Drug delivery with nanocarriers relies on the interaction of individual nanocarriers with the cell surface. For lipid-based NCs, this interaction uniquely involves a process of membrane fusion ...between the lipid bilayer that makes up the NC and the cell membrane. Cubosomes have emerged as promising fusogenic NCs, however their individual interactions had not yet been directly observed due to difficulties in achieving adequate resolution or disentangling multiple interactions with common characterization techniques. Moreover, many studies on these interactions have been performed under static conditions which may not mimic the actual transport of NCs. Herein we have observed fusion of lipid cubosome NCs with lipid bilayers under flow. Total internal reflection microscopy has allowed visualisation of the fusion event which was sensitive to the lipid compositions and rationalized by lipid diffusion. The fusion event in supported lipid bilayers has been compared with those in cells, revealing a distinct similarity in kinetics.
Herein, we demonstrate a method for the functionalization of cubic phase lipid nanoparticles (cubosomes) with a series of magnetite (Fe3O4), copper oxide (Cu2O), and silver (Ag) nanocrystals, with ...prospective applications across a wide range of fields, including antimicrobial treatments. The resulting cubosomes are characterized using small-angle X-ray scattering and dynamic light scattering, demonstrating the retention of a typical cubic phase structure and particle size following nanocrystal encapsulation at concentrations up to 20% w/w. Cryogenic transmission electron microscopy reveals significant loading and association of each nanocrystal type with both monoolein- and phytantriol-based cubosomes. The antibiotic potential of these hybrid nanoparticles is demonstrated for the first time; cubosomes with embedded silver nanocrystals display a high level of antimicrobial activity against both Gram-positive and Gram-negative bacteria, with observed minimum inhibitory concentration values ranging from 15.6–250 μg/mL. Lastly, total internal reflection fluorescence microscopy is used to visualize cubosome–bacteria interactions, suggesting the involvement of particle interactions as a delivery mechanism.
•The particle size of EWP nanoparticle increased with addition of curcumin solution.•Ethanol would induce more α-helix transferred to β-sheet during heating treatment.•At pH 3.0, EWP behaved higher ...loading efficiency than pH 3.8.•EWP nanoparticle can effectively slow down curcumin degradation rate.
This study investigated the encapsulation of curcumin into egg white protein (EWP) nanoparticles. Processing conditions used to formulate the nanoparticles, including pH and the addition of ethanol, were used to control the unfolding and aggregation behavior of EWP. The curcumin loading capacity of the EWP nanoparticles was found to be strongly linked to the structural transitions of the protein during heat denaturation, and the microscopic properties of the particles such as particle size and zeta-potential. Fibrous particles were formed at lower pH (3.0) and were associated with a higher curcumin loading than the granular particles formed at pH 3.8. Ethanol leads to an increase in β-sheet structure, and the formation of a coarser gel structure during heat denaturation, resulted in an increase in particle diameter. The highest curcumin loading capacities were 11.53 and 9.89 mg/g protein (with a final curcumin concentration of 312.5 μM and 268 μM respectively), at pH 3.0 and 3.8, respectively. Encapsulation in EWP nanoparticles was shown to both effectively slow the degradation ratio as well as protect the antioxidant activity of encapsulated curcumin.
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Cubosomes form part of the next generation of lipid nanoparticle drug delivery vehicles, enabling higher drug encapsulation efficiency, particularly for lipophilic drugs, compared to ...traditional liposome formulations. However, the mechanism of interaction of cubosome lipid nanoparticles with cells and their resultant cytotoxicity is not yet well characterised. We hypothesise that the uptake mechanism is dependent on the cell-type, and that cellular toxicity will be controlled by both the lipid composition and the uptake mechanism. The uptake of cubosomes into fibroblast and macrophage cell lines was investigated using live-cell imaging on a confocal microscope. Toxicity of the lipid particles was determined using Fluorescence-Activated Cell Sorting (FACS). Atomic Force Microscopy (AFM) provided an overview of the topography of the surface of individual cells. The cells exhibited a contrast in uptake kinetics depending on cell type attributed to varying uptake mechanisms. Cellular toxicity was dictated more by lipid composition than by the internal particle nanostructure or the uptake mechanism. Surface topography showed many surface ridges in the STO cells which could provide a location for cubosome adhesion prior to uptake. The findings provide a crucial guideline for the future engineering and application of lipid nanoparticles in drug delivery applications.
