What is the pH of solution after incubation of PP with chitosan?..Chitosan TPP nanoparticles are formed at specific concentration, pH and volume of CS TPP..Extreme acidic pH or extreme basic pH will not form nanoparticles eventhough after adding active constituent (in your case PP)..Do centrifugation at lower rpm because the gel pellets like structure will be formed while centrifuging CS-TPP nanoparticles..at higher rpm gel pellets might get ruptured..

Maybe is because you don't use a sufficient centrifugal force? If particles are small or with low density you may need an Ultracentrifuge.

Regards

The pH of the final solution should be near about 6 before adding top and your compound I.e poly phenol are acidic in nature so it may reduce the pH so make the pH adjustment...

Whe you centrifuge at lower speed around 3000 the larger particle will  settle down that can be removed and upper part can be centrifuged at higher speed 13000 to get palette of nanoparticle..

Dear Anupam Apoorva,

first, be sure that prior the mixing of the chitosan solution (plus polyphenols) and TPP solution, the concentrations and volumes must be the same as in the original preparation (possibly the same vial). If the polyphenols will be negatively charged (Ph dependent), I would suggest to mix the extract with the TPP solution first and then with the chitosan solution. Moreover, polyphenols may interfere with the ionic complexation and so, the nanoparticles may not form at all; in that case after the complexation you need to check the size of the nanoparticles in suspension and, if the size is ok, proceed with the same centrifugation procedure or increase the G-force if the nanoparticles size is smaller. If nanoparticles do not form you need to change either the concentrations ratios and/or the volume ratios.

I had the same problem when I conjugated the chitosan with FITC.

Hope you will solve your problem.

Best

Alessandro

Article Chitosan/TPP and Chitosan/TPP-hyaluronic Acid Nanoparticles:...

When you mix CS and TPP solutions, you may have three results; 1) clear solution, 2) np suspension, 3) aggregation. The second of these three corresponds to the np formation. If you don't observe the opalescense in the vial, it means you didn't get the nanopartices so you don't even need to centrifugate it. In each situation you must establish the conditions. For example in some cases, it is better to add the active substance to the CS solution prior to mixing, but it soma cases it may be just opposite. pH is also an important factor in np formation. CS/TPP ratio is very important but eventhough you keep the ratio constant an increase in the volumes may result unsuccessfully. 

Article Preparation and in vitro evaluation of chitosan nanoparticle...

Have you performed any DLS analysis of your PP-loaded samples? This would allow you to make sure that you also have a colloidal suspension of NPs in the presence of PP. I can think of some reasons that might explain the difference between the control and PP-loaded formulations: 1. Lower production yield in the presence of PP; 2. PP displaces TPP from reacting with CS; 3. pH issues in the presence of PP; 4. inadequate CS/TPP in the presence of PP. I would definitely check if there are NPs present in the PP-loaded condition and re-formulate the CS/TPP  ratio or adjust the pH if needed.

After reacting chitosan prepared in 1% acetic acid + PP please check the pH of the solution if at all the pH is alkaline the reaction won't progress. Since it is critical for NH2 terminal of chitosan to be in a protonated state so that polyions like TPP will react and form nanoparticles.

If the pH is alkaline then adjust pH to 5.5 and then try adding TPP and check for nanoparticle pellet. 

Secondly, 15000 RPM for 30min is what u generally use to pellet out chitosan nanoparticles.

Best luck.

As mentioned above the pH of solution makes a big difference in nanoparticles formation keeping all concentrations same. To avoid the ratio change due to PP in your case you can start with high molar solutions of chitosan and then finally make up the concentration desired with PP thus avoiding any change in Cs:TPP ratio for control and PP-loaded nanoparticles. But don't forgot to ensure the right pH of solutions for control as well as experimental onces. Good Luck.

I got the same when preparing 25 nm nanoparticles. Although I could see them with SEM and AFM, they never did pellet down even at speeds as high as 40K rpm.

I used centrifugation filter units, they conc. the NPs down to 200ul I then use the conc. suspension. 

How large where your control NPs? What does your polyphenol look like? It might be that the polyphenol reduced the NP size further.

I would run confirm NP formation by SEM/TEM/AFM and if all is well, use centrifugation filter units.

Maybe these can help

http://pubs.acs.org/doi/abs/10.1021/acs.molpharmaceut.5b00478

http://www.impactjournals.com/oncotarget/index.phpjournal=oncotarget&page=article&op=view&path%5B%5D=9276

https://www.ncbi.nlm.nih.gov/pubmed/26385503

I agree with Salma Tammam particles less than 50nm are very difficult to pellet.

Check for particles using DLS or Nanosight technique after synthesis for estimating particle size.

 I agree with others that the nano particles  with snall dimensions are difficult to settle down. It is important to check the particle size. 

Also  are the polyphenols  getting loaded  with chitosan  is also required to be checked by appropriate method. Since the  amt of   polymer loading  is also important to keep them in disperse state.

Dear All,

Its easy to load chitosan NPs. You may read our recent papers on chitosan NPs

Article Synthesis of chitosan based nanoparticles and their in vitro...

Article Synthesis and in vitro antifungal efficacy of Cu–chitosan na...

Chapter Synthesis of Chitosan-Based Nanomaterials

Data 2016 Cu-chitosan NP mediated sustainable approach .....maize - JAFC

Book Chitosan Based Nanomaterials in Plant Growth and Protection

I got same issues. I made chitosan nanoparticles by low molecular weight of chitosan. Mix it with surfactant and drug and then tpp. The pH still 4. And the size is around 200-400 nm. But when I centrifuge it in 11000 rpm 30 minute. it can't precipitate. I feel confused, I need to centrifuge it to separate free drug from chitosan nanoparticles for encapsulation efficiency measurement. and i am not able to find ultracentrifuge because some laboratory are closed. Do you have any suggestions how to separate it?

Other than pH, the charge between the chitosan and TPP, is also one of the key parameter. Because chitosan has the large positive charge and the TPP has the negative charge. Their electrostatic interaction forms nanoparticles. In your case, you have used same volume of polyphenol extract as chitosan. Now what happens, polyphenols also has the negative charge, so it may reduce the positive charge of chitosan and that's why after reacting with TPP, NP was not formed. Here you need to optimize the formula. I can give you two suggestions.

1. Used lesser volume of PP extract.

2. You can also mix the extract with TPP and and use it as TPP solution.