Jaradat, Abdolelah
(2020)
Synthesis of heparin conjugated silica nanoparticles for nanomedicine applications.
PhD thesis, University of Nottingham.
Abstract
Heparin has been extensively explored for cancer metastasis treatment owing to its ability to competitively inhibit metastatic cancer cells adhesion to P-selectin which is overexpressed on vascular endothelium and its inhibitory activity of platelet-tumour cells interaction. Moreover, heparin plays a key role in inhibiting the heparanase that is overexpressed during the early stage of metastasis process. However, heparin polymer is rapidly excreted through renal filtration. Detection of metastasis is of a paramount importance as it allows an early clinical intervention which could increase the survival rate of cancer patients.
In the first part of this thesis, heparin was conjugated to silica NPs surfaces in order to investigate its P-selectin blocking activity simultaneously exploiting the prolonged circulation of the NPs. Moreover, two structurally related polymers; chondroitin sulphate and sodium alginate were grafted onto the surface of silica NPs in order to be used as controls. Heparin, chondroitin sulphate and alginate were attached to NPs’ surface by coupling of the EDC-activated polymers to amine functionalised NPs. TGA, azure A and DMMB assays confirmed the attachment of these polymers to the NPs surfaces. Polymer grafted NPs had a particle diameter circa 200 nm and negative zeta potential values (~ -56 mV). Heparin, chondroitin sulphate and alginate conjugated NPs were fluorescently labelled with different fluorophores; TAMRA, FAM and Alexa 633 respectively, in order to monitor their cell surface binding under flow conditions that mimic the in vivo circulation. The study revealed that heparin and chondroitin sulphate conjugated NPs were more bound to the surface of human umbilical vein endothelial cells (HUVECs) compared to alginate conjugated NPs as indicated by fluorescence microscopy. Furthermore, silica microparticles with mean size of ~ 1.4 μm were also synthesised and cellular uptake study was conducted which has shown that heparin conjugated microparticles exhibited less cellular internalisation compared to heparin conjugated NPs. However, the latter exhibited greater P-selectin dependent binding to HUVECs’ surface under flow conditions. To conclude, heparin conjugated NPs can interfere/bind with P-selectin receptors as the competitive assay showed that these NPs were blocked by anti-P-selectin antibodies. Consequently, this could suggest that heparin conjugated NPs could be further investigated and observed in the future as potential tools to reduce the dissemination of circulating tumour cells.
In the second part of this thesis, heparin was chemically modified to produce desulphated heparin and glycol split heparin for the inhibition of heparanase as they have been reported to have less side effects and greater heparanase inhibitory activity compared to unmodified heparin. Successful synthesis of the modified heparin derivatives was confirmed by 1H-NMR. An anticoagulant assay (Anti Xa), which measures heparin activity by indirectly measuring its ability to activate antithrombin III, had indicated that glycol split heparin polymer exhibited lower anticoagulant activity compared with native heparin and thus it would have lower adverse reactions e.g. haemorrhage. Desulphated heparin is expected to have an additional clinical advantage compared to heparin such as higher antiangiogenic activity. Both heparin derivatives were attached to the surface of silica NPs using EDC coupling reaction. Heparin analogues conjugated NPs had a particle diameter around 200 nm so the EPR effect, which is a phenomenon that hypothesises a preferential accumulation of the NPs in the tumour interstitial fluid, could be exploited to accumulate the modified heparins in the extracellular space of the tumour where heparanase is overexpressed. Anti Xa assay also showed that glycol split heparin conjugated NPs exhibited lower anticoagulant activity compared with heparin conjugated NPs. Desulphated heparin conjugated NPs showed both significantly less blue shift effect on azure A λmax and lower zeta potential value compared to heparin conjugated NPs. The data together indicate successful conjugation of the modified heparin polymers to the surface of silica NPs that could be further employed to inhibit heparanase.
In the final part of this thesis, different FRET based models were designed to detect heparinase which is the bacterial form of mammalian heparanase overexpressed during metastasis, in order to enable the early diagnosis of cancer. In model I, both FRET pair were attached to heparin chain to attain fluorescence quenching using EDC/NHS coupling reaction. The fluorophore was either attached to the end or within the chain of heparin; however, the probe prepared by intrachain conjugation of the fluorophore showed significantly higher fluorescence quenching. This model showed promising results with respect to quenching efficiency (QE), unfortunately the restoration of the fluorescence signal after heparinase treatment was minimal compared with the chemical depolymerisation. Model II was then developed which adopts the use of core-shell NPs composed of 12 nm thick shell containing a fluorophore (TAMRA) and a black hole quencher conjugated heparin (BHQ2-Heparin) probe attached to NPs surfaces. Where the thinnest possible shell was used in order to maintain the FRET pair close to each other to achieve an optimum FRET energy transfer and maximal QE. The QE of this system was measured with either blank core or with a core containing 7-methoxy coumarin, however a discrepancy between the QE measurements of the core-shell nanosensors was observed with dramatically lower value obtained by coumarin based system. Due to the inconsistency of QE measurements of model II, model III was developed which comprises BHQ1 incorporated silica NPs and fluorescently labelled heparin appended to the NPs surfaces. In summation, Model III showed the best potential for bacterial heparinase detection and thus it could be further improved and utilised for detection of mammalian heparanase sampled from the blood of cancer patients for metastasis diagnosis.
In conclusion, the results highlight that heparin conjugated nanoparticles could be manipulated for versatile nanomedicine applications, thus they could be further investigated as novel potential therapeutics and diagnostics for cancer metastasis.
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