The Role of Eicosanoids in the Cardiovascular Actions of Inhaled Nanoparticles

Nanoparticles (NPs) are minute pieces of material to which we are exposed every day in the air we breathe. Some are naturally occurring and have no impact on health, whereas others are produced from urban air pollution and can worsen diseases, particularly in the lungs and blood vessels. However, there is great interest in developing new NPs because of their unique properties that are useful for many applications, such as engineering, electronics and for drug delivery. At present it is unclear exactly what effects inhaled NPs have. Our current programme of research is designed to assess whether a specialized group of fats made in the body (called eicosanoids) drive the cardiovascular effects of NPs. The changes in the profiles of these fats will provide unique fingerprints that could be used to predict the actions of new NPs.

In the proposed clinical study we shall investigate the effects of both environmental and manufactured carbonaceous NPs on the lungs, blood vessels, blood clotting, and levels of eicosanoids in blood and urine. We have previously investigated the cardiovascular effects of carbon nanoparticles after inhalation in man, and these experiments will investigate how the shape, size and composition of carbon particles influence these responses. These experiments will provide new insight into how NPs affect the body and pave the way for new ways to predict the toxic effects of NPs (reducing the need for animal experiments). The findings will enable the design of novel NP without the harmful characteristics of those found in air pollution.

This study will investigate the biological effects of inhaling different nanoparticles in healthy volunteers. We will compare 4 different types of nanoparticles: diesel exhaust nanoparticles (a nanoparticle with a complex mixture of chemicals), carbon black nanoparticles (a 'simple, clean' carbon nanoparticle) and two different sizes of graphene oxide (flat flakes of carbon), as well as inhalation of filtered air for comparison. Volunteers will be split randomly into 2 groups and each will be exposed to 3 nanoparticles, separately in a randomised order, with at least 2 weeks between the exposures (Cohort study with double blind randomised cross over design).

Screening visit 30 healthy non-smoking volunteers will be recruited. The volunteer will attend a screening visit at the Clinical Research Facility (CRF) at the Royal Infirmary of Edinburgh (RIE). Eligibility will be confirmed and consent taken. Baseline measurements of lung function (FEV1, FVC), blood pressure and blood biochemistry will be made. The participant will be asked to perform a short exercise test on a stationary bicycle to determine the bicycle workload for the main study.

Study visit On each visit, baseline measures will be taken at the CRF. The participant will be taken to a mobile facility to breathe in a set level of nanoparticles (target concentration of 200 micrograms per cubic metre) via a facemask for 2 hours while intermittently cycling. The mobile exposure chamber allows for volunteers to inhale specific air pollutants (typical of an urban environment), nanoparticles or gases at precise concentrations while exercising. Nanoparticles will be obtained as standard reference materials or by custom synthesis at the National Graphene Institute at the University of Manchester. All suspensions of nanoparticles are well characterised, free of contamination and have been extensively tested in preclinical models.

After the exposure the participant will return to the CRF and lung function and blood pressure re-measured. Blood will be withdrawn through a cannula in a large vein in the arm at set times after the exposure (15 min, 2h, 4h) for measurement of eicosanoids and other indicators of a haematological response (e.g. an inflammatory response). Blood will be slowly withdrawn (10 mL/min for 5 min) through a tube into a small piece of equipment called a Badimon Chamber which gives a measure of how readily the blood clots. The responsiveness of blood vessels in the arm will then be measured by a technique called forearm plethysmography. Here cuffs are inflated around the wrist and top of the arm and the blood flow is measured by a sensitive gauge around the forearm. Two different vasodilator drugs (acetylcholine and sodium nitroprusside) will be infused into an artery in the arm to see how the nanoparticle exposure has altered the way the blood vessels of the arm respond to these drugs. Blood will also be taken after each drug to measure substances that are release from the wall of arteries to give further indication of the health of the artery. The cannula will be removed and lung function and blood pressure will be re-measured. The participant will be allowed to rest, provided with food and drink, before leaving the hospital.

Biological samples (blood and urine) will be used to measure a range of cardiovascular parameters at the RIE and the University of Edinburgh. The primary endpoint for the study is the measurement of a panel of lipid mediators called eicosanoids, which will be performed by our collaborators at the University of Highlands and Islands who have specialised lipidomic facilities and experience in this area.

Shortened protocol An additional 12 volunteers will be recruited to perform a shortened version of the main protocol. The protocol is identical to the above with the exception that the forearm blood flow, Badimon study and the t=4 time point have been removed. These studies will be used to allow additional monitoring of the effect of nanoparticles during initial visits.

Eligibility Criteria:
Inclusion Criteria:

* Man or woman between 18 to 40 years of age, inclusive
* Judged to be in good health based on medical history, physical examination, vital signs and laboratory tests
* Body mass index (weight kg/m2) between 18 and 35 kg/m2 (inclusive), and body weight between 50 kg and 120 kg (inclusive).
* Willing/able to donate blood (and meets the site's blood donor criteria).
* No severe or significant medical condition and without intercurrent illness (e.g. viral cold or flu, chest infection)
* Not taking any regular oral or intravenous medication.
* Must be willing to abstain from food and caffeine-containing beverages for at least 12 hours beforehand, and alcohol for 24 hours beforehand.

Exclusion Criteria:

* Smoked tobacco or related products within 1 year prior to the study
* Asthmatic
* Occupation with high exposure to air pollution or other inhaled irritant
* Severe or significant medical condition
* Intercurrent illness (e.g. viral cold or flu, chest infection)
* Use of any regular oral or intravenous prescription medication, or non-prescription (including aspirin, ibuprofen, paracetamol, vitamins and herbal supplements), and not at all in the 7 days prior to the study
* Allergy or contraindication to vasodilator drugs (e.g. acetylcholine or sodium nitroprusside)
* Major or traumatic surgery within 12 weeks of screening
* Pregnant or still lactating prior to dose administration (women only)
* Given blood in the 3 months prior to the study