br Table br Particle size size distribution and zeta
Particle size, size distribution and zeta potential with respect to various initial drug concentrations (SD: standard deviation; CV variation coeﬃcient).
found to be negative in water and similar to the negative surface of the pure polymeric NPs (mean ζ value ≈ −30 mV). Neither particle size nor the quality of the polymeric suspensions varied considerably when diﬀerent drug quantities were loaded into the polymeric particles. Neither bulky sediments/aggregates nor particle aggregation were ob-served (Fig. 1A). The poor stability of PLGA NPs in an aqueous medium forms a real barrier to the clinical use of these NPs. In order to improve the long-term stability of these colloidal nanosystems, trehalose was used as cryoprotectant. An optimised concentration of 5% w/v of tre-halose was used to store the NPs at -20 °C. Particles were subject to several freeze-thaw cycles. Particle mean diameters were measured after each Okadaic acid (Fig. 1B). As can be seen in the figure, frozen particles without cryoprotectant underwent a significant increase in size (up to 600 nm, ≈ 165%) and distribution. When trehalose was used as cryo-protectant, particles suﬀered a slight size increase of 13% after the first freeze-thaw cycle. After the third cycle, the increase was about 29%, resulting in diameters of 300 nm which is acceptable for parenteral administration.
3.2. Paclitaxel entrapment into PLGA nanoparticles
A further increase in the initial drug amount (2.25; 4.5; 6.75 mg) did not translate into a significant diﬀerence in encapsulation eﬃciency (EE %) when mass of polymer was kept constant (45 mg). Due to the hy-drophobic nature of the drug and polymer, the synthesis of PTX-loaded PLGA drug delivery systems by nanoprecipitation was expected to be reproducible and yield high EE (%) values. EE (%) was above 85% in all cases (Table 2).
The highest EE (%) was obtained for a PTX concentration of 5% w/ w, probably because the high amount of polymer reduced drug loss during synthesis and slightly enhanced drug encapsulation from 85 to 91%. Drug loading values were in the range of 5–13% w/w. These re-sults demonstrate that particle size and EE% are not significantly af-fected by initial drug concentration when other formulation variables are kept constant.
3.3. Paclitaxel release from PLGA nanoparticles
In vitro drug release was evaluated in PBS at pH 7.4 and 37 °C. PTX at three diﬀerent initial concentrations was released from PLGA NPs in a biphasic process typical of this polymer. In all cases, a sustained drug-release profile was characterised by an initial burst phase of PTX release (up to ≈ 25% in 1 h), with the remaining PTX molecules released in a more sustained manner over a period of 15 days (Fig. 1C). NPs with higher drug loading (10% and 15% w/w) showed the same trend but with a faster release pattern and around 90% of PTX was released in 15 days for 15% PLGA-PTX NPs. Consequently, this evidence shows that the drug disperses uniformly from inside the nanoparticles and is mainly liberated by diﬀusion.
3.4. Haemolysis assay and cytotoxicity studies
As shown in Fig. S1, blank PLGA NPs did not produce erythrocyte lysis even at the highest concentrations tested. All haemolysis percen-tages were less than 1% and very similar to the negative control with
Encapsulation eﬃciency (EE%) and Drug Loading (DL%) related to initial drug concentration (SD: standard deviation; n = 6).
PBS. These results suggest the PLGA NPs have excellent biocompat-ibility with human erythrocytes, which is an essential characteristic for their in vivo use. Furthermore, as shown in Fig. S2, the PLGA NPs were non-toxic to all the cell lines tested, either tumor or non-tumor human and mouse, again demonstrating their safety and biocompatibility. This is one of the main advantages of our nanoparticles given that the FDA has already approved PLGA for human use [19,29]. In fact, previous studies have demonstrated the biocompatibility of similar PLGA NPs associated with 9-tetrahydrocannabinol ( 9-THC)  and other PLGA NPs associated with casein for the encapsulation of PTX .