Protein-Eye View of the in Meso Crystallization Mechanism.

For evolving biological and biomedical applications of hybrid protein?lipid materials, understanding the behavior of the protein within the lipid mesophase is crucial. After more than two decades since the invention of the in meso crystallization method, a protein-eye view of its mechanism is still lacking. Numerous structural studies have suggested that integral membrane proteins preferentially partition at localized flat points on the bilayer surface of the cubic phase with crystal growth occurring from a local fluid lamellar L? phase conduit. However, studies to date have, by necessity, focused on structural transitions occurring in the lipid mesophase. Here, we demonstrate using small-angle neutron scattering that the lipid bilayer of monoolein (the most commonly used lipid for in meso crystallization) can be contrast-matched using deuteration, allowing us to isolate scattering from encapsulated peptides during the crystal growth process for the first time. During in meso crystallization, a clear decrease in form factor scattering intensity of the peptides was observed and directly correlated with crystal growth. A transient fluid lamellar L? phase was observed, providing direct evidence for the proposed mechanism for this technique. This suggests that the peptide passes through a transition from the cubic QII phase, via an L? phase to the lamellar crystalline Lc phase with similar layered spacing. When high protein loading was possible, the lamellar crystalline Lc phase of the peptide in the single crystals was observed. These findings show the mechanism of in meso crystallization for the first time from the perspective of integral membrane proteins.

Manipulating the Ordered Nanostructure of Self-Assembled Monoolein and Phytantriol Nanoparticles with Unsaturated Fatty Acids.

Mesophase structures of self-assembled lyotropic liquid crystalline nanoparticles are important factors that directly influence their ability to encapsulate and release drugs and their biological activities. However, it is difficult to predict and precisely control the mesophase behavior of these materials, especially in complex systems with several components. In this study, we report the controlled manipulation of mesophase structures of monoolein (MO) and phytantriol (PHYT) nanoparticles by adding unsaturated fatty acids (FAs). By using high throughput formulation and small-angle X-ray scattering characterization methods, the effects of FAs chain length, cis-trans isomerism, double bond location, and level of chain unsaturation on self-assembled systems are determined. Additionally, the influence of temperature on the phase behavior of these nanoparticles is analyzed. We found that in general, the addition of unsaturated FAs to MO and PHYT induces the formation of mesophases with higher Gaussian surface curvatures. As a result, a rich variety of lipid polymorphs are found to correspond with the increasing amounts of FAs. These phases include inverse bicontinuous cubic, inverse hexagonal, and discrete micellar cubic phases and microemulsion. However, there are substantial differences between the phase behavior of nanoparticles with trans FA, cis FAs with one double bond, and cis FAs with multiple double bonds. Therefore, the material library produced in this study will assist the selection and development of nanoparticle-based drug delivery systems with desired mesophase.

Self-assembled Lyotropic Liquid Crystalline Phase Behavior of Monoolein-Capric Acid-Phospholipid Nanoparticulate Systems.

We report here the lyotropic liquid crystalline phase behavior of two lipid nanoparticulate systems containing mixtures of monoolein, capric acid, and saturated diacyl phosphatidylcholines dispersed by the Pluronic F127 block copolymer. Synchrotron small-angle X-ray scattering (SAXS) was used to screen the phase behavior of a library of lipid nanoparticles in a high-throughput manner. It was found that adding capric acid and phosphatidylcholines had opposing effects on the spontaneous membrane curvature of the monoolein lipid layer and hence the internal mesophase of the final nanoparticles. By varying the relative concentration of the three lipid components, we were able to establish a library of nanoparticles with a wide range of mesophases including at least the inverse bicontinuous primitive and double diamond cubic phases, the inverse hexagonal phase, the fluid lamellar phase, and possibly other phases. Furthermore, the in vitro cytotoxicity assay showed that the endogenous phospholipid-containing nanoparticles were less toxic to cultured cell lines compared to monoolein-based counterparts, improving the potential of the nonlamellar lipid nanoparticles for biomedical applications.

