Bebawy, George
(2022)
Novel orlistat LDL-like nanoparticles as potential anti-cancer medicines.
PhD thesis, University of Nottingham.
Abstract
Objectives: The field of Nano-therapeutics has gained vast attention over the past decades, described as the future cancer medicine. Controlling the particle size is highly crucial for many reasons including proper drug formulation, tumour targeting and enhancing bioavailability. Mostly, nanoparticles within the range of 100 to 200 nm are the most easily manufactured, yet they may not be the best option for optimum drug targeting and tissue accumulation which are in favour of smaller particles (< 50nm). Thus, preparing nanoparticles of small size (<50nm) in an easy way and with high concentrations of the required drug still requires work.
Additionally, fabrication of effective anticancer nanomedicine has taken a new pathway over the last decades, where new active pharmaceuticals ingredients (API) were introduced as novel therapeutics for cancer therapies. One of the recently studied drugs is orlistat, a weight loss medication that showed remarkable activity against many tumours through inhibiting cellular fatty acid synthesis and hindering cellular division and growth. However, orlistat is highly hydrophobic, and formulation of nanoparticles of small size to ensure cellular accumulation and yet having high drug content is still a hurdle. Therefore in this thesis, a straightforward solvent shift technique was adopted for preparation of phospholipids coated orlistat nanoparticles of controlled size, high stability, and immense drug content.
Various naturally existing organic and inorganic nanoparticles have been present before they were synthesized in labs. Among the naturally occurring nanoparticles is the low-density lipoprotein (LDL), a normal blood constituent, serving as a means of cholesterol delivery to tissues. Cholesterol is one of the main constituents of the cell membranes. Cells get their cholesterol requirement either through making it themselves, taking it up from LDL, or both. Distinctly, being of high proliferation rate, tumour cells need large amounts of cholesterol obtained chiefly from LDL to establish new membranes. Thus, LDL could be used as a vehicle to carry antitumor drugs, consequently acting as an excellent targeting modality.
Methods: The present thesis involved the preparation of nanoparticles of hydrophobic materials through rapid solvent exchange technique, including coating of the particles to control the size of the prepared nanoparticles. Investigating the effect of different concentrations of the materials used and the rate of nanoprecipitation on particle size and surface charge was conducted using dynamic light scattering and transmission electron microscopy. Afterwards, the results were compared to each other to find the inflection point in the particle size growth with the concentration of used material and effect of different coating on particle ripening.
This thesis as well involved the preparation of phospholipids coated orlistat nanoparticles through nanoprecipitation by solvent exchange technique, together with stabilising of the particles with surfactants to control the size of the prepared nanoparticles. Characterisation of the prepared nanoparticles was done using dynamic light scattering, transmission electron microscopy, differential scanning calorimetry (DSC) and polarised light microscopy (PLM). Prepared nanoparticles were further freeze dried to assess for shelf stability. Moreover, encapsulation efficiency and drug release patten were assayed.
Finally, preparation of novel LDL-like nanoparticles of the recent anti-cancer medication orlistat (ORL) was done through the simple straightforward solvent exchange technique, this includes coating of the particles with a mixture of phospholipids including POPC, DSPC, Cholesterol and DSPE PEG (5000) maleimide. An 11-mer peptide moiety, which resembles the active site of the apoprotein B of the natural LDL, is attached to the NPs through simple click chemistry. The prepared formulation was evaluated for its physico-chemical properties through size analysis and conjugation efficacy. Finally, the efficacy of the formulation was evaluated against breast cancer through cell studies on various breast cancer cell lines.
Key findings: Results showed that the prepared nanoparticles of different materials used (triolein, trihexanoin, tricaprin, and orlistat) shared a common pattern of particle size growth upon using high concentrations of the material. Yet, coating the nanoparticles during the solvent exchange resulted in controlling the size at high concentrations, near to the therapeutic doses, keeping it below 50nm. Moreover, the slow rate of nanoprecipitation (0.11ml/min), which is mostly adopted, enhanced the particles ripening, unlike the rapid solvent shift technique (1 ml/s). The prepared nanoparticles were of suitable zeta potential to ensure surface stability against further aggregation.
Moreover, the prepared coated orlistat nanoparticles showed a small controlled size (< ~50 nm) even at high drug loading concentrations, with a proper zeta potential of up to (-70 mV) which ensure stability against aggregation. Orlistat was completely coated with the phospholipid used according to DSC thermograms and PLM. Freeze drying the NPs did not affect its size upon storage for up to 6 months. The prepared formulation showed entrapment efficiency of 77% and a controlled release pattern over 1 week period.
Concerning the novel LDL-like ORL NPs, the prepared formulation was in size range of around 50 nm, and of spherical intact structure with a stable average zeta potential of -25 mV. The click chemistry employed for peptide attachment was successful with % free sulfhydryl groups in the conjugated peptide drop to 0.7%. The formulation showed enhanced cytotoxicity in all the employed breast cancer cell lines, resulting in significant decrease in the IC50 compared to other orlistat NPs formulation with no peptide conjugated. Moreover the rate of internalisation of the LDL-like ORL NPs was significantly enhanced in breast cancer cell lines, due to the combined effect of active and passive targeting.
Conclusions: In conclusion, coating of the particles and controlling the rate of solvent shift in our study resulted in a suitable and easy way of controlling particle size and avoiding undesired particle size growth. Although orlistat is highly hydrophobic, yet it could be formulated into nanoparticles assemblies, using a simple method and with high stability, small controlled size and immense drug concentration. Finally, LDL-like ORL NPs is a promising formulation for combating breast cancer, with significant efficacy and targeting ability.
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