Design and development of drug co-delivery platform for enhanced anti-tumour efficacy

Abstract

Combination therapy, often referring to the administration of two or more therapeutic agents with different functional sites in cancer cells, has been used to address the efficacy of chemotherapy in clinical application. However, the clinical outcomes of conventional combination therapies with the cocktail mixture of multiple free drugs often suffers from unideal therapeutic outcomes due to the variations in drug properties such as different solubility, individual pharmacokinetic fate, as well as inconsistent tissue distribution and cell permeability. The work presented in this thesis aimed to promote the synergism of the therapeutics with carrier-free co-delivery nanoplatforms. To achieve this purpose, three different nano-delivery systems (nanoDDSs) were designed and fabricated with different combinations of anti-tumour therapeutic agents. Physicochemical characterisations and in vitro anti-tumour activity of each nanoDDS were performed to evaluate their suitability for the drug co-delivery to realise an augmentation in the anti-tumour efficacy.

In the first part of this work, a self-assembled carrier-free nanoDDS was prepared with cisplatin (CDDP, an alkylating agent) and dactolisib (BEZ, a PI3K inhibitor). The suitable formulation process for the nanoDDS was firstly explored by the preparation with different drug ratios, from which a modified formulation was selected through the comparison of particle size and morphological features. The as-prepared nanoparticles, denoted as CDDP-BEZ nanoparticles (NPs), were uniform spheres with a dynamic diameter of 136.5 ± 0.7 nm.

Based on the coordination bond between the hydrophilic CDDP and BEZ, the driving force for the formation of CDDP-BEZ NPs was majorly hydrophobic interaction and electrostatic interaction, which further led to a pH-responsive drug release profile. By simultaneously affecting DNA and inhibiting PI3K signalling axis, CDDP-BEZ NPs exhibited potent anti-proliferative effect on cancer cells. Besides, the increased lipophilicity due to the coordination with BEZ facilitated the accumulation of CDDP in mitochondrion, resulting in the activation of mitochondrion-dependent intrinsic apoptosis and subsequently leading to a further elevated synergistic efficacy in killing cancer cells.

Additionally, CDDP-BEZ NPs successfully restraint the cell migration and invasion induced by chemo therapy. With a 3-dimensional (3D) tumour spheroid model, we further confirmed that CDDP-BEZ NPs maintained all the superiorities in combating tumour development and progression under complicated physiological condition.

In the second part of this work, the feasibilities of addressing the negative insulin feedback by the co-administration of PI3K inhibitors with anti-hyperglycaemia agents were explored. To realise this goal, two nanoDDSs were designed and fabricated, one for the co-delivery of BEZ and glucose oxidase (GOx), and the other one of the co-delivery of BEZ and metformin. An albumin-based nanoDDS was prepared for the co-delivery of BEZ and GOx through the hydrophobic interaction-induced co-assembly of chemically modified BEZ (Lx-BEZ), GOx, and bovine serum albumin (BSA). For the modification of the formulation process, nanoparticles prepared with different ratios among BEZ, GOx, and BSA at various mixing time were compared in the aspect of particle size and morphology, from which a nanoDDS denoted as LBGB was selected. The as-prepared LBGB was uniform quasi-sphere with a diameter of around 70 nm, with preservation of the catalytic activity of GOx and a pH-responsive drug release profile of BEZ. Enhanced synergy between GOx and BEZ was observed with LBGB in cancer cells compared with the physical mixture of dual agents, even with the existence of insulin. Besides, LBGB exhibited a more potent restraint on the migration property of cancer cells compared with the mixed administration of GOx and BEZ. Consistent anti-tumour activity of LBGB was also observed on 3D tumour spheroids, indicating its excellent therapeutic efficacy even under complicated environment. For the co-delivery of BEZ and metformin, a nanoDDS based on Lx-BEZ and the polymeric prodrug of metformin (pMet) was proposed and established through the π-π stacking interaction. The modified formulation process was obtained by the screening of nanoparticle prepared with different ratios of Lx-BEZ and pMet at different mixing time, from which a suitable nanoDDS (pMet-LB) was selected by the comparison of particle size and morphology.

Despite unsatisfactory pH-responsive drug release profile, pMet-LB exhibited excellent anti-tumour efficacy in 2-dimensional (2D) monolayer-cultured cancer cells, and maintained its potency in killing cancer cells with the presence of insulin. However, no superiority was observed in the anti-tumour activity from pMet-LB on the 3D tumour spheroids, likely due to its lack of pH-responsiveness. This failed intention emphasised the importance of using suitable 3D tumour models to better simulate the actual tumour microenvironment, through which an outcome better reflecting the real physiological process could be obtained.

In the third part of this work, the feasibilities of establishing a nanoDDS for the steady delivery of Ca2+ and other anti-tumour therapeutic agents was explored. Both CaCO3- and CaO2-based nanoparticles were explored for this purpose, through which a CaO2-based nanoDDS modified with curcumin-grafted-hyaluronic acid and transferrin (CaO2@HC-Fe-Tf) was selected for further study. With excellent lyophilisation and serum stability, CaO2@HC-Fe-Tf was uniform sphere of around ~250 nm in diameter with a clear core-shell structure, holding the capacity to enter cancer cells, release H2O2, and initiate Ca2+ overloading, and resulting in its anti-cancer activity.

Despite the relatively mild potency in killing cancer cells, CaO2@HC-Fe-Tf exhibited obvious suppression on the migration of cancer cells from the spheroid. Results from this experiment suggested that further combination with other therapeutic agents might be necessary for an ideal therapeutic outcome.

Collectively, this work demonstrated that carrier-free co-delivery nanoDDSs represented a promising strategy in addressing the issues related with the combination therapy currently applied in clinical. With the precise delivery of both therapeutic agents into the same cell with minimum assistance of vector materials, the carrier-free co-delivery nanoDDSs implemented a way for anti-tumour agents with different targets to simultaneously exert their functions, thus achieving a facilitated anti-tumour efficacy. Through the design and fabrication of three nanoDDSs based on different combinations of therapeutic agents, this work explored the feasibility of building stable carrier-free co-delivery nanoDDSs via different strategies, while evaluating the potential of each nanoDDSs in the aspects of physicochemical properties such as particle size and morphology, as well as in vitro anti-tumour efficacy on 2D monolayer-cultured cells or 3D tumour spheroids. With the discrepancies observed between the results from 2D monolayer study and those from the 3D tumour spheroid study, this work also proposed the application of tumour spheroids as in vitro model in evaluating the therapeutic profile of the established nanoDDSs, which could better simulate the complicated in vivo environment for the actual pharmacokinetic evaluation.

The results obtained from this work suggested that with proper design, the carrier-free co-delivery nanoDDS held the potential to serve as versatile platform, realising the simultaneous delivery of a wide range of anti-tumour agents including chemotherapeutics, enzymes, as well as inorganic substance. Apart from the augmentation in the anti-tumour efficacy, carrier-free co-delivery nanoDDS also provided a cost-friendly alterations, for there was a significant cut-down in the development lifecycle with the combination of two FDA-approved therapeutic agents. In addition, the recently emerged computational screening greatly accelerated the progress of the rational design of optimal combination, paving the way for the industrialisation of the carrier-free co-delivery nanoDDSs and the subsequent clinical translation. Collectively, the work presented in this thesis aimed to exploit the possibility of using carrier-free co-delivery nanoDDS to augment the therapeutic efficacy and minimise the vector-driven side effects in the anti-tumour combination therapies, exploring a potent and affordable way to benefit the cancer patients worldwide.