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•The first study to monitor gastric devolution of casein gels using USANS and SANS.•Gels prepared in D2O exhibit earlier onset of gelation, are firmer yet more brittle compared to ...H2O.•Rennet-induced gel structure is sensitive to the acidic environment of the stomach.•Pepsin induces micelle swelling and promotes higher levels of protein digestion in the stomach.•Acidification and shear govern early gastric digestion kinetics.
This study aimed to understand the structural devolution of 10% w/w rennet-induced (RG) and transglutaminase-induced acid (TG) gels in H2O and D2O under in vitro gastric conditions with and without pepsin. The real-time devolution of structure at a nano- (e.g. colloidal calcium phosphate (CCP) and micelle) and micro- (gel network) level was determined using ultra-small (USANS) and small-angle neutron scattering (SANS) with electron microscopy. Results demonstrate that gel firmness or elasticity determines disintegration behaviour during simulated mastication and consequently the particle size entering the stomach. Shear of mixing in the stomach, pH, and enzyme activity will also affect the digestion process. Our results suggest that shear of mixing primarily results in erosion at the particle surface and governs gel disintegration behaviour during the early stages of digestion. Pepsin diffusivity, and hence action, occur more readily in the latter stages of gastric digestion via access to the particle interior. This occurs via the progressively larger pores of the looser gel network and channels created within the larger, less dense casein micelles of the RG gels. Gel firmness and brittleness were greater in the D2O samples compared to H2O, facilitating gel disintegration. Despite the higher strength and elasticity of RG compared to TG, the protein network strands of the RG gels become more compact when exposed to the acidic gastric environment with comparatively larger pores observed through SEM imaging. This led to a higher degree of digestibility in RG gels compared to TG gels. This is the first study to examine casein gel structure during simulated gastric digestion using scattering and highlights the benefits of neutron scattering to monitor structural changes during digestion at multiple length scales.
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Non-lamellar lyotropic liquid crystal nanoparticles (LLCNPs) are gaining significant interest in the fields of drug delivery and nanomedicine. Traditional, top-down formulation ...strategies for LLCNPs are typically low-throughput, can lack controllability and reproducibility in the particle size distribution, and may be unsuitable for loading more fragile therapeutics. The development of a controllable, reproducible, scalable, and high-throughput strategy is urgently needed.
Monoolein (MO)-based LLCNPs with various stabilizers (F127, F108, and Tween 80) and phytantriol (PT)-F127 cubosomes were produced at various flow conditions via a bottom-up method using a microfluidic platform.
This simple enabling strategy was used to formulate LLCNPs with lower polydispersity compared to the traditional top-down homogenization method. Significantly, particle size could be quantitatively controlled by varying the overall flow-rate; a scaling law was identified between nanoparticle mean size and the total flow rate (Q) of meansize∼Q-0.15 for MO cubosomes and meansize∼Q-0.19 for PT cubosomes (at a fixed flow rate ratio). Effective size control was achieved for a range of cubosome formulations involving different lipids and stabilizers. The formulation of stable, drug-loaded cubosomes with high encapsulation efficiency using this method was exemplified using calcein as a model drug. This work will further promote the utilisation of LLCNPs in nanomedicine and facilitate their clinical translation.
Lipid nanoparticles of internal cubic symmetry, termed cuboplexes, are potential nonviral delivery vehicles for gene therapy due to their “topologically active” nature, which may enhance endosomal ...escape and improve delivery outcomes. In this study, we have used cationic cuboplexes, based on monoolein (MO) doped with a cationic lipid, for the encapsulation and delivery of antisense green fluorescent protein (GFP)small interfering RNA (siRNA) into Chinese Hamster Ovary (CHO)GFP cells. Agarose gel electrophoresis has confirmed the successful encapsulation of siRNA within cationic cubosomes, while synchrotron small-angle X-ray scattering (SAXS) demonstrated that the underlying cubic nanostructure of the particles was retained following encapsulation. The cationic cubosomes were shown to be reasonably nontoxic against the CHO-GFP cell line. Fluorescence-activated cell sorting (FACS) provided evidence of the successful transfection to CHO-GFP cells. Knockdown efficiency was strongly linked to the type of cationic lipid used, although all cubosomes had essentially the same internal nanostructure. The gene knockdown efficiency for some cationic cubosomes was shown to be higher than lipofectamine, which is a commercially available liposome-based formulation, while the controlled release of the siRNA from the cubosomes over a 72 h period was observed using confocal microscopy. This combination exemplifies the potential of cationic cuboplexes as a novel, nonviral, controlled-release delivery vector for siRNA.
