Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.684457
Title: The interaction of amino-functionalised multi-walled carbon nanotubes with the blood-brain barrier in vitro and brain delivery to mouse brain in vivo
Author: Kafa, Houmam
ISNI:       0000 0004 5921 3190
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2016
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Abstract:
The blood brain barrier (BBB) plays an important role in maintaining the integrity of the brain by regulating the flux of molecules into the brain parenchyma. Only molecules of certain size, charge or lipophilicity are allowed to cross the BBB and to enter the brain. The BBB is comprised of a thin layer of endothelial cells lining the brain capillaries and forming an interface between circulating blood and brain parenchyma. Drug delivery to the brain has always been a challenge due to the low drug uptake at the BBB level, which prevents therapeutics from reaching their potential targets in the brain. Carbon nanotubes (CNTs) are novel colloidal nanoparticles with interesting physical and chemical properties making them an ideal platform for designing drug delivery vectors. CNTs exhibit high surface area to size ratio allowing relatively high doses of the drug to be loaded on the surface, along with other targeting ligands to specifically target affected tissues. In this work, we investigate the ability of functionalised multi-walled carbon nanotubes (f-MWNT) to cross the BBB in vitro using a co-culture BBB model comprised of primary porcine brain endothelial cells (PBEC) and primary rat astrocytes. Brain uptake following systemic administration of radiolabelled f-MWNT in mouse was also investigated. f-MWNT has shown the ability to cross biological membranes both actively and passively, so we hypothesise that it has intrinsic ability to cross the BBB. Two types of f-MWNT were studied; wide f-MWNTs (w-MWNT: 20-30 nm diameter) and thin f-MWNT (t-MWNT: 8 nm diameter). w-MWNT were functionalised using 1,3-dipolar cycloaddition reaction whereas the t-MWNT were oxidised to increase dispersibility and then functionalised using 1,3-dipolar cycloaddition and amidation reactions. Angiopep-2 (ANG) peptide has shown promising results in targeting nanoparticles to the brain via the interaction with lowdensity lipoprotein receptor-related protein 1 (LRP1). ANG was synthesised by solid-phase peptide synthesis (SPPS), and was conjugated to w- and t-MWNT using a maleimide-thiol click reaction yielding w-MWNT-ANG and t-MWNT-ANG, respectively. The degree of functionalisation on f-MWNT constructs was quantified using thermogravimetric analysis (TGA) while the length and diameter were measured using Transmission Electron microscopy (TEM). The synthesised ANG was characterised with high performance liquid chromatography (HPLC) and mass spectrometry, and the amount of ANG loading on the f-MWNT was measured using the bicinchoninic acid assay (BCA). w-MWNT was used as a prototype to study the interaction with the co-culture BBB model using TEM. TEM examination confirmed that w-MWNT crossed the PBEC monolayer via energy-dependent transcytosis. w-MWNT were observed within endocytic vesicles and multi-vesicular bodies after 4 and 24 hours. A complete crossing of the in vitro BBB model was observed after 48 hours, which was further confirmed by the presence of w-MWNT within the astrocytes. Incubating the cells at 4 °C inhibited the transcytosis process. w-MWNT was observed on the apical side of the PBEC monolayer with no evidence of vesicular uptake, which proves that the transport of f-MWNT was energy-dependant. The interaction of f-MWNT with the BBB co-culture was assessed using the radioactive tracer 111In. Radiolabelled f-MWNT constructs were incubated with the co-culture model and the transport rate was measured by gamma counting. The transport of w-MWNT and t-MWNT reached 15.6±1.1% and 7.6±0.3% after 72 hours, respectively. In comparison, targeted w-MWNT-ANG and t-MWNT-ANG reached 20.3±0.9% and 11.6±0.9% after 72 hours, respectively. This showed that the conjugation of ANG to the f-MWNT led to a significant increase in transport across the PBEC monolayer, and that w-MWNT and w-MWNT-ANG achieved significantly higher transport across the endothelial cells compared to t-MWNT and t-MWNT-ANG, respectively. The transport of f-MWNT constructs was also investigated in vivo. Radiolabelled f-MWNT was injected via tail-vein in mouse and brain accumulation was measured using gamma counting. w-MWNT-ANG showed significant brain uptake (2.0±0.5% injected dose/g) at 5 min after intravenous injection in mouse, after whole body perfusion with heparinized saline. This value was significantly higher than the precursor w-MWNT (1.1±0.3% injected dose/g) at 5 min postinjection. Capillary depletion confirmed the presence of w-MWNT and w-MWNTANG in both brain capillaries and parenchyma fractions. Moreover, w-MWNT-ANG resulted in higher parenchyma accumulation (0.3±0.07 % injected dose/total brain parenchyma) compared to w-MWNT (0.09±0.01 % injected dose/total brain parenchyma). t-MWNT and t-MWNT-ANG showed higher brain accumulation than w- MWNT constructs following i.v. administration. Brain accumulation reached (2.4±0.9 % injected dose/g) and (2.7±0.3 % injected dose/g) for t-MWNT and t- MWNT-ANG, respectively. However, there was no significant difference between t MWNT and t-MWNT-ANG. This shows that the thin diameter of the t-MWNT constructs resulted in a higher brain accumulation than w-MWNT. However, the targeting effect of ANG was only observed with the w-MWNT derivatives. In case of t-MWNT constructs, the effect of the diameter on the brain accumulation seemed to be more prominent compared to the effect of ANG targeting. The findings presented in this thesis show the ability of f-MWNT to cross the BBB in vitro and to accumulate in mouse brain following systemic administration. Conjugating ANG resulted in improved transport across the co-culture model for the w-MWNT and w-MWNT-ANG, and led to greater brain accumulation in vivo. t- MWNT showed greater brain accumulation compared to w-MWNT constructs. However, ANG conjugation did not enhance brain accumulation compared to the precursor t-MWNT. These results could pave the way for the use of f-MWNT as nanocarriers for delivery of drugs and biologics to the brain, after systemic administration.
Supervisor: Abbott, Nancy Joan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.684457  DOI: Not available
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