy whilst sequestering proinflammatory cytokines as a proof-of-concept for atherosclerosis treatment. In addition, by comparing the use of whole macrophages as NP carriers versus macrophage membrane-coated NPs they have also been able to provide additional insight into the mechanistic action of plaque removal.
Atherosclerosis is often initiated by dysfunction of endothelial layers, where oxidised forms of low-density lipoproteins (LDLs) accumulate and lead to local inflammation and overproduction of reactive oxygen species (ROS). This inflammation attracts monocytes which then differentiate into macrophages, engulfing the LDLs which often results in the macrophages dying or rupturing which, in turn, creates a positive feedback loop to the immune system, recruiting more immune cells.
As a starting point here the team theorised that ROS-responsive NPs either loaded inside macrophages or encapsulated by macrophage membranes could be used as effective delivery systems. As statins are a first-line treatment used to prevent cardiovascular disease, the team selected the statin, atorvastatin (AT), as the cargo drug to be delivered to sites of atherosclerosis.
ROS-responsive NPs were first prepared via self-assembly of amphiphilic oxidation-sensitive chitosan oligosaccharide (Oxi-COS). The NPs were then loaded with hydrophobic AT (AT-NPs) and when these NPs were tested, they were shown to be ROS responsive demonstrating ~80% drug release under atherosclerotic ROS-simulated conditions and only ~20% drug release under low ROS conditions.
Macrophage membrane coated NPs
The next step was to coat the AT-NPs with macrophage membranes (MM-AT-NPs) and to test the effects of these on inflammation. Initial tests here demonstrated that the MM-AT-NPs successfully attenuated inflammation in vitro and resulted in reduced levels intracellular ROS, nitric oxide (NO) production and apoptosis.
The team then wanted to assess the biocompatibility of the system, so they injected ‘drug free’ MM-NPs into mice. Mice were injected every four days – in total four times – and compared with control saline-injected animals, none of the animals showed any obvious signs of organ damage and organ function biomarkers also appeared unchanged.
‘Live’ macrophages as a delivery system
With the biocompatibility of the system proved, the big test was to then target delivery in an atherosclerotic mouse model. Here, the team wanted to compare their MM-AT-NPs delivery system with live macrophages as a delivery tool. Macrophages are immune responsive and proactively migrate to inflammatory sites, therefore whole macrophages may provide a better ‘targeting’ strategy than the membrane alone. To achieve this, the NPs (either drug loaded – or drug free) were internalised into macrophages (AT-NPs/MA and NPs/MA respectively).
To compare the targeting ability of both systems (MM-NPs and NPs/MA) and to assess the safety for the NPs/MA, they fluorescently labelled both with a synthetic dye in the absence of AT. Again by injecting mice every 4 days, the group showed that the NPs/MA were tolerated well and were deemed biocompatible. Strong fluorescence was seen in the aorta of the mice injected with either MM-NPs or NPs/MA however, enhanced targeting efficiency was seen with NPs/MAs likely due to active recruitment of macrophages in the development process of atherosclerosis. With both systems now exhibiting biocompatibility and efficient targeting the team were ready to move to the ultimate experiment.
Atherosclerotic plaque reduction
In order to determine therapeutic efficacy in an atherosclerotic mouse model, mice deficient in apolipoprotein E (ApoE−/−) that had been pre-fed a high-fat for a month, were injected once a week for two months with formulations of saline alone, AT alone, AT-NPs, MM-AT-NPs, or AT-NPs/MAs. Following this treatment regime, the mice were assessed for aortic plaques. The saline-treated control group showed a plaque area of ∼20% of the total aorta tissue area. The AT alone treated mice showed a moderately reduced plaque area of ∼15%, however, mice treated with AT-NPs or AT-NPs/MAs showed reduced plaque areas of ~14%, and those treated with MM-AT-NPs exhibited markedly reduced plaque area down to ∼8% of the total aorta tissue area.
Interestingly, in mice treated with MM-AT-NPS, a number of molecular indicators showed additional promise of this treatment. In these mice, the necrotic core of the plaques was reduced, high accumulation of vascular smooth muscle cells (which are involved in the inhibition of atherogenesis) was observed along with reduced endothelial proliferation and reduced perivascular neovessels suggesting that overall plaque formation had been stabilised from further progression.
The team also observed the lowest expression of major pro-inflammatory cytokines in both the aorta tissues (TNF-α, IL-1β, and IL-6) and the blood serums (TNF-α and IL-6) in the MM-AT-NPs treated group. This suggested that both systemic and localised inflammation was reduced in these mice. Indeed, further work revealed that MM-AT-NPs may have actually sequestered these proinflammatory cytokines.
Conclusions and future applications:
The study here described a macrophage-biomimetic drug delivery system, in which ROS responsive NPs were coated with macrophage membranes. These MM-NPs were shown to accumulate at the targeted inflammatory tissues, and their cargo drugs released in response to locally overproduced ROS, leading to effective pharmacotherapy in mouse models. Furthermore, it is likely that the macrophage membranes retain antigens residing on the membrane, and can efficiently bind and sequester, multiple proinflammatory cytokines, and chemokines. The synergistic effects of selective pharmacotherapy and sequestering of proinflammatory molecules makes this platform a potentially powerful tool for atherosclerosis treatment.
Previous work from Nicholas Leeper and Bryan Smith from Michigan State University and Stanford University also exploited macrophages for the treatment of atherosclerosis. Plaques often remain ‘stealthy’ to the immune system, and in this previous work the group used NPs to carry a chemical inhibitor that blocks the ‘stealth signals’. The NPs were injected into mice and selectively taken up by the monocytes, which then differentiated into macrophages. When tested in atheroprone mice, this nanotherapy approach reduced plaques by 40% in mice with less advanced plaques, and by 20% in mice with more advanced plaques. The expression of inflammatory genes linked to cytokine and chemokine pathways in macrophages were also reduced.
Taken together biohijacking macrophages appears to be a promising strategy for the delivery of drugs that can treat atherosclerosis. Whether the use of membranes, or as live whole cells is optimal, remains a question, however the work presented here suggests that the membrane strategy is the most beneficial. This may, however, be cargo dependant, or even vary with differing NPs (here ROS-responsive), but it certainly warrants a more thorough side-by-side investigation. With around a 60% plaque reduction seen using the MM-AT-NPs, this could represent a powerful tool in the clinic. Perhaps, a worthy investigation would be to trial the Leeper/Smith NP method and the MM-AT-NP system together, in the hope of a synergistic effect.
It is also hoped that the ROS responsive NPs may become a promising platform for other inflammatory diseases. The membranes of other immune cell types could be used, and the cargo adapted for other diseases. There is also current interest in biohijacking immune cells in attempts to design new treatments for diseases such as cancer. Whilst these are still in the early stages of development and testing it is hoped they will translate into clinical therapies in the not so distant future.
Gao, C., Q. Huang, C. Liu, C. H. T. Kwong, L. Yue, J.-B. Wan, S. M. Y. Lee and R. Wang (2020). “Treatment of atherosclerosis by macrophage-biomimetic nanoparticles via targeted pharmacotherapy and sequestration of proinflammatory cytokines.” Nature Communications 11(1): 2622.