A promising method to obtain PLGA nanoparticles is by nanoprecipitation, a procedure that was developed by Fessi and coworkers and enables production of particles in the 100–300 nm range [12]. Advantages of this method include that it is a single step not requiring extended shearing/stirring rates, sonification, or high temperatures. The method is characterized by the absence of an oil–aqueous interface which is detrimental to protein structure and function [13,14].

However, the nanoprecipitation method, as developed, is mostly suitable for hydrophobic compounds that are soluble in ethanol or acetone, but display limited solubility in water. For example,

Barichello et al. obtained encapsulation efficiencies close to 100% with the lipophilic BMS-907351 chemical structure drugs cindomethacin and cyclosporine A, but less than 15% for the hydrophilic drugs vacomycin and phenobarbital [15]. In order to overcome Carfilzomib datasheet these limitations, the original nanoprecipitation method was modified by Bilati et al. using a wide range of water-miscible organic solvents [13]. This work provided evidence that nanoprecipitation could occur with solvents other than acetone or ethanol, and that an accurate solvent and non-solvent selection could be extended to enable nanoprecipitation of more hydrophilic drugs. It remains difficult to identify two suitable solvents, because one of them must be able

to dissolve both drug and polymer (solvent or diffusing phase), Cobimetinib while the polymer should be insoluble in the second solvent (non-solvent or dispersing phase). In a second study, they selected the water-miscible organic solvent DMSO as the diffusing phase and tested the encapsulation using the model proteins lysozyme and insulin [16]. The authors were able to load nanospheres efficiently with lysozyme, but not with insulin. Note that the study by Bilati et al. [13] did not include protein stability experiments. This is troublesome because DMSO is reported to irreversible unfold most proteins [17,18] and it is therefore unlikely that the developed method is generally applicable. We set out to overcome the aforementioned problems by developing a new nanoencapsulation procedure. Overcoming the obstacles in protein encapsulation by one-step nanoprecipitation is challenging. First, it is difficult to find a common solvent for the quite hydrophobic PLGA and the hydrophilic protein. Second, the organic solvent can induce deleterious protein structural and functional loss. We therefore designed a novel two-step nanoprecipitation method ( Fig. 1) and tested its capability to encapsulate two model proteins, lysozyme and a-chymotrypsin, into PLGA nanospheres.