Modeling the Influence of Fatty Acid Incorporation on Mesophase Formation in Amphiphilic Therapeutic Delivery Systems.

Dispersed amphiphile-fatty acid systems are of great interest in drug delivery and gene therapies because of their potential for triggered release of their payload. The mesophase behavior of these systems is extremely complex and is affected by environmental factors such as drug loading, percentage and nature of incorporated fatty acids, temperature, pH, and so forth. It is important to study phase behavior of amphiphilic materials as the mesophases directly influence the release rate of the incorporated drugs. We describe a robust machine learning method for predicting the phase behavior of these systems. We have developed models for each mesophase that simultaneous and reliably model the effects of amphiphile and fatty acid structure, concentration, and temperature and that make accurate predictions of these mesophases for conditions not used to train the models.

High-Throughput Screening of Saturated Fatty Acid Influence on Nanostructure of Lyotropic Liquid Crystalline Lipid Nanoparticles.

Self-assembled lyotropic liquid crystalline lipid nanoparticles have been developed for a wide range of biomedical applications with an emerging focus for use as delivery vehicles for drugs, genes, and in vivo imaging agents. In this study, we report the generation of lipid nanoparticle libraries with information regarding mesophase and lattice parameter, which can aid the selection of formulation for a particular end-use application. In this study we elucidate the phase composition parameters that influence the internal structure of lipid nanoparticles produced from monoolein, monopalmitolein and phytantriol incorporating a variety of saturated fatty acids (FA) with different chain lengths at varying concentrations and temperatures. The material libraries were established using high throughput formulation and screening techniques, including synchrotron small-angle X-ray scattering. The results demonstrate the rich polymorphism of lipid nanoparticles with nonlamellar mesophases in the presence of saturated FAs. The inclusion of saturated FAs within the lipid nanoparticles promotes a gradual phase transition at all temperatures studied toward structures with higher negative surface curvatures (e.g., from inverse bicontinuous cubic phase to hexagonal phase and then emulsified microemulsion). The three partial phase diagrams produced are discussed in terms of the influence of FA chain length and concentration on nanoparticle internal mesophase structure and lattice parameters. The study also highlights a compositionally dependent coexistence of multiple mesophases, which may indicate the presence of multicompartment nanoparticles containing cubic/cubic and cubic/hexagonal mesophases.

Amphiphilic brush polymers produced using the RAFT polymerisation method stabilise and reduce the cell cytotoxicity of lipid lyotropic liquid crystalline nanoparticles.

Self-assembled lipid lyotropic liquid crystalline nanoparticles such as hexosomes and cubosomes contain internal anisotropic and isotropic nanostructures, respectively. Despite the remarkable potential of such nanoparticles in various biomedical applications, the stabilisers used in formulating the nanoparticles are often limited to commercially available polymers such as the Pluronic block copolymers. This study explored the potential of using Reversible Addition-Fragmentation chain Transfer (RAFT) technology to design amphiphilic brush-type polymers for the purpose of stabilising phytantriol and monoolein-based lipid dispersions. The synthesised brush-type polymers consisted of a hydrophobic C12 short chain and a hydrophilic poly(ethylene glycol)methyl ether acrylate (PEGA) long chain with multiple 9-unit poly(ethylene oxide) (PEO) brushes with various molecular weights. It was observed that increasing the PEO brush density and thus the length of the hydrophilic component improved the stabilisation effectiveness for phytantriol and monoolein-based cubosomes. Synchrotron small-angle X-ray scattering (SAXS) experiments confirmed that the RAFT polymer-stabilised cubosomes had an internal double-diamond cubic phase with tunable water channel sizes. These properties were dependent on the molecular weight of the polymers, which were considered in some cases to be anisotropically distributed within the cubosomes. The in vitro toxicity of the cubosomes was assessed by cell viability of two human adenocarcinoma cell lines and haemolytic activities to mouse erythrocytes. The results showed that phytantriol cubosomes stabilised by the RAFT polymers were less toxic compared to their Pluronic F127-stabilised analogues. This study provides valuable insight into designing non-linear amphiphilic polymers for the effective stabilisation and cellular toxicity improvement of self-assembled lipid lyotropic liquid crystalline nanoparticles.

