Another critical control test is to determine the endotoxin concentration in nanoparticle solutions. Endotoxins are components of the outer membranes of bacteria, most commonly gram negative bacteria, that are released when cells are disrupted. When the concentration is high, they can produce a toxic response. Endotoxin contamination can come from the reagent materials used in nanoparticle fabrication or be introduced during subsequent fabrication and processing steps. At nanoComposix, all BioPure nanoparticle formulations are prepared using aseptic protocols and each starting reagent is tested to ensure they contain extremely low endotoxin levels. Using synthesis reagents with low endotoxin concentration is critical since, due to their larger size, endotoxins will be concentrated along with the nanoparticles during the tangential flow filtration concentration step.
Silver nanoparticles have unusual size, shape, and agglomeration dependent optical properties that interfere with standard endotoxin tests. Initial attempts to use a chromogenic endotoxin assay failed due to the strong optical properties of silver nanoparticles. The chromogenic endotoxin assay utilizes a synthetic chromophore substrate that becomes strongly colored when exposed to endotoxins. The color intensity is linear with respect to endotoxin concentration over a wide range, allowing the endotoxin concentration to be calculated through comparison with a standard curve. While this is commonly considered the simplest of endotoxin tests and is highly sensitive, with a lowest limit of detection (LLoD) of ~0.005 EU/mL, the chromogenic color intensity is measured at 405 nm, which overlaps with the silver nanoparticle extinction peaks (Figure 1). Due to the large optical response of the silver nanoparticle solutions, the additional colorimetric signal from the assay is not accurately measured and results in erroneous values.
In 2011, we modified a test developed by the National Characterization Laboratory (NCL) at the National Cancer Institute (NCI) that accurately measures endotoxin levels in the presence of silver nanoparticle solutions using a Pyros Kinetic Turbidity Assay Instrument. As of 2012, all BioPure solutions sold by nanoComposix are guaranteed to have an endotoxin concentration of less than 2.5 EU/mL, a threshold selected in collaboration with the NCL. This corresponds to an endotoxin mass concentration of < 0.3 ng/mL for 1 mg/mL solution of silver nanoparticles. We believe that the Kinetic Turbidity Assay is the most accurate method for measuring endotoxin levels in the presence of silver nanoparticles. However, there are some important modifications to the standard procedure that need to be made in order to obtain accurate results and these are discussed below.
The Kinetic Turbidity Assay is based upon the reaction between endotoxin and a lysate. If endotoxin is present, the lysate reagent causes a clotting reaction which increases the solution optical density. A spectrometer measures the OD at 660 nm, as a function of time, and monitors the time required to reach 20 mAbs (0.2 OD higher than the initial sample baseline as time= zero, Figure 8). This data is compared to a standard curve to quantitatively calculate the endotoxin concentration.
The assay is highly sensitive, with a lowest limit of detection of ~0.005 EU/mL, and has been verified by the NCI’s Nanotechnology Characterization Laboratory and nanoComposix to work extremely well with nanoparticle formulations. Two excellent resources to learn more about endotoxin assays that can be used for nanomaterials can be found at the NCL’s website as well as in this journal article.
The protocol for the Kinetic Turbidity LAL Assay essentially follows the procedure outlined in NCL Method STE-1.2 that is available online. The main challenge in adapting this procedure to the testing of silver nanoparticles is the change in the optical properties of the silver nanoparticles due to destabilization of high concentrations of silver nanoparticles in the presence of the KT reagents. This can result in the two following issues:
Baseline OD issues: The assay detects OD at 660 nm. Larger silver nanoparticle formulations have significant extinction at 660 nm. If the background OD is relatively high (e.g. >0.1 or 0.2), then this can reduce the dynamic range of the assay. For samples with high OD at 660 nm, the sample must be diluted to reduce the baseline OD, or the supernatant must be tested in the absence of nanoparticles.
Changing baseline issues: This assay typically collects sample baseline OD data for the 1st 30 seconds. If the lysate causes the particles to aggregate, this can significantly increase the baseline OD at 660 nm (Figure 2), which can result in a false positive detection of endotoxin. The solution is to reduce the duration of the baseline collection. If the base collection reduction is still not sufficient to obtain accurate results, the sample concentration needs to be reduced to lower the impact on the baseline.
To avoid both Baseline and Changing Baseline issues, we recommend that nanoComposix BioPure materials be tested using the dilutions listed in Table 5. Alternatively, a higher limit of detection can be obtained by spinning out the silver nanoparticles and measuring the concentration of endotoxin in the supernatant. In internal tests at nanoComposix, more than 85% of the measured endotoxin content remains in the solution compared to that bound to the nanoparticle surface. We also provide endotoxin testing of each batch of BioPure nanoparticles, and provide this information to our customers in the specification sheets provided with each order.
|Nanoparticle Diameter||Suggested Dilution of BioPure Formulations|
Table: Suggested dilutions for BioPure formulations using Kinetic Turbidity Assay. NanoXact formulations can be tested without dilution.
At nanoComposix we guarantee that our concentrated Biopure formulations have a measured endotoxin level that is less than 2.5 EU/mL for particle sizes >20 nm and less than 5 EU/mL for particles sizes 20 nm and less. When diluted to standard working concentrations (such as 0.02 mg/mL NanoXact), the endotoxin levels are near the limit of detection in our assay system and are considered "endotoxin free". If you have any questions about our endotoxin measurements or want more information about the measured endotoxin value for a specific batch please contact us.