Self-assembled Lipid Nanoparticles for Targeted Cancer Therapeutics
Creators: name: Cai, Xudong
Publisher: name: RMIT University
Year: 2025
Subjects: classificationCode: 320604
subject: Nanomedicine
classificationCode: 400302
subject: Biomaterials
classificationCode: 340603
subject: Colloid and surface chemistry

description: In recent decades, lipid nanoparticles (LNPs) have gained significant attention as innovative drug delivery systems due to their therapeutic potential. Among these, non-lamellar LNPs, such as cubosomes and hexosomes, have garnered particular interest. Cubosomes are characterized by bicontinuous lipid bilayers that form two distinct, non-intersecting water channels, resulting in a large internal surface area. Hexosomes, on the other hand, possess an internal mesophase compartmentalized by lipid bilayers arranged parallel to the plane of a hexagonal lattice. The unique structures of cubosomes and hexosomes offer several advantages, including enhanced drug encapsulation, controlled release, and improved cell fusion, all of which contribute to greater pharmaceutical efficacy. Their substantial internal surface areas allow for higher payload loading, making them ideal carriers for a wide range of therapeutic compounds, including cancer drugs, proteins, peptides, and RNA. 

In aqueous environments, amphiphilic lipids and stabilizers are the primary structural components of non-lamellar LNPs. Monoolein (MO) and phytantriol (PT) are the most extensively studied lipids in this context. While amphiphilic lipids can self-assemble into bulk mesophases, their exposure to the external aqueous environment often leads to lipid particle aggregation due to the presence of hydrophobic domains. Stabilizers play a crucial role in preventing this by providing electrostatic or steric barriers that enhance particle stability. Among the various stabilizers available, Pluronic® block copolymers, particularly F127, have been widely used over the past few decades. However, despite its effectiveness in stabilizing these structures, Pluronic® F127 does not impart MO/PT-based LNPs with responsiveness to biological stimuli, limiting its applicability in dynamic biological environments.

Increasing efforts are being made to develop stimuli-responsive LNPs that exploit physiological differences between healthy and cancerous tissues to reduce side effects on healthy cells. For example, the pH of normal cells and tissues is around 7.4, while the pH of cancerous sites typically ranges from 5.5 to 6.5. Additionally, the concentration of hydrogen peroxide (H2O2) in cancer cells is about 100 times higher than in normal cells.

This thesis focuses on the design and synthesis of amphiphilic block copolymers using Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization. These copolymers offer dual functionality: they stabilize MO/PT-based cubosomes and introduce additional properties, such as structural responsiveness to pH and H2O2. The copolymers include pH-responsive groups like 2-(dimethylamino)ethyl methacrylate (DMAEMA) and diethylaminoethyl methacrylate (DEAEMA), H2O2-responsive groups like 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acrylate (TBA), and a fluorescent probe, tetraphenylethylene (TPE). Additionally, they feature a hydrophilic segment with 113 PEG units. The hydrophobic blocks interact with lipid bilayers, while the hydrophilic PEG block extends into the aqueous environment to prevent particle aggregation through steric hindrance. By engineering the surface of LNPs with these multifunctional stabilizers, this work aims to enhance controlled drug release, cell targeting, and bioimaging capabilities.

To investigate the internal mesophases of LNPs stabilized by newly developed polymers, high-throughput synchrotron SAXS was employed. The SAXS results reveal that the internal mesophases of these LNPs are influenced by the copolymer structure, the concentration of the added polymer, and the types of lipids used (Chapter 2 and Chapter 5). Notably, the pH responsiveness of the MO/PT-based LNP systems shows a transition from cubic (Q2) to hexagonal (H2) mesophases as the pH increases. This behavior is attributed to the pH-sensitive nature of DMAEMA and DEAEMA functional blocks, whose solubility changes due to the protonation or deprotonation of their amine groups at different pH values (Chapter 2). These findings underscore the potential of these LNPs in the development of stimuli-responsive drug delivery systems for various biomedical applications.

In terms of controlled drug release, encapsulation studies of anticancer drugs were conducted using developed responsive LNPs. Chapter 3 details the encapsulation of the highly hydrophobic drug Paclitaxel (PTX), while Chapter 4 covers Temozolomide (TMZ) and the hydrophilic drug Cisplatin (CDDP). The LNPs demonstrated high encapsulation efficiency for both hydrophilic and hydrophobic drugs. Notably, LNPs functionalized with DMAEMA and TBA groups effectively controlled drug release rates in response to pH and H2O2 variations. Specifically, PTX was released more rapidly under acidic conditions (pH 5.5) and/or in the presence of 50 mM H2O2, compared to physiological pH 7.4. The release behavior conformed well to the Higuchi model (Chapter 3).

For cell targeting, Chapter 4 describes the development of Angiopep-2 (Ang2)-functionalized cubosomes designed to cross the blood-brain barrier (BBB) and target glioblastoma therapy. In vitro models, including the BBB hCMEC/D3 cell monolayer, 3D BBB cell spheroids, and microfluidic BBB/GBM-on-a-chip models, were used to assess the efficiency of these cubosomes. Results indicated that Ang2-functionalized cubosomes penetrated the BBB more effectively than unfunctionalized particles. In vivo biodistribution studies showed a threefold increase in brain accumulation of Ang2-functionalized cubosomes compared to their unfunctionalized counterparts. Additionally, these cubosomes exhibited significantly higher uptake by U87 glioblastoma cells in both the BBB/GBM-on-a-chip model and 3D U87 cell spheroids.

In Chapter 5, the polymeric surface stabilizer of LNP was modified to provide bioimaging capability to the system. This was done through the introduction of TPE groups into amphiphilic block and random copolymers with varying lengths of DMAEMA segments. The TPE groups in the LNPs showed aggregation-induced emission (AIE) property, which were significantly influenced by copolymer concentration and pH value. Enhanced TPE fluorescence observed at higher copolymer concentrations and elevated pH levels. Cellular uptake studies confirmed that TPE-functionalized LNPs are effectively tracked in vitro, with endocytosis identified as the primary internalization pathway. 

This thesis presents the development of multifunctional amphiphilic block copolymers incorporating pH- and H2O2-responsive groups and bioimaging functionalities. These innovative stabilizers enable controlled drug release, precise cancer targeting, and real-time tracking, advancing the design of next-generation LNP systems for biomedical applications.