Highlighting Cubosome Research at Diamond

Nanomedicine is a rapidly growing field that uses nanoparticles to diagnose and treat diseases. Nanoparticles are tiny particles smaller than 100 nanometres, about 1/1000th the width of a human hair. Nanoparticle formulations can be made from a variety of materials, including proteins, polymers, lipids, metals, and inorganic elements. 

Figure: Engineered lipid nanoparticles (cubosomes), loaded with the drug copper acetylacetonate (blue), and with their surfaces functionalized with affimers that bind carcinoembryonic antigen (red). This potential nanomedicine binds specifically to colorectal cancer cells, restricting tumour growth.

A long-standing challenge that these nanoparticle formulations could help tackle is the focused delivery of chemotherapy drugs. Next-generation nanostructured liquid crystalline nanoparticles (NPs) called cubosomes, could help to better target drugs at tumour sites and increase the effectiveness and treatments while reducing side effects. 

Cubosomes have high loading capacity, biocompatibility and thermostability, and have been shown to effectively deliver therapeutics to tumour sites in the patient's body. Cubosomes can be designed for active targeting, to direct the therapeutics to the tumour cells, and protect healthy cells from systemic side-effects. Active targeting can be achieved by attaching antibodies, peptides or aptamers that recognise cancer cells. Furthermore, cubosomes can be designed to respond to external stimuli such as magnetic fields, temperature or pH offering further control over drug delivery. Nanoparticles can also deliver drugs or other therapies to areas of the body that are difficult to reach with traditional methods. Research has been conducted at Diamond on several beamlines from the Soft Condensed Matter group involving cubosomes. 

Lipid nanoparticles (LNPs) have attracted enormous interest as drug delivery vehicles; for example, LNPs were essential to the development of COVID-19 mRNA vaccines. LNPs with an internal cubic symmetry, termed cubosomes, are an emerging class of such nanoparticles that offer several advantages, such as high encapsulation of cargo and biocompatibility. However, to date, cubosomes have mainly been used for passive targeting, which often leads to off-target toxicity. Their cytotoxicity and biodistribution in vivo are largely underexplored, hindering clinical translation. 

Researchers from the University of Leeds attached a synthetic antibody, known as an affimer, to the surface of engineered cubosomes that were loaded with a model chemotherapeutic drug to actively target colorectal cancer cells. They used a range of biophysical techniques to characterise the cubosomes and studied their therapeutic efficacy extensively in colorectal cancer models both in vitro (2D cell culture and 3D spheroid models) and in vivo in tumour xenograft bearing mice.  

After collecting preliminary Small Angle X-ray Scattering (SAXS) data on the Diamond-Leeds offline SAXS instrument (DL-SAXS), the team used Diamond's I22 beamline to characterise the internal nanostructure adopted by the LNPs upon surface functionalisation and drug encapsulation. Using SAXS was essential, as the internal nanostructure strongly correlates to their in vivo performance and greatly impacts the LNP's formation-structure-function relationship. The high flux, tuneable energy and spatial/temporal resolution on I22 were crucial, giving diffraction patterns from the weakly scattering dilute samples and resolving the Bragg reflections expected. 

The results showed that surface functionalisation and drug encapsulation didn't alter the internal nanostructure symmetry of the LNPs. The cubosomes exhibited preferential accumulation in cancer cells compared to normal cells both in vitro and in vivo, whilst showing low non-specific absorption and toxicity in other vital organs. Mice subjected to targeted drug-loaded cubosomes experienced: increased drug accumulation in the tumour tissue compared to other vital organs, a decrease in tumour growth, and increased survival rates compared to control groups, demonstrating the exciting potential for affimer-tagged cubosomes in therapeutic applications. 

Understanding how the nanostructure of LNPs leads to function is key to their successful clinical translation. This work focused on engineering LNPs for colorectal cancer treatment. However, understanding LNP structure-function relationships is essential for the development of novel drug delivery vehicles to target a multitude of diseases, vaccines and gene therapy.  

Figure: Structure and characteristics of functional cubosomes. (a) Schematic representation of functional cubosome structures and their active targeting towards tumour cells, (b) SAXS diffraction profiles of (i) empty cubosomes and (ii) drug-laden cubosomes.

Researchers from the University of Oxford evaluated an active tumour-targeting cubosome system directed towards rhabdomyosarcoma (RMS) cells. They employed a top-down synthesis approach to produce blank cubic phase NPs, which were subsequently functionalised with hyaluronic acid (HA), anti-CD221 half-sized antibodies (ha-Abs) and superparamagnetic iron oxide nanoparticles (SPIONs). These triple-functionalised cubosomes can be controlled via an external magnetic field. 

Small-Angle X-ray Scattering (SAXS) measurements on Diamond's B21 beamline investigated the lipid lattice patterns and verified the formation of cubic phases. Both SAXS and cryo-Electron Microscopy (cryo-EM) performed at the Electron Bio-Imaging Centre (eBIC) played an important role in optimising the cubosome synthesis method and validating the structures throughout the investigation.  

The researchers found that all the cubic phase NPs possessed the primitive (Im3m) cubic phase, lattice parameters of 126-155 Å, and water channel diameters of 22-30 Å. The well-organised lattice patterns, however, were compromised by incorporating more than 3% SPIONs. The stability analysis showed that antibody-conjugated cubosomes were able to maintain the Im3m structure for 40 days under ambient conditions, while the integrity of triple-functionalised cubosomes were preserved for 30 days under the same conditions. 

The cubosome-based drug delivery platform constructed in this study can encapsulate large quantities of hydrophilic, lipophilic, or amphiphilic therapeutics and confine the chemotherapy to tumour tissues in an active manner. In addition, the cubosome synthesis and functionalisation procedures the team has established may also be useful in structural biology, vaccine, transfection, cosmetics, or biomedical imaging. 

To find out more about the I22 beamline or discuss potential applications, please contact Principal Beamline Scientist Nick Terril: [email protected] 

To find out more about the B21 beamline or discuss potential applications, please contact Principal Beamline Scientist Nathan Cowieson: [email protected] 

Pramanik, A. et al. Affimer tagged cubosomes: Targeting of carcinoembryonic antigen expressing colorectal cancer cells using in vitro and in vivo models. ACS Applied Materials & Interfaces 14, 11078-11091 (2022). DOI: 10.1021/acsami.1c21655 

Mun, H. et al. CD44 and CD221 directed magnetic cubosomes for the targeted delivery of helenalin to rhabdomyosarcoma cells. Nano Research 16, 2915–2926 (2023). DOI:10.1007/s12274-022-5037-4