Date: 10th July 2020
Sepsis is a life-threatening condition caused by a dysregulated host response to infection which can lead to the damage of multiple organ systems. It is extremely challenging to detect and treat as the heterogeneous features of the host and bacterial interactions create a complex and dynamic disease. Now scientists have developed a nanotrap (NT) to efficiently capture and attenuate a group of inflammatory mediators for effective sepsis treatment.
In 2017, there were an estimated 48.9 million cases of sepsis recorded worldwide and a staggering 11 million sepsis-related deaths. The current treatment for sepsis is antibiotic administration, fluid resuscitation, and vasopressors if required to maintain organ perfusion. Prompt treatment is critical for sepsis, as the condition can rapidly deteriorate, often leaving survivors with long-term damage and disabilities.
During sepsis, there is an accumulation of septic molecules such as lipopolysaccharides (LPS), cytokines and damage- or pathogen-associated molecular patterns (DAMPs/PAMPs). These are derived from the invading bacteria and bind to receptors on the host immune cells, which stimulate the production of inflammatory cytokines, triggering severe systemic inflammation. These molecules have been targets for developing new treatments for sepsis, but have proved mostly ineffective likely to due to the limited design of just targeting one mediator.
Now a team of scientists from State University of New York Upstate Medical University, US, have developed a telodendrimer (TD) nanotrap that can adsorb multiple sepsis mediators, resulting in 100% survival of severe sepsis in murine models.
A telodendrimer is a unique class of polymer, with tuneable aggregation properties, which have previously been used in the development of nanocarriers. The team here had previously shown that protein encapsulation and delivery by TD nanocarriers was based on the synergistic combination of multiple charge and hydrophobic interactions. In this work therefore the team hypothesised that TD nanocarriers composed of positively charged and hydrophobic moieties would be able to capture and carry LPS which are negatively charged.
The team started by creating a TD nanocarrier, a so-called nanotrap, consisting of a linear hydrophilic polyethylene glycol (PEG) tail tethered to an oligolysine dendron or telodendrimer ‘head’ – generating an ‘octopus-like’ flexible dendritic scaffold. Basic amino acids or derivatives were conjugated on to the TD periphery together with hydrophobic moieties to create a positively charged nanocarrier.
- The team tested the TD nanocarriers in vitro showing that they could bind fluorescently labelled LPS and assemble them into nanoparticles.
- LPS isolated from different gram negative bacteria were also efficiently loaded into the TD forming nanoparticles.
- LPS and most cytokines have relatively small molecular weights (10–30 kDa). Therefore, the team conjugated the TD nanotrap on different size-exclusive hydrogel resins to selectively capture the inflammatory mediators and exclude the abundant large serum proteins.
- A PEGA resin with a molecular weight cut off ~50 kDa exhibited an optimal size-exclusive effect against large proteins and allowed diffusion of the LPS throughout the nanotrap resin so was selected for further studies.
- To test the system in biological fluids, foetal bovine serum (FBS) and whole blood from healthy human volunteers were spiked with fluorescently labelled LPS (FITC-LPS) and incubated with the TD nanotrap-resin. The resin nanotrap removed the FITC-LPS from FBS with ~91% efficiency, and ~96% efficiency in the whole blood after a 2 hours incubation.
- Charge interactions were crucial for nanotrap protein binding, and the team noticed that most proinflammatory cytokines had a negative charge while the most common anti-inflammatory cytokines had a positive charge. By changing the charge groups in the TD nanotrap the team were able to target a specific group of cytokines for precise immune modulation. Positively charged nanotrap (NT+) adsorbed proinflammatory cytokines and anti-inflammatory cytokines would be sequestered by the negatively charged nanotrap (NT-).
- The team then wanted to test the nanotrap for sepsis treatment, they used plasma from sepsis model mice and incubated it with the resin NT+. Incubation significantly reduced the levels of IL-1β and IL-6 (negatively charged proinflammatory cytokines) from septic plasma with up to 98% efficiency.
- Next the team implanted the resin nanotrap into the abdominal cavity of the sepsis mice. The septic mortality was~ 63% after 48 hours post sepsis induction in control mice. Surprisingly, mice implanted with NT+ showed an increased mortality rate to 77.8%, suggesting that proinflammatory molecules had been targeted which was likely to dampen the initiation of the immune response. However, the team found that delayed implantation post sepsis induction increased survival rate, perhaps allowing time for the immune system to activate, but preventing it from being over-activated.
- Sepsis is usually treated with antibiotics, so the team used a combination of NT+ and a moderate antibiotic treatment (50% of the full dose) to treat the sepsis induced mice. This yielded a 100% survival at day 42, compared with the control group which showed mortality >80% on day 7.
- A combination of NT- and antibiotics also significantly reduced the acute-phase mortality however, severe abdominal abscess formation, indicative of prolonged illness were observed.
- The analysis for both cytokines and inflammatory signalling pathways revealed a significant reduction in local, systemic, and remote organ inflammation, which correlated with the reduced organ failure in the NT+ and antibiotic treatment group.
Conclusion and future applications:
Sepsis is a major and very challenging medical problem, the nanotrap offers a unique and innovative technology to attenuate the host’s inflammatory response to infection by scavenging a broad range of PAMPs and DAMPs. The nanotrap demonstrated here is also flexible and can be designed to target specific pro- or anti-inflammatory signaling molecules by changing the charge group composition.
As next steps here the team are now exploring translation to the clinical with large animal models being tested, and initial biocompatibility testing has been favourable. Intraperitoneal implantation in mice for 6 months showed no noticeable acute or chronic toxicity, and normal body weight and blood counts were observed.
One area which does require work, however, is application of the NT. Whilst the NT resin suspension was directly applied into the abdominal cavity of the mice in this study, this method would not be a relevant form of administration for septic patients. However, the nanotrap technology can be applied systemically via nanoparticles, or locally at the site of infection (nanogel and hydrogel); the team are also exploring packaging the NT resin into haemoperfusion cartridges at different stages of infection or sepsis to modulate the host’s immune response.
From the experiments here the application of NT resins with different charges at different times after sepsis induction resulted in different survival benefits in sepsis mice. This is likely due to their different mode of action and adsorbing different cytokines. By harnessing this potential and applying different charged NTs at different times of infection therefore there is a potential to increase the effectiveness of the treatment yet further. As pro- and anti-inflammatory signalling pathways are involved in many diseases and infections the range of nanotrap applications is likely to grow and expand into effective treatments for many other conditions.
The sequestering of disease-inducing molecules is becoming a growing area of interest for translation research. We recently reported the development of cellular nanosponges as an effective medical countermeasure to SARS-CoV-2 which can attract, sequester and neutralise the virus in vitro. Furthermore, these nanosponges may also sequester inflammatory cytokines, which cause many of the most lethal manifestations of COVID-19.
It is anticipated that these novel technologies will make crucial breakthroughs and fulfill the unmet clinical need for many devastating diseases, and it is hoped this work is the next step in the drive towards clinical trials in the not so distant future.
Shi, C., X. Wang, L. Wang, Q. Meng, D. Guo, L. Chen, M. Dai, G. Wang, R. Cooney and J. Luo (2020). “A nanotrap improves survival in severe sepsis by attenuating hyperinflammation.” Nature Communications 11(1): 3384.
https://doi.org/10.1038/s41467-020-17153-0
