I prepared chitosan nano particle using following step
Low Molecular weight Chitosan (Sigma) was dissolved at a concentration of 3 mg/ml in 0.5% acetic acid solution, then raised to pH 4.6 with 10 N NaOH and filtered the solusion using 0.45 filters.
A solution of TPP at the concentration of 1.0 mg/ml was prepared with deionized water and filtered the solusion using 0.45 filters Then, 10ml of TPP solution was added drop wise under constant stirring to 25 ml of the chitosan solution.
After that opalescent suspension were separated by centrifugation at 16,000 rpm for a period of 30min at 14 °C.
The supernatant was discarded and the wet pellet of CS/TPP nanoparticles was collected.
The pellet was washed with deionized water and freeze drying, and stored at 4 °C for further studies
Dear Sajith,
When chitosan is reacted with TPP, the amine moieties of the chitosan can be cross-linked with the phosphine residues of TPP, resulting to a nanoparticle form. During the event, the molecular structure of the chitosan shall be modified, which the solubility in an acid solution can be changed as well as they turn to swelling or a gel-like compound, rather than a sol form. Theses forms are usually insoluble in any aqueous medium neither acidic or neutral.
Dear Kim
Thank you very much your explanation. I attempt many time with different CS/TPP ratio and different pH.But i got the same result in every time. How i avoid above condition during the chitosan nano particle synthesis.
When you crosslinked the chitosan by TPP or other cross linker then its become like a complex structure so its solublity further reduces (obviously).
First be sure why you want to cross linked the chitosan ?
There are no doubt the complex would be form more the solubility or its release would be retarded.
Chitosan are more preffered for sustained or control release or retardation of release purpous.
Dear Javed
Thank you very much for your answer. Can you further describe above situation or send me any reference link with more information
I believe an excessive amount of TPP shall be thoroughly removed from the supernatant so take more washing steps (>5times) and immediately freeze-drying. Then, dissolve it into water and sonicate it, if possible. Sonication/homogenization often provides us desirable water solubility of CS nanoparticles, as an essential emulsion method. Good luck and stay warm in Korea ^_^
Hello Sajith,
I would suggest that you try complexing your polyelectrolites at lower concentrations (5 mg/mL for chitosan and 1 mg/mL for TPP seems like a lot to me).
You can always consider re-suspending the freeze-dried nanoparticles cake in a smaller volume to get a suspension with the targeted concentration afterwards.
If so, freeze-drying itself is a process worth optimizing for your personal purposes, as it does not often yield satisfactory results at once.
Best!
Charles
Dear Fortier
Thank you very much for your explanation.Can you further describe your explanation?
Absolutely. From my experience with polyelectrolite complexes, concentrations higher than 0.5 mg/mL tend to yield bigger and less stable nanoparticles.
If however you need final concentrations higher than what such low starting concentrations yield, freeze-drying can fix your problem.
If so, pay attention to add agents that will allow your sample to be processed best: a bulking agent can be very helpful in order to have a nice cake that will allow your nanoparticles to be resuspended at almost the same size as they were formed.
Pay attention to the ionic strength of your complexing solution, as those ions will get concentrated as well upon freeze-dry and rehydration at a smaller volume.
Almost everything is worth looking into: glassware, mixing technique, annealing, buffering, etc.
You will find more accurate and rigorous details in the literature, here is the find of paper that helped me.
Article Recent advances and further challenges in lyophilization
Dear Sajith,
When chitisan is used for controlled release reason sometimes it is neccesery to joint physicaly with drugs with negative charge so we use cross linkers with positive charge like TPP and producing a complex. With using related method like ionic gelation as you described nanoparticles are created. You can use some agent like tween to enhance a nanoparticles stability.
Chitosan concentration is usualy between 1-3 and 0.5 mg/ml for TPP for more effective result about nanoparticle.
The supernatent then freez dried and after that you can disperse a proper amount of it in 1 ml water and then sonicate.
Dear Shahsavari
Thank you very much for your answer. I will follow above steps
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