First Direct Observation of Stable Internally Ordered Janus Nanoparticles Created by Lipid Self-Assembly.

We present the first observation of Janus nanoparticles consisting of stable, coexisting ordered mesophases in discrete particles created by lipid self-assembly. Cryo-TEM images provided visual identification of the multicompartment Janus nanoparticles and, combined with SAXS data, confirmed the presence of mixed cubic phases and mixed cubic/hexagonal phases within individual nanoparticles. We further investigated computer visualization models to interpret the potential interface between the interconnected coexisting nanostructured domains within a single nanoparticle.

Lipid-PEG conjugates sterically stabilize and reduce the toxicity of phytantriol-based lyotropic liquid crystalline nanoparticles.

Lyotropic liquid crystalline nanoparticle dispersions are of interest as delivery vectors for biomedicine. Aqueous dispersions of liposomes, cubosomes, and hexosomes are commonly stabilized by nonionic amphiphilic block copolymers to prevent flocculation and phase separation. Pluronic stabilizers such as F127 are commonly used; however, there is increasing interest in using chemically reactive stabilizers for enhanced functionalization and specificity in therapeutic delivery applications. This study has explored the ability of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with poly(ethylene glycol) (DSPE-PEGMW) (2000 Da ≤ MW ≤ 5000 Da) to engineer and stabilize phytantriol-based lyotropic liquid crystalline dispersions. The poly(ethylene glycol) (PEG) moiety provides a tunable handle to the headgroup hydrophilicity/hydrophobicity to allow access to a range of nanoarchitectures in these systems. Specifically, it was observed that increasing PEG molecular weight promotes greater interfacial curvature of the dispersions, with liposomes (Lα) present at lower PEG molecular weight (MW 2000 Da), and a propensity for cubosomes (QII(P) or QII(D) phase) at MW 3400 Da or 5000 Da. In comparison to Pluronic F127-stabilized cubosomes, those made using DSPE-PEG3400 or DSPE-PEG5000 had enlarged internal water channels. The toxicity of these cubosomes was assessed in vitro using A549 and CHO cell lines, with cubosomes prepared using DSPE-PEG5000 having reduced cytotoxicity relative to their Pluronic F127-stabilized analogues.

Characterization and bioactive properties of zirconia based polymeric hybrid for orthopedic applications.

Zirconia is a transition metal oxide with current applications to orthopedic implants. It has been shown to up-regulate specific genes involved in bio-integration and injury repair. This study examines the effects of zirconia and polydimethylsiloxane (PDMS) hybrids on the proliferation and viability of human primary osteoblast and fibroblast cells. In this study, zirconia-PDMS hybrid coatings were synthesized using a modified sol gel process. The hybrid material was characterized using optical microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and contact angle analysis. This study demonstrates that Zr-PMDS surface materials display hydrophobic surface properties coupled with a preferential deposition of polymer near the surface. Primary osteoblast and fibroblast proliferation and viability on hybrid coated surfaces were evaluated via a rapid screening methodology using WST-1 and calcein AM assays. The cells were seed at 5,000 cells per well in 96-well plates coated with various composition of Zr-PDMS hybrids. The results showed increasing cell proliferation with increasing zirconia concentration, which peaked at 90 % v/v zirconia. Proliferation of osteoblasts and fibroblasts displayed similar trends on the hybrid material, although osteoblasts displayed a bi-phasic dose response by the calcein AM assay. The results of this current study show that Zr-PDMS may be used to influence tissue-implant integration, supporting the use of the hybrid as a promising coating for orthopedic trauma implants.

