Designing a safe dendronised polymeric nanocarrier for hydrophobic drugs or gene delivery in cancer therapy

A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of PhilosophyThe hardship of cancer is continuously increasing and is rapidly spreading globally. At present, almost one-third of newly discovered potential therapeutics have poor pharmacokinetics and biopharmaceutical properties. Chemotherapeutic agents known to be the most effective treatment, lack tumour specificity and suffers from poor solubility. The lack of specificity results in severe side effects in off-target tissues, whereas poor soluble drugs exhibit short half-life in the bloodstream and high overall clearance rate. Amphiphilic block copolymers based on hydrophobic dendrons have shown to be a promising strategy to enhance the solubility of hydrophobic drugs, prolong circulation time, minimise non-specific uptake, and allow for specific tumour-targeting through the EPR effect. Herein, we have proposed the development of a new safe and more specific non-viral vector system based on peptide dendronised polymeric micelles to enhance the delivery of hydrophobic drugs into liver cancer cells. G3(PLLA and OGPLLL) arginine dendron of third generation bearing eight peripheral hydrophobic or cationic groups (PBF and BOC or guanidine groups) were synthesised in high yield, identified and tested for purity using NMR, MS and TLC. A series of three amphiphilic system characterised by different hydrophilic pullulan derivatives segments were then synthesised using Huisgen azide-alkyne 1,3-dipolar cycloaddition between pullulan (P), lactosylated pullulan (P(Lac)) and pullulan bearing disulphide linkage (PSS) with the G3 dendron to lead amphiphilic block copolymers P-PLLA, P(Lac)-PLLA and PSS-PLLA, respectively. Hydrophilic pullulan and lactobionic acid were selected for amphiphilic modification, aiming at specific asialoglycoprotein receptors recognition onto hepatocytes cells in the liver. Macromolecular structures of amphiphilic P-PLLA, P(Lac)-PLLA and PSS-PLLA were able to self-assemble spontaneously into spherical nanoarchitectures of sizes less than 90nm with low polydispersity in the aqueous media, which was confirmed by CAC, DLS and TEM. Furthermore, the polyaromatic anticancer drug Doxorubicin (Dox) was selectively encapsulated in the hydrophobic core through multiple interactions with the dendron, including π-π interactions, hydrogen bonding and hydrophobic interactions. Such multiple interactions had the merits of enhanced drug loading capacity (>16%), excellent stability against dilution, and excellent sustained release property. Results showed that a high number of hydrophobic segments within a micellar core enhance higher loading efficiency of hydrophobic drugs, whereas, an increase of surface hydrophilicity or an increase in the length of the hydrophobic segment, both have an effect in reducing the micellar size and CAC value. The disulfide-containing PSS-PLLA micelles were able to co-encapsulate both hydrophobic drugs Dox and Curcumin (Cur) which could simultaneously be co-released in high rate from the carrier (>80wt% in 60hours) in response to the high redox potential environment. Most importantly, the release of Dox from the carrier at pH 5 enhanced the release of curcumin, whereas curcumin, in turn, would improve the efficiency of Dox anticancer activity by overcoming Dox MDR. Such a delivery system provides a promising approach for combination therapy in cancer. The cell viability assay presented that the blank micelles had excellent biocompatibility both in the normal and tumour cells. Moreover, loaded drugs nanoparticles could be effectively internalised into the hepatoma carcinoma cells, and Dox-Cur-PSS-PLLA dramatically inhibited cell proliferation. Also, cationic dendron conjugated pullulan (P-OGPLLL) could efficiently condense DNA with excellent hemocompatibility and high gene transfection in Hela and Hek293T cells. Thus, this work offers an effective strategy of designing a non-viral system and the P-PLLA, P(Lac)-PLLA, PSS-PLLA and P-OGPLLL nanocarriers serve as a reliable drug/gene delivery nanoplatform to enable the improvement of the bioavailability, targetability, efficacy and overcome MDR of therapeutic agents

Tailoring the supramolecular structure of amphiphilic glycopolypeptide analogue toward liver targeted drug delivery systems

