Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

This method can help answer in the medical field about safety of nano medicines. The advantages of this technique is that it is standard, and is used worldwide for evaluation of products, To nano medicines. Demonstration of the procedure, will be Barry Neun, at the Nano Technology Characterization Lab.

For calibration standard preparation, use 900 microliters of limulus amebocyte lysate, or LAL grade water. And 100 microliters of control standard endotoxin, to prepare as many intermediate dilutions as needed, to enable the preparation of a calibration standard with a concentration of one endotoxin unit per milliliter. Then using 900 microliters of LAL grade water, and 100 microliters of the one endotoxin unit per milliliter calibration standard, prepare a second calibration standard, at a concentration of 0.1 endotoxin unit per milliliter.

Then repeat the serial tenfold dilution to prepare two lower calibration standards. To obtain a total of four calibration standards, ranging from 0001 to one endotoxin unit per milliliter. To prepare a 05 endotoxin unit per milliliter quality control, combines 50 microliter of the one endotoxin unit per milliliter of control standard endotoxin solution, with 950 microliters of LAL grade water.

Next in the software select the instrument settings, and create the template. Select collect data, and enter the test ID and data group in the general tab. Under the hardware tab, select the instrument type, and the LAL method, and verify that a serial number, the system ID, and the serial port information appear on the screen.

Click okay two times to confirm the instrument selection, and the communication with the instrument, and enter the sample ID's in the same order that the samples will be tested. Then use the default buttons to enter the negative control standard curve, and test samples. Add the appropriate volume of negative control calibration standards, and quality control into duplicate pre labeled glass tubes.

And add the appropriate volume of LAL to the first test vile. Vortex the vile briefly, and place the vial into the appropriate tests slots in the instrument carousel. Before loading the samples, perform several dilutions of the test nano material within the maximum valid dilution as demonstrated.

To prepare a 05 endotoxin unit per milliliter of inhibition enhancement control, combine 25 microliters of the one endotoxin unit per milliliter of control standard endotoxin solution, with 475 microliters of the test nano material at a given dilution. After vortexing, load the appropriate innovation enhancement controls, and the unspiked study samples into the instrument. Then aliquot unspiked and the spiked nano materials at a specific dilution into pre labeled tubes.

Before vortexing, and loading into the instrument carousel. To perform a Gel-Clot LAL, prepare control standard endotoxin to a final concentration of four lambda, and combine 100 microliters of standard, with 100 microliters of water, or test sample to achieve a final concentration of control standard endotoxin two lambda. Add 100 microliters of Lysine to each lambda dilution.

Vortex the tubes briefly, and place the rack of tubes into a 37 degree Celsius water bath for one hour. At the end of the incubation, invert the tubes with a smooth motion, and manually record the results using plus for a firm clot, and minus for no clot, or a loose clot. In this representative analysis, PEGylated liposomal doxorubicin interfered with chromogenic LAL at dilution five, however this interference was overcome at higher dilutions.

Spike recovery was between 50 and 200%when the formulation was tested at dilutions 50 and 500 in the turbidity and chromogenic LAL, as well as at dilution five, in the turbidity LAL. When adjusted by the dilution factor, the results were consistent between delusions in both essays. Moreover, the results were consistent between the three essay formats.

Remember that each sample has its own range of dilutions, each LAL format uses its control standard in the doxin and lysate, and that the tubes used to prepare the dilutions, and to perform a reaction at different for each LAL essay. For this procedure other methods like Can be performed to answer additional questions about material After its development, this technique paved the way for the sutures and the field of nano medicine, to explore the medical applications of nano technology formulated drugs in humans, and clinical models including but not limited to non human primates, rabbits, dogs, and rats. Don't forget that working with doxin can be extremely hazardous and the precautions such as wearing personal protective equipment, should always be taken while performing this procedure.