Cell interactions with lipid nanoparticles possessing different internal nanostructures: Liposomes, bicontinuous cubosomes, hexosomes, and discontinuous micellar cubosomes.

HYPOTHESIS: 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. EXPERIMENTS: 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. FINDINGS: 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.

Angiopep-2-Functionalized Lipid Cubosomes for Blood-Brain Barrier Crossing and Glioblastoma Treatment.

Glioblastoma multiforme (GBM) is an aggressive brain cancer with high malignancy and resistance to conventional treatments, resulting in a bleak prognosis. Nanoparticles offer a way to cross the blood-brain barrier (BBB) and deliver precise therapies to tumor sites with reduced side effects. In this study, we developed angiopep-2 (Ang2)-functionalized lipid cubosomes loaded with cisplatin (CDDP) and temozolomide (TMZ) for crossing the BBB and providing targeted glioblastoma therapy. Developed lipid cubosomes showed a particle size of around 300 nm and possessed an internal ordered inverse primitive cubic phase, a high conjugation efficiency of Ang2 to the particle surface, and an encapsulation efficiency of more than 70% of CDDP and TMZ. In vitro models, including BBB hCMEC/D3 cell tight monolayer, 3D BBB cell spheroid, and microfluidic BBB/GBM-on-a-chip models with cocultured BBB and glioblastoma cells, were employed to study the efficiency of the developed cubosomes to cross the BBB and showed that Ang2-functionalized cubosomes can penetrate the BBB more effectively. Furthermore, Ang2-functionalized cubosomes showed significantly higher uptake by U87 glioblastoma cells, with a 3-fold increase observed in the BBB/GBM-on-a-chip model as compared to that of the bare cubosomes. Additionally, the in vivo biodistribution showed that Ang2 modification could significantly enhance the brain accumulation of cubosomes in comparison to that of non-functionalized particles. Moreover, CDDP-loaded Ang2-functionalized cubosomes presented an enhanced toxic effect on U87 spheroids. These findings suggest that the developed Ang2-cubosomes are prospective for improved BBB crossing and enhanced delivery of therapeutics to glioblastoma and are worth pursuing further as a potential application of nanomedicine for GBM treatment.

Understanding magnetic nanoparticle osteoblast receptor-mediated endocytosis using experiments and modeling.

Iron oxide nanoparticles are promising candidates for controlling drug delivery through an external magnetic force to treat a wide range of diseases, including osteoporosis. Previous studies have demonstrated that in the presence of hydroxyapatite coated magnetite (Fe3O4) nanoparticles, osteoblast (or bone forming cell) proliferation and long-term functions (such as calcium deposition) were significantly enhanced. Hydroxyapatite is the major inorganic component of bone. As a further attempt to understand why, in the current study, the uptake of such nanoparticles into osteoblasts was experimentally investigated and mathematically modeled. Magnetite nanoparticles were synthesized using a co-precipitation method and were coated with hydroxyapatite. A cellular uptake experiment at low temperatures indicated that receptor-mediated endocytosis contributed to the internalization of the magnetic nanoparticles into osteoblasts. A model was further developed to explain the uptake of magnetic nanoparticles into osteoblasts using receptor-mediated endocytosis. This model may explain the internalization of hydroxyapatite into osteoblasts to elevate intracellular calcium levels necessary to promote osteoblast functions to treat a wide range of orthopedic problems, including osteoporosis.

Paclitaxel-loaded cubosome lipid nanocarriers stabilised with pH and hydrogen peroxide-responsive steric stabilisers as drug delivery vehicles.

