I prepared PLGA nanoparticles. Then I loaded anticancer drugs in PLGA nanoparticles. Then I attached targeting ligands like folic acid (FA) and hyaluronic acid (HA) separately to target the cancer cells. I want to quantify the number of targeting ligands per nanoparticles. Is is possible to measure?
To determine the concentration of ligand loaded on nanoparticles,
1. If the ligand has fluorescence, we can use fluorescence spectroscopy,
2. using dynamic light scattering
3. Zeta potential measurement
5. polarization spectroscopy
6. UV-visible spectroscopy
depend on ligand properties.
Many of the aforementioned methods will tell you if you have got the ligand on the surface but will be very qualitative especially if your particles or drugs have a high background. In terms of quantification, or at least an estimation, an indirect depletion measurement would probably be the most suitable. That is by measuring the amount of ligand left in solution after attachment to and separation of the particles you can infer the amount of ligand on the particle surface UV/Vis would be suitable for folic acid. Depending on the amount of HA you are using/attaching you might be able to use NMR after spiking with a known reference. For larger particles that you can dry down I've also had some success with XPS, that said the calculations are not straight forward for particles and it's easy to interpret poorly.
You could get average but not the exact number of targeting ligands per nanoparticles. @Kye: by measuring the amount of ligand left in solution after attachment to and separation of the particles you can infer the amount of ligand on the particle surface.
Kye Robinson Thanks for your detailed explanation. I came to know from your answer that UV could be useful to find the amount of folic acid on the surface of the nanoparticles and NMR to find the amount of Hyaluronic acid.
You can check this out, did for targeting peptides and later on using the same approach for folic acid and other small molecules
https://pubs.acs.org/doi/abs/10.1021/acs.molpharmaceut.5b00478
Number of targeting ligands attached can be counted, but when you talk about per particle I am not sure if you will have a correct estimate about it. The size of the particles varies, similarly, the functional groups over their surface do.
Here, the bonding between the FA and PLGA or HA and PLGA is important. Focus on that particular bond and respective functional groups. NMR, Mass, XPS are very strong techniques for such quantification. Crosscheck the results with UV-vis and FL spectroscopy. So you need to know:
1. Number of the particular functional group (that attach to ligand)- (weight or weight%)
2. The attached/left-out ligands (weight or weight%)
Dear Researcher,
You can use some of spectroscopic techniques such as TGA and HNMR in order to quantify exact values of conjugated materials in nanoparticles. The titles of two related manuscripts are attached below:
1. Targeting delivery of oxaliplatin with smart PEG-modified PAMAM G4 to colorectal cell line: In vitro studies
2. ynthesis and evaluation of polyethylene glycol-and folic acid-conjugated polyamidoamine G4 dendrimer as nanocarrier
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