Preparation, characterisation and in vitro evaluation of curcumin loaded PLGA-DSPE hybrid nanoparticles

It is a very common method to use polymer and lipid structured biological materials in the preparation of nano drug carrier systems. Poly (D, L Lactic-co-Glycolic acid) (PLGA) is a biocompatible, biodegradable, and FDA-approved biopolymer. It is very easy to produce nano-micelles from this polymer and is suitable for transporting hydrophobic drugs, distinguishing it from other biocompatible polymers. Apart from this, Curcumin is a natural compound isolated from the rhizomes of the Curcuma longa plant, also known as turmeric, and has many important bioactivities for humans. It has been reported that curcumin has cell cycle-inhibiting, proliferation-interfering, anti-inflammatory and apoptosis-inducing effects on cancer cells. Among these bioactivities, the inhibition of cancer cell proliferation and increase their tendency to apoptosis are first. In addition, Curcumin is known to reduce the level of proteins associated with chemotherapy resistance. Reducing the expression level of these proteins prevents the development of chemotherapy resistance in cancer cells and makes the chemotherapy drug more effective. Our group had previously conducted many studies with PLGA nano-micelles, and in one of them, they loaded 125 g/mL Curcumin into the nano-micelle and examined the change in NF-κB levels in cancer cells it administered. As a result of this study, it was noticed that curcumin was successful in reducing the level of NF-κB, but the amount required to reduce all subunits of this protein was insufficient. This problem could be caused by two reasons. Curcumin is a compound that cannot reduce the level of all subunits of NF-κB or the amount of curcumin carried by PLGA is too small to achieve this effectiveness. Similarly, the reason why the in vivo activity of curcumin is not found to be high enough is due to the insufficient bioavailability of the molecule and consequently the molecule accumulates in the target area in too low amounts. In order to fully measure the relationship between curcumin-loaded nanocarriers and resistance, a high amount of curcumin must first be delivered to the target. In this thesis, we tried to increase the hydrophobic capacity of PLGA to transport curcumin with DSPE, which is a biocompatible and FDA-approved phospholipid. For this purpose, PLGA-DSPE hybrid nano-micelles were prepared with two different methods, namely "Emulsification-Solvent Evaporation" and "Film Preparation-Rehydration Method" and their physicochemical characterization was completed. Afterward, PLGA, with enhanced hydrophobic capacity for carrying curcumin, was used with 5-FU, a chemotherapy agent, and the healing behaviors regarding biological effects were examined in-vitro. It has been successfully shown that the amount of curcumin carried by nano micelles prepared by the "Film Formation-Rehydration Method" method increases up to 250 g/mL. Physicochemical characterization of curcumin-loaded nano-micelles was performed with Dynamic Light Scattering (DLS) FT-IR, Differential Scanning Chalorimetry (DSC), and HPLC. It was determined that curcumin was successfully encapsulated in the core of the nano-particle and DSPE interacted with all functional groups of PLGA by DSC and FT-IR methods in nano-micelles whose size was determined as 120 nm (number%) by the DLS method. The percent encapsulation efficiency (%EE) of the amount of curcumin trapped in the hybrid nano-micelle formed by HPLC method was measured as 92.006% and the drug loading capacity (DL%) as 7.301%. The release mechanism of curcumin from the hybrid nano-micelle was investigated and the Korsmeyer–Peppas model was found to be the most suitable one. Accordingly, it was revealed that the release of curcumin from hybrid nano micelles occurs by diffusion due to swelling of the nanocarrier. The stability of the obtained optimized formulation was examined by the above-mentioned analysis methods, in various media (room temperature, after lyophilization, and in the medium) and at various times, and it was determined that the prepared formulation was suitable for marketing during high-scale production. In addition, Computational Molecular Modeling for curcumin-loaded DSPE-PLGA hybrid nano micelles was performed with Quantum chemical cluster models, Periodic DFTB+ calculations, and Molecular dynamics (MD) simulations. As a result, it has been determined that curcumin interacts not only with each DSPE or PLGA individually but with both, DSPE has a great effect on keeping the PLGA chains together, and the triple system together creates a very stable system in the formed PLGA, DSPE and curcumin matrix. This result is compatible with stability studies. Finally, the in vitro biological effect of optimized DSPE-PLGA hybrid nano micelles carrying 250 µg/mL curcumin was investigated on transfected LoVo human colorectal cancer cell line (LoVo-Luc) and Healthy colon cell line CCD-1072. For this, firstly, the IC50 value of 5-FU was calculated as 440.9 M, and in further experiments, this concentration of 5-FU was used together with optimized hybrid nano-micelles loaded with curcumin. The apoptosis effect of the optimized formulation on cancer cells was performed by Acridine orange (AO) / ethidium bromide (EB) double staining, DAPI staining, fluorescence microscopy, and Annexin V-FITC and PI staining on flow cytometry. The effect of the optimized formulation on apoptosis was statistically compared to free curcumin and was found significant. Following this, apoptotic protein increase and resistance-related beta catenin protein were examined by Western Blot method. It was concluded that the protein expression levels of the mentioned proteins resulting from the combination application of drug-loaded nanoparticles and 5-FU were significantly higher than the application of nanoparticles alone. In line with this, a significant increase in intracellular reactive oxygen species (ROS) rates were observed in the combined use of chemotherapy and curcumin carried by the hybrid nanocarrier. Finally, the effect of optimized DSPE-PLGA hybrid nano-micelles on the intracellular entry of curcumin was investigated. As a result, It was determined that the optimized DSPE-PLGA hybrid nano-micelle successfully doubled the curcumin carrying capacity of PLGA and increased the efficacy of chemotherapy in combination treatment with 5-FU. Examining the effectiveness of the optimized hybrid nano-formulation prepared by in vivo studies was designed as the next step of the research.