5-Florourasil Yüklü PLGA Nanopartiküllerinin Toroidal Mikroakışkan Sistemler ile Üretimi ve Proses Değişkenlerinin Deney Tasarımı ile Optimizasyonu

Abstract

In recent decades, microfluidics has presented new opportunities for the production of nanoparticles (NPs). However, microfluidic devices that produce NPs at laboratory scale require more advanced technologies to enable larger scale production for the pharmaceutical industry. One solution is the parallelization of microfluidic devices. However, scale-up of microfluidic devices through parallelization requires complex fluid flow distribution and the use of a separate set of pumps or pressure controllers for each fluid inlet. This approach is more complex, expensive, and less reliable. On the other hand, high-flow micromixers, such as staggered herringbone mixers, are difficult to manufacture using traditional methods when miniaturisation is required for lab-scale research. The new toroidal (bifurcating) micromixer, which operates at high flow rates of 2-10 mL/min, is well suited for production of NPs for both research and the pharmaceutical industry. Additionally, it can be fabricated more cost-effectively compared to other micromixers. Despite all these advantages, the production of 5 Fluorouracil (5FU) loaded poly(lactic-co-glycolic acid) (PLGA) NPs using a toroidal micromixer and the effect of critical process parameters on NP properties have not yet been investigated. The aim of this study was to produce of reproducible and stable 5FU loaded PLGA (5FU-PLGA) NPs using an innovative toroidal microfluidic system for cancer therapy. To achieve this, the critical process parameters of the new toroidal microfluidic device, the flow rate ratios (FRR, the organic to aqueous phases) and the total flow rate (TFR) were evaluated. The effects of these process parameters on the physicochemical properties of both PLGA nanoparticles (NPs) and 5FU-PLGA NPs were analyzed. Furthermore, a Design of Experiments (DoE) approach was used to identify critical process parameters and predict optimal process conditions. For the preliminary formulation trials, different solvents (acetone and acetonitrile), stabilizers (Tris buffer and 1% PVA), and PLGA concentrations (0.5%, 1%, and 2%) were tested at constant process parameters. The formulation using 1% PLGA/acetone as the organic phase and Tris buffer as the aqueous phase was selected from the preliminary studies due to its smallest size and lowest PDI values. Subsequently, 1% 5FU (w/w) was loaded onto the PLGA NPs.In this formulation, the effects of process parameters with TFR between 5 and 15 mL/min and FRR between 1:3 and 1:7 on PLGA NPs and 5FU-PLGA NPs were investigated. The resulting PLGA and 5FU-PLGA NPs were found to be monodisperse and spherical with sizes between 100-150 nm. The PDI values of both drug-loaded and drug-free NPs were less than 0.2, and the zeta potential ranged from approximately -65 to -45 mV. The drug encapsulation efficiency (~52%) and concentration (~9.50×10¹¹ NPs/ml) of the NPs were not affected by variations in these process parameters. A systematic assessment of critical process variables for the production of PLGA and 5FU-PLGA NPs was conducted using Design of Experiment (DoE). According to the response surface design, the size and zeta potential values of both PLGA and 5FU-PLGA NP were directly influenced by TFR but not by FRR. In addition, the size and zeta potential values of PLGA and 5FU-PLGA NPs were affected differently by different interactions of the independent variables (TFRxTFR, FRRxFRR, TFRxFRR). Furthermore, based on the full factorial design, there was no significant correlation between encapsulation efficiency or PDI and the three parameters (TFR, FRR, and TFRxFRR). As a result, TFR had been identified as the most significant parameter influencing NP properties, and this finding is known to be closely related to the design of the micromixer. After PLGA NPs and 5FU-PLGA NPs were purified by centrifugation, they retained their overall size, PDI and zeta potential values. In order to determine the shelf life of the drug loaded formulations, the NPs were stored at +4°C for 3 months and remained stable. To assess the effect of temperature on the NPs, they were stored at 25°C and 37°C for 21 days. It was found that the stability of the NPs was negatively affected by the increase in temperature. It was observed that NP size and encapsulation efficiency (~50%) did not change when different ratios of 5FU (1%, 5%, 10%, 15%, 20% (w/w)) were loaded onto PLGA NPs. All formulations were found to be monodisperse and highly stable even after the purification process. When the release profile of 5FU-loaded (%1 and %20 (w/w)) PLGA NPs was examined, after an initial burst release within the first 8 hours, 5FU exhibited a controlled release over 5 days. This release pattern was found to be consistent with the Higuchi kinetic model. When evaluating the cytotoxicity results of 5FU-PLGA NPs, it was determined that the 5FU-PLGA NPs with a drug loading capacity of 20% achieved an IC50 value, indicating a toxic effect on human squamous carcinoma cells (A-431). In conclusion, the toroidal microfluidic system with single microchannel successfully enabled the production of stable 5FU-loaded PLGA NPs for laboratory and pharmaceutical industry applications without the need for scale-up.