Investigation of supercooled lipid melts as foundation of cyclosporine A loaded nanostructured lipid carrier design

Abstract

Peptides are a rapidly growing therapeutic class, where their advantage versus small molecules is their high specificity for target sites which results in lower side effects. However, peptide activity is dependent on their specific structure which is prone to denaturation from biological and chemical factors. As a result, about half of the peptides in current clinical use exhibit short half-lives, and peptide research has attempted to address this issue by peptide cyclization, D-amino modifications, lipidation and use of nanoparticles. The requirements for suitable peptide delivery are protection of peptide from biological/chemical denaturation, high drug loading and controlled release of cargo.

Nanostructured lipid carriers (NLCs) are a lipid nanoparticle system that incorporates both solid and liquid lipids within the inner matrix. Compared to a lipid emulsion, the solid matrix provides enhanced stability in storage, and the carrier may utilize lipids that are associated with biodegradability and established safety profiles. This formulation was established in 1990s, yet despite described advantages it has not produced a viable product in market for pharmaceutical use. A reason for this may relate to established research practices for production of NLCs predominately use long chain lipids that requires production with multiple high enthalpy processes that risks peptide degradation and increased production costs. This provides an opportunity for innovation in nanocarrier design, where the projects aim is to deconstruct the NLC design and utilize overlooked excipients to design a nanoparticle bespoke to peptide delivery. The peptide model chosen for this project was cyclosporine A because its structure is cyclic and lipophilic. This allows for efficient solubilization in lipids and is a representation of peptide engineering approaches.

Our first objective is to establish the saturation solubility of cyclosporine A in various selection of lipids. The choice of lipids varies from their aliphatic chain length, physical state and is representation in previous NLC research. Liquid lipids and supercooled lipids solid melts were measured with mixture of lipids and cyclosporine A in thermomixer, and its saturation solubility was measured with high performance liquid chromatography. For crystalline solid lipids, cross polarised light microscopy was used to measure peptide saturation solubility. The results highlighted a medium chain solid lipid glyceryl caprylate with the highest 344.5 ± 79.5 mg/ml. This was six times greater than equivalent liquid lipid propylene glycol monocaprylate at 131.7 ± 25.7 mg/ml and thirty-four times greater than long chain crystalline solid glyceryl dipamitate/distearate measured < 10 mg/ml. The monoglyceride supercooled lipid melts showed good compatibility with all liquid lipid tested and therefore was of interest to test.

The second objective was to analyse how the use of supercooled lipid melts effects NLC production. For our project, we focused on NLC production of high shear homogenization. The advantage of this manufacture methodology is omission of organic solvent and continuous manufacture potential. This process typically used in NLC research is to melt the lipids under temperature, the use of high shear homogenizer to produce a pre-emulsion, after which a second process such as ultra sonication can be utilized to form nano emulsions. Compared to typical NLCs production process that would utilize long chain lipids, the use of supercooled lipid melts enabled production that utilized 50% less homogenization power, ~20°C less temperature and elimination of second process such as ultrasonication or high-pressure homogenization. The produced NLCs had sub 200 nm in particle size and low polydisperse system of <0.2 in PDI. This showed the advantage of supercooled lipid melts to be used as solid matrix of NLC is its lower melting point, thus allowed for production of NLC with reduced enthalpy.

The next objective required was to use supercooled lipid melts to produce NLC designs described in research. These are NLC type I imperfect crystal model, which describes liquid lipid that creates imperfections within solid lipid matrix. Type II structureless NLCs describes the lipid core that lacks the solid characteristics, but rather a viscous melt. Type III multiple type model is where liquid lipid droplets are encased within solid lipid shell. Previous research that utilized supercooled lipid melts produced type II structureless NLCs. Our research showed that the use of surfactants that were solid at room temperature nucleate the nanoparticle surface and form Type I imperfect crystal NLCs. This was observed with differential scanning calorimetry, where a NLC sample was with surfactant PEG 100 ester stearate, we increased the crystallization temperatures of glyceryl caprate and glyceryl caprylate NLCs to 27°C and 22.5°C compared to polysorbate 80 which produced NLCs that had crystallization temperatures of -6.5°C and N/A respectively. The PEG 100 ester stearate NLCs had encapsulation efficiency and drug loading of ~70% and 3.75% respectively. The NLCs were stable in 4 weeks storage in multiple conditions, spherical in structure and an in vitro release study of 6 hours showed 16.8% extra release for glyceryl caprate NLCs and 25% extra release for glyceryl caprylate NLCs. In chapter 5, type III core shell (multiple type) NLC was successfully produced with supercooled lipid melt trilaurin in one homogenization process. The result with sub 200 nm particles and < 0.2 PDI. This work establishes potential for the use of supercooled lipid melts to produce various NLC structures that utilize milder processing conditions suited for peptide delivery.

Date of Award	Jul 2026
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy
Supervisor	Sheiliza Carmali (Supervisor) & Gavin Andrews (Supervisor)