Nanostructured lipid carriers for controlled and triggered drug delivery : synthesis and advanced characterisation
MetadataVis full innførsel
The emerging field of nanomedicine allows development of targeted drug delivery systems that improves the efficiency and bioavailability of important therapeutics in the clinic. Translation of engineered nanoparticles for medicine applications to medicinal products is dependent on clinical testing and several preclinical characterisation steps, including physiochemical characterisation, in vitro characterisation and in vivo characterisation. Nanomedicine is a new therapeutic option and the characterisation methods applied to conventional small molecule drug formulations are generally insufficient or not applicable for nanomedicines. The establishment of methods and structures to provide preclinical characterisation data on nanomedicines is therefore of clinical importance for bringing promising nanomedical drugs to the market. Nanostructured lipid carriers (NLCs) have attracted increasing scientific and commercial interest during the last decade due to their biocompatible and biodegradable properties, facile synthesis and potential as drug carriers for oral delivery. In this study, NLCs based on two different chemical platforms were developed with separate encapsulation of four different payloads: the potentially anti-cancer agent curcumin, the anti-tubercular antibiotic bedaquiline, the anti-fungal antibiotic amphotericin B (AmB) and superparamagnetic iron oxide nanoparticles (SPIONs). Concomitantly, a comprehensive characterisation cascade was developed for characterisation of these and future NLCs. This involved size measurements by dynamic light scattering (DLS) and Field Flow Fractionation (FFF), quantitation of NLC components and payload by LC-MS/MS and ICP-MS, and surface chemistry-based particle separation by monolithic chromatography. The studies also involved co-encapsulation of curcumin and SPIONs to study hyperthermia-controlled release by the application of an external magnetic field. In vitro studies explored cellular effects of drug loaded NLCs, including cellular uptake studies by flow cytometry and a high-throughput screening assay for detection of nanoparticle toxicity in relevant cell lines. The DLS and FFF size measurement techniques used in this work are based on different principles and showed distinctive advantages in nanoparticle size determination. The quick DLS batch analysis is efficient for quality control of newly synthesised NLCs, but is unable to provide specific size determination of polydisperse samples. FFF offers an additional separation-step that allows size measurement of sample populations of different size, but is more time consuming and resource demanding compared to DLS. Highly sensitive nanoparticle component quantification by LC-MS/MS and ICP-MS was found to provide valuable information about the particle composition of the NLCs, including drug vi and SPION loading capacities. A screening of NLC analysis by monolith columns with different chemical functionalities allowed surface chemistry-based separation, and showed that this is a technology with potential for large-scale up-concentration, purification and fractionation of nanoparticle batches. Curcumin and magnetic SPIONs were co-encapsulated in NLCs, allowing use in imaging (theranostic) applications and for triggered drug delivery by magnetic field exposure. Exposure experiments with this novel NLC platform and alternating magnetic fields were performed in this work, using a magnetic field generator designed and built for this purpose. Some early results indicated that AMF induced drug release might have occurred in this set-up. These indications are, however, not clear and need further investigation. The in vitro uptake study showed cellular uptake of curcumin loaded NLCs by epithelial cancer cells after 3 hours of incubation. This observation shows promise for cytosolic drug delivery. The cell line cytotoxicity study included AmB loaded particles prepared by the two NLC formulations. These results showed dose-dependent toxicity for all the tested samples. A significant difference in cell viability could be stated between the different NLC formulations, indicating a surfactant-specific toxicity. AmB encapsulation by the original NLC formulation was shown to induce lower toxicity in LLC-PK1 kidney cells compared to similar concentrations of free AmB.