Responsive nanoparticle delivery systems hold great potential for next-generation chemotherapeutic treatment with reduced off-target side effects. In this work, we formulated responsive lipid-based cubosomes loaded with paclitaxel (PTX) as a model drug and stabilised by novel amphiphilic block copolymers (ABCs) containing the pH-responsive poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) and/or the hydrogen peroxide (H2O2)-responsive poly(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acrylate) (PTBA) blocks. The results showed that these cubosomes with a particle size of around 250 nm exhibited excellent PTX encapsulation efficiency of up to 60% and had the ability to control the release rate of the drug in response to pH and H2O2 changes. Specifically, compared to the physiological pH of 7.4, PTX was released faster from the cubosome carriers when exposed to pH 5.5 and/or 50 mM H2O2 conditions, which are pathological conditions found in a tumour microenvironment. In vitro cytotoxicity and cell uptake studies further investigated the cellular interactions of these cubosomes. It was found that cubosomes containing PTX had more toxic effects than the control free PTX sample. Compared to cubosomes stabilised by the non-responsive block copolymer Pluronic® F127, the ABC-stabilised cubosomes also had higher cell internalisation efficiency demonstrated by the cytoplasmic fluorescence intensities using confocal microscopy. These results demonstrated that ABCs containing responsive moieties can stabilise lipid cubosomes and enhance controlled release of poorly soluble chemotherapeutics and cellular uptake.

Nanomaterial-based treatments for medical device-associated infections.

Bacterial infections remain one of the biggest concerns to our society. Conventional antibiotic treatments showed little effect on the increasing number of antibiotic-resistant bacteria. Advances in synthetic chemistry and nanotechnology have resulted in a new class of nanometer-scale materials with distinguished properties and great potential to be an alternative for antibiotics. In this Minireview, we address the current situation of medical-device-associated infections and the emerging opportunities for antibacterial nanomaterials in preventing these complications. Several important antimicrobial nanomaterials emergent from advances in synthesis chemistry are introduced and their bactericidal mechanisms are analyzed. In addition, concerns regarding the biocompatibility of such materials are also addressed.

Recent advances in research applications of nanophase hydroxyapatite.

Hydroxyapatite, the main inorganic material in natural bone, has been used widely for orthopaedic applications. Due to size effects and surface phenomena at the nanoscale, nanophase hydroxyapatite possesses unique properties compared to its bulk-phase counterpart. The high surface-to-volume ratio, reactivities, and biomimetic morphologies make nano-hydroxyapatite more favourable in applications such as orthopaedic implant coating or bone substitute filler. Recently, more efforts have been focused on the possibility of combining hydroxyapatite with other drugs and materials for multipurpose applications, such as antimicrobial treatments, osteoporosis treatments and magnetic manipulation. To build more effective nano-hydroxyapatite and composite systems, the particle synthesis processes, chemistry, and toxicity have to be thoroughly investigated. In this Minireview, we report the recent advances in research regarding nano-hydroxyapatite. Synthesis routes and a wide range of applications of hydroxyapatite nanoparticles will be discussed. The Minireview also addresses several challenges concerning the biosafety of the nanoparticles.

Mechanisms of enhanced osteoblast gene expression in the presence of hydroxyapatite coated iron oxide magnetic nanoparticles.

Hydroxyapatite (HA) coated iron oxide (Fe(3)O(4)) magnetic nanoparticles have been shown to enhance osteoblast (bone forming cells) proliferation and osteoblast differentiation into calcium depositing cells (through increased secretion of alkaline phosphatase, collagen and calcium deposition) compared to control samples without nanoparticles. Such nanoparticles are, thus, very promising for numerous orthopedic applications including magnetically directed osteoporosis treatment. The objective of the current study was to elucidate the mechanisms of the aforementioned improved osteoblast responses in the presence of HA coated Fe(3)O(4) nanoparticles. Results demonstrated large amounts of fibronectin (a protein known to increase osteoblast functions) adsorption on HA coated Fe(3)O(4) nanoparticles. Specifically, fibronectin adsorption almost doubled when HA coated Fe(3)O(4) nanoparticle concentrations increased from 12.5 to 100 µg ml(-1), and from 12.5 to 200 µg ml(-1), a four fold increase was observed. Results also showed greater osteoblast gene regulation (specifically, osteocalcin, type I collagen and cbfa-1) in the presence of HA coated Fe(3)O(4) nanoparticles. Collectively, these results provide a mechanism for the observed enhanced osteoblast functions in the presence of HA coated iron oxide nanoparticles, allowing their further investigation for a number of orthopedic applications.