Amphiphilic glycopolypeptide analogues have harboured great importance in the development of targeted drug delivery systems. In this study, lactosylated pullulan-graft-arginine dendrons (LP-g-G3P) was synthesized using Huisgen azide-alkyne 1,3-dipolar cycloaddition between lactosylated pullulan and generation 3 arginine dendrons bearing Pbf and Boc groups on the periphery. Hydrophilic lactosylated pullulan was selected for amphiphilic modification, aiming at specific lectin recognition. Macromolecular structure of LP-g-G3P combined alkyl, aromatic, and peptide dendritic hydrophobic moieties and was able to self-assemble spontaneously into core-shell nanoarchitectures with small particle sizes and low polydispersity in the aqueous media, which was confirmed by CAC, DLS and TEM. Furthermore, the polyaromatic anticancer drug (doxorubicin, DOX) was selectively encapsulated in the hydrophobic core through multiple interactions with the dendrons, including π-π interactions, hydrogen bonding and hydrophobic interactions. Such multiple interactions had the merits of enhanced drug loading capacity (16.89 ± 2.41%), good stability against dilution, and excellent sustained release property. The cell viability assay presented that LP-g-G3P nanoparticles had an excellent biocompatibility both in the normal and tumor cells. Moreover, LP-g-G3P/DOX nanoparticles could be effectively internalized into the hepatoma carcinoma cells and dramatically inhibited cell proliferation. Thus, this approach paves the way to develop amphiphilic and biofunctional glycopolypeptide-based drug delivery systems.the European Commission Research and Innovation (PIRSES-GA-2011-295218

Tailoring the supramolecular structure of guanidinylated pullulan toward enhanced genetic photodynamic therapy

In the progress of designing a gene carrier system, what is urgently needed is a balance of excellent safety and satisfactory efficiency. Herein, a straightforward and versatile synthesis of a cationic guanidine-decorated dendronized pullulan (OGG3P) for efficient genetic photodynamic therapy was proposed. OGG3P was able to block the mobility of DNA from a weight ratio of 2. However, G3P lacking guanidine residues could not block DNA migration until at a weight ratio of 15, revealing guanidination could facilitate DNA condensation via specific guanidinium-phosphate interactions. A zeta potential plateau (∼+23 mV) of OGG3P complexes indicated the nonionic hydrophilic hydroxyl groups in pullulan might neutralize the excessive detrimental cationic charges. There was no obvious cytotoxicity and hemolysis, but also enhancement of transfection efficiency with regard to OGG3P in comparison with that of native G3P in Hela and HEK293T cells. More importantly, we found that the uptake efficiency in Hela cells between OGG3P and G3P complexes was not markedly different. However, guanidination caused changes in uptake pathway and led to macropinocytosis pathway, which may be a crucial reason for improved transfection efficiency. After introducing a therapeutic pKillerRed-mem plasmid, OGG3P complexes achieved significantly enhanced KillerRed protein expression and ROS production under irradiation. ROS-induced cancer cells proliferation suppression was also confirmed. This study highlights the guanidine-decorated dendronized pullulan could emerge as a reliable nonviral gene carrier to specifically deliver therapeutic genes

Self-assembly of pH-sensitive fluorinated peptide dendron functionalized dextran nanoparticles for on-demand intracellular drug delivery.

In this study, the amphiphilic fluorinated peptide dendrons functionalized dextran (FPD-HZN-Dex) via an acid-sensitive hydrazone linkage was successfully designed and prepared for the first time. We demonstrated a spontaneous self-assembly of amphiphilic FPD-HZN-Dex into the well-defined nanoparticles with the core-shell architecture in aqueous media, which is attributed to the efficient amphiphilic functionalization of dextran by the hydrophobic fluorinated peptide dendrons. The spherical morphology, uniform particle size and good storage stability of the prepared FPD-HZN-Dex nanoparticles were characterized by dynamic light scattering and transmission electron microscopy, respectively. In vitro drug release studies showed a controlled and pH dependent hydrophobic drug release profile. The cell viability assays show excellent biocompatibility of the FPD-HZN-Dex nanoparticles for both normal cells and tumor cells. Moreover, the FPD-HZN-Dex self-assembled systems based on pH-sensitive hydrazone linkage also can serve as stimulus bioresponsive carriers for on-demand intracellular drug delivery. These self-assembled nanoparticles exhibit a stimulus-induced response to endo/lysosome pH (pH 5.0) that causes their disassembly over time, enabling controlled release of encapsulated DOX. This work has unveiled a unique non-covalent interaction useful for engineering amphiphilic dendrons or dendrimers self-assembled systems.The European Commission Research and Innovation (PIRSES-GA-2011-295218