Abstract
Effective cancer treatment is often impacted by side effects and low drug concentrations at the target site, placing a burden on patients and their quality of life. The treatment of cancer with nanoparticles and treatment delivery systems is a very exciting, challenging and promising avenue. Over the last few decades, a significant amount of research has been done on developing nanoparticulate drug delivery systems (DDSs) in targeting severe diseases such as cancer. Nanocarriers have attracted considerable attention, due in part, to the ability to self- assemble into nanoparticles (NPs) with a carried payload.In the following project the polysaccharide pullulan (PUL) was used as starting material for developing biocompatible and biodegradable NPs as carriers with the opportunity for modification. Commercial, high molecular weight (HMW) PUL (Mn = 100 3 200 kDa) was modified with octyl/decyl glycidyl ether (OGE) and triphenyl phosphine (TPP). A series of various molar ratios of OGE modified PULs (PUL:OGE ratio = 1:0.25 3 1:5) and different chain length of TPP deivatives (6-12 carbons) were prepared and reacted with PUL to yield the polymers PUL-OGE(0.25), PUL-OGE(0.5), PUL-OGE(1), PUL-OGE(2), PUL-OGE(5), PUL-
TPP(6), PUL-TPP(8), PUL-TPP(10) and PUL-TPP(12). The successful chemical modifications were confirmed via different methods using spectrometric (FT-IR, NMR, DLS, pyrene fluorescence assay), chromatographical (GPC), thermal analytical (TGA) and microscopical (TEM) techniques. Depending on the degree of substitution (DS) of attached OGE and the corresponding degree of conjugation (DC) of attached TPP moieties, the mean diameters showed values ranging from 131 and 192 nm, in case of PUL-OGE NPs, while values of 450 3 740 nm were achieved regarding PUL-TPP polymers. The DS and DC values were 15.68 3 100.92% and 29.38 - 68.65%, respectively. Overall, the particle sizes increased following modifications with higher OGE:PUL feed ratios and longer hydrocarbon chain lengths of TPP moieties. The DS and DC values showed with a few exceptions an inverted relationship to the measured critical aggregation concentration (CAC), which was 5.65 3 18.70 µg ml-1 (PUL-OGE NPs) and 0.71 3 2.31 (PUL-TPP NPs). Chemically and enzymatically degraded PUL polymers with number average molecular weights of ~ 7, 9 and 18 kDa 3 PUL[7], PULl[9] and PUL[18] - were used in 1:1 molar ratio reactions with OGE and yielded particles sizes of 123.0 to 161.5 nm, average DS values between 41 and 49% and CACs from 6.76 to 9.80 µg ml-1. While the particle size of low molecular weight (LMW) PUL-OGE polymers increased with higher polymer molecular weight, the CAC decreased. All modified polymers were colloidal stable with values of -21.7 to -25.7 mV (HMW PUL-OGE), -35.3 to -27.5 mV (LMW PUL-OGE) and -19 to -31 mV (PUL-TPP).
The modified PULs (HMW PUL-OGE, LMW PUL-OGE and PUL-TPP) were loaded with the anticancer drug docetaxel (DTX) using nanoprecipitation method. The encapsulation efficiencies, determined via HPLC method, were 33.63 3 40.25% (HMW PUL-OGE-DTX), around 45% (LMW PUL-OGE-DTX) and 29.48 3 67.40% (PUL-TPP-DTX).
Cell viability studies of unmodified PUL polymers in PC-3 human prostate cancer cells and PNT-2 endothelial cells revealed no toxicity, which changed after modification. High reduction in cell viabilities were addressed to the compound with the highest OGE modification, namely PUL-OGE(5). The lowest OGE modified and highest TPP modified compound, in specific PUL-OGE(0.25) and PUL-TPP(12), had visibly higher toxicity in PC-3 cancer cells than PNT-2 endothelial cells. In LMW PUL-OGE polymers the order of increased toxicity in both cell lines was PUL[9]-OGE < PUL[18]-OGE < PUL[7]-OGE. PUL-OGE and PUL-TPP encapsulated DTX formulations revealed in some cases synergistic cancerous effects against PC-3 cancel cells, which outperformed the effect of the free drug alone as shown in the compounds PUL- OGE(1), PUL-OGE(2), PUL[7]-OGE and PUL[9]-OGE, PUL-TPP(6), PUL-TPP(10) and PUL-TPP(12). The impact of drug delivery on mitochondria and cytotoxic properties were facilitated with the studied PUL-TPP components. These polymers were especially of interest, since they gave an indication of the targeting ability of mitochondrial cancer cells.
In vitro haemolysis assays in red blood cells (RBCs) following 1 h incubation at 37 °C conferred the non-toxicity of low and high molecular weight PUL polymers, which increased upon grafting OGE and TPP moieties. The assays assessed the highest haemolysis in the studied compound for PUL-TPP(12), accounting for 44.70% at 2 mg ml-1. The 18 kDa LMW PUL and commercial PUL showed no haemolytic activity even after grafting of OGE. This was in contrast to the polymer PUL-OGE(5), exhibiting 7.51% haemolysis at a concentration of 2 mg ml-1.
The synthesised and analysed PUL-OGE and PUL-TPP polymers were regarded as a promising platform for the delivery of DTX to PC-3 prostate cancer cells. It can be assumed that the developed drug delivery system (DDS) is also suitable to be applied onto other cancer cells. The presented work offers the opportunity to expand the findings on the design of new therapeutics with other chemotherapeutic cargos. Furthermore, PUL-TPP polymers open up the possibility to be used in targeting of mitochondrial cancer cells.
| Date of Award | 31 Jul 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Helen Louise Fillmore (Supervisor), Aikaterini Lalatsa (Supervisor) & Eugen Barbu (Supervisor) |