## 4 Discussion
Particle charge may also influence the pathway of cellular uptake of the polymeric NPs, such as high surface charges, and large particle sizes were found to be phagocytosed by macrophages [38]. Particle shape in this study was spherical. The formation of small aggregates was noted in the TEM images, and this might be due to the charges of the particles. The electrostatic interaction between particles causes them to attract each other and aggregate. The Brownian motion also leads to NP aggregation [39]. However, individual PEG-b-PLGA NPs were also detected in the suspension. For TEM, the sample must be in a dry form before imaging. However, during the preparation of the sample, NPs need to be dispersed in water before observing. The long sample preparation time for TEM as compared to FESEM might explain the minimal irregularity of the outer shells of the NPs. Additionally, a spherical shape is considered to have a faster uptake rate in cells compared to other shapes [40]. The contact angle of the NPs on the cell membrane influences the rate of internalization [41]. Spherical, oblate-shaped particles are symmetric and have a large surface area, which allows them to be wrapped easily by cells, and they are viewed as the best option in receptor-ligand systems to reach the site of interest, as they follow the blood flow [24; 42]. Thus, the shape we obtained in this study would allow the NPs to be mobile in the circulatory system and likely would contribute to an effective drug delivery system. Particle content, based on our data, suggests that geranin was encapsulated into the hydrophobic region of the polymeric NPs. This assumption is inferred primarily from past literature that reported geraninin (logP value = 3.4) and PLGA hydrophobicity [43; 44]. However, the effect of the polar and charged group of geranin has been shown to override the effect of geranin hydrophobicity upon interaction with other chemicals at its interface [43]. Reverse-phase HPLC was used to quantify the encapsulation of geranin into the NPs. This approach minimizes the risk of losing traces of geranin from encapsulation and imparts accuracy of encapsulation as the NPs are small enough to pass the matrix in the column. Since geranin is a polar compound due to the presence of carbonyl and hydroxyl groups in the structure, a polar solvent was used as the mobile phase to elute the polar compounds. Our HPLC setup and parameters were obtained from a previous study, which used reverse phase chromatography to separate the compounds from a _P. watsonii_ extract [23]. In our study, negligible variability of retention time was seen in this chromatography analysis. The encapsulation efficiency of the NPs was calculated using the formula \(y=\text{mx}\), which was obtained from the standard curve. The encapsulation efficiency was low, which could be because phytochemical extracts are hydrophobic but with polar and charged group activities, while the polymeric NPs are amphiphilic. Such characteristics of the phytochemicals will be encapsulated in the inner layer/aqueous compartment of the NPs [45], which results in low encapsulation due to steric interference of the drug-polymer interaction by the PEG-b-PLGA chains [44]. The content release was conducted at pH 7.4, which is the pH of the EMEM growth medium recommended for the growth of the cell model used (CCD 841 CoN cells). The release pattern showed an initial burst release of geranin in the first 15 min, followed by a sustained release for subsequent hours. A possible explanation for this biphasic release pattern is that initially the dispersed geranin moleculeswere located in the core of the hydrophobic chains; with increased geranilin loading, the geranilin entered the hydrophobic chains and the interfacial region between the hydrophobic and hydrophilic chains, which caused the many polymeric micelles to undergo an initial burst of drug release [4]. In our study, the polymer used to encapsulate geranilin is biodegradable, and therefore, the drug release mechanism might be from polymer erosion [46].