Search

Parkinson’s disease (PD) is a neurodegenerative disorder where formation of Lewy Bodies (LBs) in the dopaminergic neurons of the substantial nigra pars compacta (SN) cause progressive cell death, resulting in a dopamine (DA) deficiency which manifests itself in motor symptoms including tremors and bradykinesia. Current PD treatments focus on symptom reduction through oral delivery of levodopa (L-DOPA), a precursor of DA. However, L-DOPA delivery to the brain is inefficient due to low oral bioavailability, degradation in the gastrointestinal (GI) tract and the liver, short systemic half-life, systemic L-DOPA decarboxylation into DA and limited brain uptake due to the blood brain barrier (BBB). Additionally, due to the buildup of L-DOPA tolerance by the PD brain, increased dosages are required as the disease progresses, resulting in increased systemic DA concentrations causing serious side effects like dyskinesias. To improve PD treatment efficiency and to reduce side effects, recent research focuses on the encapsulation of L-DOPA in nanoparticles (NPs), the most popular of which include the polymeric- and lipid-based NPs. Both formulations are able to protect L-DOPA from systemic decarboxylation into DA and increase L-DOPA delivery to the central nervous system (CNS). Additionally, these NPs can be modified with proteins and antibodies specifically targeting the BBB, not only improving targeting to-, but also the crossing of the BBB, thereby reducing required dosages and free systemic DA. An alternative treatment strategy is to avoid the BBB altogether through direct intranasal delivery of NP encapsulated L-DOPA to the brain. Through intranasal delivery, L-DOPA can be directly delivered to the brain via the olfactory and trigeminal nerves, reducing free systemic DA and avoiding the BBB-associated problems. These polymeric- and lipid-based NPs can be additionally modified to improve mucoadhesion and cell penetration, resulting in increased therapeutic concentrations o

Human natural killer (NK) cells have received much interest due to significant progress in understanding their function and some promising findings in tumor therapy. NK cells are cytotoxic lymphocytes that have the ability to kill malignant or virally infected cells. Several receptors control their activity, transmitting either activating or inhibitory signals, triggering NK cell responses while assuring self-tolerance. The genetic enhancement of their effector capabilities is needed to increase the tumor-killing activity of NK cells. Many techniques of transfection have been examined for this purpose. This study describes the process of optimizing mRNA delivery into commonly accessible NK cell lines, NK-92 and KHYG-1, using electroporation and lipofection as transfection techniques. Based on the outcomes, there is a vital difference between the two transfection approaches, with lipofection being an inefficient transfection method for both tested cell lines and with electroporation enabling over 90% and 51% efficiency in the KHYG-1 and NK92 cells line, respectively. Finally, an optimized protocol for electroporation and lipofection was used to transfect primary NK cells.

Natural Killer (NK) cells are a promising approach for adoptive cell therapy (ACT) since they can initiate a strong cytotoxic anti-tumor response, as the result of a balanced interplay between activating and inhibiting receptors. Although NK cells are considered less toxic and possibly also more effective than T cells, making them a potent alternative for immunotherapy, clinical efficacy of NK cells may be limited due to a lack of solid tumour infiltration and tumour editing causing suppression of NK cytotoxicity. These issues may be overcome by optimizing the cellular phenotype with additional activating receptors or silencing inhibitory receptors, through genetic manipulation. Yet, current methods for gene transfection of NK cells are lacking in efficiency or they induce undesired toxicity or phenotypical changes in the cells. A well-characterized nonviral method for NK cell transfection is thus desired. Towards this end, this research describes the synthesis of cationic polyplexes and lipid nanoparticles (LNPs) for the efficient delivery of enhanced Green Fluorescent Protein (eGFP) mRNA to NK cells. Although positively charged mRNA-polyplexes were unsuccessful for NK cell transfection, even when combined with cellular uptake and endosomal escape enhancing peptides, LNPs proved to be a suitable mRNA delivery platform. Upon optimization of the LNP lipid composition, including the use of Lipid 5 as ionizable lipid and -sitosterol (instead of cholesterol) as steroid, as well as microfluidics optimization, transfection efficiency up to ~85% eGFP expression was reported for the KHYG-1 NK cell line. Ultimately, the optimized LNP formulation presented ~75% transfection efficiency in umbilical cord blood derived NK cells, with a 5.5-fold increase in fluorescence from eGFP expression compared to the next-best nonviral alternative for NK cell transfection, electroporation. The reported LNP formulation is an effective non-toxic method for gene delivery in clinically relevan

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease known. PD is characterised by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) due to the formation of Lewy Bodies (LB). LB disturb essential cellular pathways and induce neural cell death, resulting in dopamine (DA) deficiencies which manifests itself in (non-)motor symptoms including tremors, rigidity and loss of smell. At the time of writing, no treatment exist that cures or slows down the progressivity of PD. Current treatments aim at the delivery of L-3,4-dihydrophenylalanine (L-DOPA), a DA precursor, to the brain. L-DOPA is generally taken orally as this is convenient for patients, but is poorly taken up systemically, resulting in inefficient drug delivery. Moreover, rapid enzymatic and oxidative conversion of L-DOPA systemically cause a low half-life (50 minutes), leading to the need of high and frequent drug intake. As PD progresses, the SNpc lose the ability to store L-DOPA which further increases the need of excessive L-DOPA intake. As 1% of the administered L-DOPA reaches the brain, the majority is present systemically resulting in severe side effects including dyskinesia. To improve current treatments, L-DOPA is encapsulated in liposomes to protect against rapid degradation, lead to sustained drug release, lower the systemic concentration and result in increased (indirect) targeting. The preparation, characterisation and L-DOPA loading of the liposomes have been formulated by previous students. This report continues on the project by demonstrating the L-DOPA retention kinetics in liposomes in relevant biological media to mimic in vivo. These retention studies have been performed with an innovative method, based on the biotin and streptavidin interaction. The retention kinetics in blood plasma demonstrated the presence of a burst release in the first 2h of incubation in which close to 40% of the original content is rele