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Lyotropic liquid crystalline nanoparticles (LLCNPs) with complex internal nanostructures hold promise for drug delivery. Cubosomes, in particular, have garnered interest for their ...ability to fuse with cell membranes, potentially bypassing endosomal escape challenges and improving cellular uptake. The mesostructure of nanoparticles plays a crucial role in cellular interactions and uptake. Therefore, we hypothesise that the specific internal mesophase of the LLCNPs will affect their cellular interactions and uptake efficiencies, with cubosomes exhibiting superior cellular uptake compared to other LLCNPs.
LLCNPs with various mesophases, including liposomes, cubosomes, hexosomes, and micellar cubosomes, were formulated and characterised. Their physicochemical properties and cytotoxicity were assessed. Chinese Hamster Ovarian (CHO) cells were treated with fluorescently labelled LLCNPs, and their interactions were monitored and quantified through confocal microscopy and flow cytometry.
The non-lamellar LLCNPs showed significantly higher cellular interactions compared to liposomes, with cubosomes exhibiting the highest level. However, there was no significant difference in relative cell uptake between cubosomes, hexosomes, and micellar cubosomes. Cell uptake experiments at 4 °C revealed the presence of an energy-independent uptake mechanism. This study provides the first comparative analysis of cellular interactions and uptake efficiencies among LLCNPs with varying mesophases, while maintaining similar size, composition, and surface charge.
Antibiotic-resistant bacteria pose a significant threat to humanity. Gram-negative strains have demonstrated resistance to last resort antibiotics, partially due to their outer membrane, which ...hinders transport of antimicrobials into the bacterium. Nanocarrier (NC)-mediated drug delivery is one proposed strategy for combating this emerging issue. Here, the uptake of self-assembled lipid nanocarriers of cubic symmetry (cubosomes) into bacteria revealed fundamental differences in the uptake mechanism between Gram-positive and Gram-negative bacteria. For Gram-positive bacteria, the NCs adhere to the outer peptidoglycan layers and slowly internalize to the bacterium. For Gram-negative bacteria, the NCs interact in two stages, fusion with the outer lipid membrane and then diffusion through the inner wall. The self-assembled nature of the cubosomes imparts a unique ability to transfer payloads via membrane fusion. Remarkably, the fusion uptake mechanism allowed rapid NC internalization by the Gram-negative bacteria, overcoming the outer membrane responsible for their heightened resilience. Here this is demonstrated by the marked reduction in the minimal inhibition concentration required for antibiotics against a pathogenic strain of Gram-negative bacteria, Escherichia coli. These results provide mechanistic insight for the development of lipid NCs as a new tool to combat bacteria.
An improved vaccine is urgently needed to replace the now more than 100-year-old Bacillus Calmette–Guérin (BCG) vaccine against tuberculosis (TB) disease, which represents a significant burden on ...global public health. Mycolic acid, or cord factor trehalose 6,6′ dimycolate (TDM), a lipid component abundant in the cell wall of the pathogen Mycobacterium tuberculosis (MTB), has been shown to have strong immunostimulatory activity but remains underexplored due to its high toxicity and poor solubility. Herein, we employed a novel strategy to encapsulate TDM within a cubosome lipid nanocarrier as a potential subunit nanovaccine candidate against TB. This strategy not only increased the solubility and reduced the toxicity of TDM but also elicited a protective immune response to control MTB growth in macrophages. Both pre-treatment and concurrent treatment of the TDM encapsulated in lipid monoolein (MO) cubosomes (MO–TDM) (1 mol %) induced a strong proinflammatory cytokine response in MTB-infected macrophages, due to epigenetic changes at the promoters of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in comparison to the untreated control. Furthermore, treatment with MO–TDM (1 mol %) cubosomes significantly improved antigen processing and presentation capabilities of MTB-infected macrophages to CD4 T cells. The ability of MO–TDM (1 mol %) cubosomes to induce a robust innate and adaptive response in vitro was further supported by a mathematical modeling study predicting the vaccine efficacy in vivo. Overall, these results indicate a strong immunostimulatory effect of TDM when delivered through the lipid nanocarrier, suggesting its potential as a novel TB vaccine.
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