I want to know why the PLGA nanoparticle prepared by double emulsion method is not solubilizing in water or PBS.
I have used 100 mg of PLGA in DCM and mixed with 7 mg of protein (O/W1) and sonicated for 30 secs at 38% amplitude and then added the mixture in 0.5% PVA and sonicated for 30 secs at 38% amplitude. Then this whole mixture is mixed with 100 ml of 0.3% PVA again and stirred for 3 hrs to remove DCM. Then centrifuged at 12000 RPM for 40 min and washed with water thrice.
The particle should be soluble in water or PBS. But its not. Can any one please tell me what could have went wrong in this or what can be done to solubilize this?
Dear Santhosh,
Regardless of the hydrophobic nature of PLGA and so its nanonoparticles, you should be aware that Double Emulsion method is a very challenging technique, as numerous parameters play key roles in overall efficiency, yield and quality of the particles.
You are using a high quantity of PLGA copolymer, while it's been demonstrated that the more polymer concentration, the higher portion of larger particles as well as microparticle and a large volume of aggregates. As you didn't mention and I guess, you do not filter the final mixture of particles. As a result, presence of the macro-sized aggregates could be another reason of poor water solubility, as the aggregates make the emulsion opaque and are easily seen suspended and unsolved with bare eyes!
Another point is the sonication intensity and timing which is VERY VERY VERY.. to an unimaginable extent important !!! your sonication timing and intensity sounds inadequate!
But technical suggestions:
1. Instead of 100 mg, use 25 mg PLGA in a volume of 1300 microliter of DCM(as it rapidly evaporates during sonication time!)
2. Use 1 mg of the protein in only 100 microliter(10 mg/ml water solution)
3. Do the 1st emulsification step drop-wise! Use the "20 microliter-10 sec" method. Begin with 30w amplitude and finish with 60w for a total 60 sec time period
4. Add the 1st emulsion to 2ml of 0.25% PVA solution and do this stream-wise under sonication: Use my "150 microliter-20 sec" method for a total of 180 sec begining with 60w amplitude and finishing with 100w and finally a few sec of the maximum amplitude.
5. add 2 ml of 0.1% PVA solution to the the 2nd emulsion and pipet the mixture, letting it thinly emulsify. Then, transfer the emulsion gently and gradually to 30 ml of 0.1% PVA solution. For solvent evaporation, half an hour of stirring followed by 15 min of vacuum evaporation at 40 oC is more preferred.
6. You can set aside a low volume of the preparation for size and morphology characterization (DLS or SEM) without filtering, and filter the rest with a 0.45 um millipore filter. Comparing the resulted data of these two, you would figure out how much your preparation was successful in Nano-particle preparation rather than Micro-particle preparation!
7. Centrifugation at 12000 rpm for 40 min is ok, but would be better if used 15000 for 30 min
8. THE FINAL very important suggestion: Give up with PLGA and proceed through PLGA-PEG! I promise lots of setbacks would be resolved this way as PEG moiety offers a hydrophilic corona to the NPs, resulting in enhanced solubility and colloidal stability, as well as stealth properties in body system leading to longer serum life time, and beyond..
AND PLEASE don't forget UPVOTING!
Cheers!
PLGA is hydrophobic, so thats difficult to soluble in water. Try to dissolve with tetrahydrofuran or acetone, it should be fine. But if there is no choice, in case for some analysis you may need to use water to dissolve the PLGA. Just sonicate the sample once its been mixed with water/PBS and use syringe filter to filter the solution.
Dear Santosh,
As they are made of PLGA which is very hydrophobic polymeric material. if you really want to dissolve your particle for encapsulation efficiency or for other characterisation, you can also use tetrahydrofuran as solvent or digest your particle with 1 N NaOH so that all your protein may come out from the particles and you can estimate the encapsulated molecule.
Dear Santhosh,
Regardless of the hydrophobic nature of PLGA and so its nanonoparticles, you should be aware that Double Emulsion method is a very challenging technique, as numerous parameters play key roles in overall efficiency, yield and quality of the particles.
You are using a high quantity of PLGA copolymer, while it's been demonstrated that the more polymer concentration, the higher portion of larger particles as well as microparticle and a large volume of aggregates. As you didn't mention and I guess, you do not filter the final mixture of particles. As a result, presence of the macro-sized aggregates could be another reason of poor water solubility, as the aggregates make the emulsion opaque and are easily seen suspended and unsolved with bare eyes!
Another point is the sonication intensity and timing which is VERY VERY VERY.. to an unimaginable extent important !!! your sonication timing and intensity sounds inadequate!
But technical suggestions:
1. Instead of 100 mg, use 25 mg PLGA in a volume of 1300 microliter of DCM(as it rapidly evaporates during sonication time!)
2. Use 1 mg of the protein in only 100 microliter(10 mg/ml water solution)
3. Do the 1st emulsification step drop-wise! Use the "20 microliter-10 sec" method. Begin with 30w amplitude and finish with 60w for a total 60 sec time period
4. Add the 1st emulsion to 2ml of 0.25% PVA solution and do this stream-wise under sonication: Use my "150 microliter-20 sec" method for a total of 180 sec begining with 60w amplitude and finishing with 100w and finally a few sec of the maximum amplitude.
5. add 2 ml of 0.1% PVA solution to the the 2nd emulsion and pipet the mixture, letting it thinly emulsify. Then, transfer the emulsion gently and gradually to 30 ml of 0.1% PVA solution. For solvent evaporation, half an hour of stirring followed by 15 min of vacuum evaporation at 40 oC is more preferred.
6. You can set aside a low volume of the preparation for size and morphology characterization (DLS or SEM) without filtering, and filter the rest with a 0.45 um millipore filter. Comparing the resulted data of these two, you would figure out how much your preparation was successful in Nano-particle preparation rather than Micro-particle preparation!
7. Centrifugation at 12000 rpm for 40 min is ok, but would be better if used 15000 for 30 min
8. THE FINAL very important suggestion: Give up with PLGA and proceed through PLGA-PEG! I promise lots of setbacks would be resolved this way as PEG moiety offers a hydrophilic corona to the NPs, resulting in enhanced solubility and colloidal stability, as well as stealth properties in body system leading to longer serum life time, and beyond..
AND PLEASE don't forget UPVOTING!
Cheers!
You did not mention what the ratio of PLGA was used!
Polyester PLGA is a copolymer of poly lactic acid (PLA) and poly glycolic acid (PGA). PLGA physical properties depend upon multiple factors including the ratio of lactide to glycolide (e.g., 50:50, 65:35, 75:25, 85:15). In water, PLGA biodegrades by hydrolysis of its ester linkages and presence of methyl side groups in PLA makes it more hydrophobic than PGA. Therefore, lactide rich PLGA copolymers are less hydrophilic, absorb less water and subsequently degrade more slowly. PLGA containing less than 50% glycolyl units (glycolic acid units) is soluble in most common organic solvents, such as halogenated hydrocarbons (chloroform and dichloromethane), ethyl acetate, acetone, dioxan, and tetrahydrofuran. PLGA rich in glycolyl units (50% and higher) is insoluble in most organic solvents, and uncommon solvents. Generally, the higher the percentage of lactide units, the longer the polymer lasts before degrading in the presence of water.
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