## 2 Materials and Methods
### In Vitro Cell Viability and Cytotoxicity  
Several studies have demonstrated in vitro and in vivo studies that PGS, or PTX, has anticancer activity against breast cancer [11; 12; 50; 51]. In this context, to investigate the potential anticancer effect of these drugs (PGS and PTX), Alamar blue assay was carried out on MDA-MB-231 breast cancer cells. The percentage of Alamar blue reduction was used to measure the cell viability with or without treatment with drug-loaded microparticles. A higher percent reduction value implies a higher cell growth and, by extension, a higher cell viability. The breast cancer cell line (MDA-MB-231) was obtained from American Type \begin{table}
\begin{tabular}{l c c} \hline \hline
**S/N** & **Kinetic Model** & **Equation** \\ \hline
**1** & Zeroth order & \(Q_{t}=Q_{0}+K.t\) \\ \hline
**2** & First order & \(\log Q_{t}=\log Q_{0}+\frac{Kt}{2\text{,}\text{M}S}\) \\ \hline
**3** & Higuchi & \(Q_{t}=K.t^{\frac{1}{2}}\) \\ \hline
**4** & Korsmeyer–Peppas (K–P) & \(\frac{M_{t}}{M_{\infty}}=Kt^{n}\) \\ \hline \hline \end{tabular} where \(Q_{0}\) is the initial amount of drug in the solution, \(Q_{t}\) is the cumulative amount of drug released at time \(t\), and \(K\) is the kinetic constant, \(t\) is the time, \(\frac{M_{t}}{M_{\infty}}\) is a fraction of drug released after time \(t\), and \(n\) is the release exponent.  
\end{table} 
Table 1: Kinetic models and their respective equations. \begin{table}
\begin{tabular}{l c c} \hline \hline
**Serial Number** & **Name of Equation** & **Mathematical Expression** \\ \hline
1 & [PERSON] (1 st form) & \(K_{t}=D_{f}e^{\frac{Kt}{H}}\) \\ \hline
2 & Arrhenius (2 nd form) & \(lnK_{t}=lnD_{f}-\frac{E_{\text{a}}}{R}\times\frac{1}{T}\) \\ \hline
3 & Eyring & \(\ln\frac{K_{t}}{T}=-\frac{\Delta H}{R}\frac{1}{T}+\ln\frac{K_{\text{a}}}{H}+ \frac{\Delta S}{R}\) \\ \hline
4 & Change in Gibbs free energy & \(\Delta G=\Delta H-T\Delta S\) \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Thermodynamic parameters and their respective mathematical expressions. Culture Collection (ATCC) (Manassas, VA, USA). The passage number of used cell culture was 10, and the cells were maintained under standard cell culture conditions to prevent contamination by mycoplasma, bacteria, fungi, and virus. The materials and supplements used for the cell culture were sterile and all cell culture procedures were carried out in a Laboconco Delta Series Purifier Class II Biosafety Cabinet (Laboconco Corporation, Kansas City, MO, USA). The dosage levels used was based on prior studies [11; 12; 50]. The cells were incubated with drug-loaded PLGA-CS-PEG microspheres or nonloaded PLGA-CS-PEG microsphere suspension at 10 mg/mL, respectively, at time intervals 0, 6, 24, 48, and 72 h. In addition, the cells were treated with free PTX and PGS at 15 \(\upmu\)M concentration at the time intervals. In this study, a complete cell culture medium referred to as Leibovitz's 15+ (L15+) was used. L15+ contains Leibovitz's 15 (L15), 10% FBS, and 2% penicillin/streptomycin. MDA-MB-231 cells were cultured in a T75 flask using L15+ medium and incubated in a humidified incubator set at 37 \({}^{\circ}\)C. The cells were harvested at 70-80% confluence (log phase of growth) using trypsin-EDTA. Alamar blue cell viability assay was used to perform the in vitro cell viability and drug-induced cytotoxicity studies on MDA-MB-231 cells, as described in our previous work [11]. Approximately 10\({}^{4}\) cells per well were seeded in 24-well plates (\(n=4\)) [11]. Prior to the cell culture experiments, the drug-loaded microspheres were exposed to UV light under sterile conditions. Three hours post cell seeding, the L15+ medium was replaced with 1 mL of L15+ containing 10 mg/mL of PLGA-CS-PEG microparticles loaded with drugs and the plates were incubated at 37 \({}^{\circ}\)C and 5% CO\({}_{2}\) for 3 h in a humidified incubator. At predetermined time intervals (0, 6, 24, 48, and 72 h after the incorporation of the drug-loaded microspheres), the L15+ medium was replaced with L15+ containing 10% Alamar blue reagent.