Two-step nanoprecipitation for the production of protein-loaded PLGA nanospheres

Abstract

One of the first methods to encapsulate drugs within polymer nanospheres was developed by Fessi and coworkers in 1989 and consisted of one-step nanoprecipitation based on solvent displacement. However, proteins are poorly encapsulated within polymer nanoparticles using this method because of their limited solubility in organic solvents. To overcome this limitation, we developed a two-step nanoprecipitation method and encapsulated various proteins with high efficiency into poly(lactic-co-glycolic)acid (PLGA) nanospheres (NP). In this method, a protein nanoprecipitation step is used first followed by a second polymer nanoprecipitation step. Two model enzymes, lysozyme and α-chymotrypsin, were used for the optimization of the method. We obtained encapsulation efficiencies of >70%, an amount of buffer-insoluble protein aggregates of typically <2%, and a high residual activity of typically >90%. The optimum conditions identified for lysozyme were used to successfully encapsulate cytochrome c(Cyt-c), an apoptosis-initiating basic protein of similar size, to verify reproducibility of the encapsulation procedure. The size of the Cyt-c loaded-PLGA nanospheres was around 300–400 nm indicating the potential of the delivery system to passively target tumors. Cell viability studies, using a human cervical cancer cell line (HeLa), demonstrate excellent biocompatibility of the PLGA nanoparticles. PLGA nanoparticles carrying encapsulated Cyt-c were not efficient in causing apoptosis presumably because PLGA nanoparticles are not efficiently taken up by the cells. Future systems will have to be optimized to ascertain efficient cellular uptake of the nanoparticles by, e.g., surface modification with receptor ligands.

Development and Validation of a Stability-Indicating Method for Perillyl Alcohol Incorporated in Poly(lactide-co-glycolide) Nanoparticles and Stress Degradation Studies

“…Acetonitrile was chosen because it is already used in the mobile phase and also because there have been reports in the literature confirming the solubility of polymer in this solvent. 39,40 The sum of the results observed for the clear supernatant and recovered nanoparticles coincide with the theoretical value used in the formulation, indicating that in this case the indirect determination through the clear supernatant can be used to quantify the encapsulation efficiency. Since there is not consumption of nanoparticles by indirect determination, this procedure was chosen as a way to improve the yield of nanoparticles per batch.…”

Optimization of the encapsulation efficiency of a novel oral insulin delivery nanosystem

“…Higher speed rate can unbalance the osmotic equilibrium between the internal and the external phase and decreasing the EE [41]. Insulin can pass to the external phase or may also denature, thus affecting the drug stability [42]. With 1000 rpm, we got a more stable and homogeneous system.…”

“…Além disso, uma elevada agitação pode interferir com o equilíbrio osmótico entre a fase interna e externa e diminuir a EE [41]. A insulina pode passar para a fase externa aquosa através de um processo de difusão ou mesmo poderá desnaturar, afectando assim a sua estabilidade [42]. A 1000 rpm, obtivemos um sistema mais estável e homogéneo.…”

Sustained release of TGF-β1 from biodegradable microparticles prepared by a new green process in CO2 medium

“…On the other hand, protein precipitation from an aqueous solution by the addition of water-miscible organic solvents has emerged as an alternative to avoid the disadvantages of the techniques mentioned above. This non-denaturing approach was used to prepare protein particles and thus to ensure its stability upon subsequent encapsulation into polymeric microparticles (Giteau et al, 2008a;Morales-Cruz et al, 2012;Tran et al, 2012).…”