Abstract
Background: As opposed to conventional cancer therapies (i.e. chemotherapy, radiation or surgery) that target cancer cells directly, immunotherapies stimulate the immune system to release effectors that kill cancer cells. Immunotherapies can offer safer and more effective approaches for certain cancer types. Intratumoral delivery of immunotherapies that utilises the tumour as a source of tumour antigens holds promise for inducing anti-tumour immune responses without the need for tumour antigen identification. Intratumoral administration of the immunostimulatory cytosine-phosphate-guanine (CpG) alone or in combination with other immunostimulatory agents has shown some promise in pre-clinical and clinical studies. However, previous bioimaging studies have reported that CpG administered intratumorally diffused rapidly from the tumour into circulating blood, which reduced its anti-tumour efficacy and caused systemic side effects. It was hypothesized that conjugation of CpG to a dextran-based nanocarrier would enhance retention of the CpG at the tumour resulting in enhanced anti-tumour activity of intratumoral CpG. The thesis aim was to test this hypothesis using the two nanoplatforms prepared from amino-dextran, (i) amino-dextran nanoparticle (aDNP) and (ii) dextran polymeric conjugate.
Methods: Amino-dextrans with a molecular weight of either 20, 40, 70 or 110 kDa were used to prepare the dextran-based nanocarriers. aDNP was prepared by desolvation of amino-dextran of 70 kDa followed by the chemical crosslinking of amino groups. Class-B CpG-1668 oligonucleotide was immobilised onto aDNP either by adsorption (CpG-adsorbed-aDNP) or conjugation to aDNP (CpG-conjugated-aDNP). To prepare the CpG-dextran conjugate, CpG was conjugated to amino-dextran of different molecular weight using either stable or redox-responsive bis-aryl hydrazone linkages. Amino-dextrans and CpG-loaded dextran nanocarriers were characterised for their physicochemical properties such as particle size, shape, and surface charge, by dynamic light scattering and transmission electron microscopy. Conjugation of CpG to amino-dextran was confirmed by size-exclusion chromatography and agarose gel electrophoresis. An in vitro cleavage assay was conducted by incubating the CpG-dextran conjugate with the cellular reducing agent, glutathione (GSH), at extracellular (10 or 20 μM) or intracellular concentrations (5 mM) for 2 h at 37 °C. In vitro cellular uptake by antigen-presenting cells (APCs) and immunostimulatory activity of CpG formulations to activate bone-marrow-derived dendritic cells (BMDCs) was determined by flow cytometry. The uptake by BMDCs was visualised by either fluorescence or confocal microscopy. An in vivo therapeutic tumour study was undertaken using an MC-38 murine colorectal cancer cell line injected subcutaneously into the left flank of C57BL/6 mice. Free CpG or the CpG-dextran conjugate was administered intratumorally when the tumour reached 50 mm2 (day 0), followed by three other doses given on days 3, 6, and 9.
Results: The hydrodynamic size of amino-dextran polymers ranged from 10 to 20 nm and the zeta potential ranged from -2 to +10 mV. Amino-dextrans were more readily internalised by splenic dendritic cells or macrophages compared to B-cells. The incorporation of amino groups significantly enhanced the uptake of the high-molecular-weight dextran (110 kDa) by BMDCs compared to native dextran, while for the low molecular weight dextran (20 kDa) there was no significant difference in uptake for native and amino-dextrans.
The size and zeta potential of aDNP could be controlled to produce uniform spherical particles in the size range of 50 to 300 nm and surface charge of -16.5 to +14 mV. Different formulation parameters such as the anti-solvent, water-to-anti-solvent ratio, level of amine functionality of dextran, and crosslinker content, were shown to impact the particle size and surface charge of aDNP. CpG-conjugated-aDNP showed higher uptake by APCs and stimulated a higher level of cytokines compared to CpG-adsorbed aDNP; both formulations had similar particle size, surface charge and CpG loading capacity.
CpG-dextran conjugates were 15 to 30 nm in size with a net negative charge. The in vitro cleavage assay showed that the reversible conjugate was stable in the simulated extracellular reductive environment but cleaved in the intracellular environment, while the non-reversible conjugate was stable in both reductive environments. The CpG-dextran conjugate of the low dextran molecular weight (20 kDa) showed enhanced uptake by BMDCs compared to free CpG. The reversible and non-reversible conjugates of the same dextran molecular weight demonstrated no significant difference in the uptake by BMDCs. Non-reversible CpG-dextran conjugates (20 or 40 kDa) produced a higher level of cytokines compared to free CpG in vitro. The reversible CpG-dextran 40 kDa conjugate showed an enhancement in both tumour growth inhibition and survival of mice in vivo compared to the non-reversible counterpart or free CpG.
Conclusion: This thesis highlights the potential of dextran-based nanoplatforms for intratumoral delivery of CpG. The results demonstrate that conjugation of CpG to the dextran carrier via a reversible linkage improves the in vivo anti-tumour activity of intratumoral CpG compared to the non-reversible conjugate or free CpG. This new dextran nanoplatform could be applied to other vaccine formulations for the delivery of immunostimulatory agents.