Novel pH-Responsive Cubosome and Hexosome Lipid Nanocarriers of SN-38 Are Prospective for Cancer Therapy.

pH-responsive nanoparticles enable the selective delivery of a chemotherapeutic agent to tumours while reducing adverse effects. Herein we synthesised four novel aminolipids and developed pH-responsive nanostructured lipid nanoparticles (LNP), which exhibited a slow-releasing hexagonal structure (H2) at physiological pH and quick release bicontinuous cubic phase (Q2) at the acidic tumour pH. The nanoparticles were used to encapsulate and control the release of the chemotherapeutic agent SN-38. High-throughput formulation techniques were employed to fabricate LNP by mixing various amounts of aminolipid with monoolein (MO). The effect of aminolipids on MO self-assembled structures was studied using small-angle X-ray scattering (SAXS) at various pH values. Out of the four studied aminolipid-MO LNP systems, the nanoparticles containing N-(Pyridin-4-ylmethyl) oleamide (OAPy-4) or N-(2(piperidin-1yl)ethyl) oleamide (OAPi-1) exhibited a pH-induced H2 to Q2 phase transition in a tumour-relevant pH range (pH 5.5-7.0). SN-38 is 1000 times more efficacious than the commercially available prodrug irinotecan. However, low solubility in water and instability at physiological pH makes it unsuitable for clinical use. SN-38 was loaded into LNP containing MO and aminolipid OAPy-4. The drug loading and entrapment efficiency were determined, and the results indicated that the aqueous solubility of SN-38 loaded in LNP dispersions was ~100 times higher compared to the solubility of the pure drug in aqueous solution. Furthermore, we demonstrated that the in vitro SN-38 release rate from LNPs was faster at lower pH (pH 5) than at neutral pH. Therefore, pH-responsive LNPs developed in this study can potentially be employed in delivering and controlling the release of the potent drug SN-38 to tumour sites.

Controlling the pH dependent transition between monoolein Fd3m micellar cubosomes and hexosomes using fatty acetate and fatty acid additive mixtures.

HYPOTHESIS: Cubosomes made from the inverse micellar cubic mesophase (I2) with Fd3m symmetry possess a unique structure of closely packed inverse micelles. These have prospective functionality in sustained drug release. In this study, we hypothesised that similar to fatty acids, various fatty acetate compounds can induce the formation of micellar Fd3m cubosomes in monoolein (MO) nanoparticles. They are different to micellar cubosomes made of MO and a fatty acid, which are pH responsive and can transition from an Fd3m phase to an inverse hexagonal phase (H2) as pH increases. We hypothesised that by co-doping a fatty acetate and fatty acid into MO, precise control of the Fd3m-H2 phase transition pH in nanoparticles can be achieved. EXPERIMENTS: Five unsaturated fatty acetates with hydrocarbon chain lengths between 18 and 24 were added to MO at a weight ratio of 0.45 - 0.60 to form nanoparticles. The nanoparticles were prepared using high-throughput formulation and characterised with synchrotron small angle X-ray scattering (SAXS). MO nanoparticles doped with vaccenyl acetate and vaccenic acid were used to demonstrate the fine control over Fd3m-H2 phase transition pH. FINDINGS: Micellar cubosomes (Fd3m phase) were found in MO nanoparticles doped with fatty acetates. The Fd3m structure was stable in a wide pH range of 2.6 - 8 and at temperatures up to 45 °C. In MO nanoparticles doped with the acetate/acid mixture, the Fd3m-H2 phase transition pH was tuned between pH 5 and pH 7 by adjusting the ratio of vaccenyl acetate and vaccenic acid. As a H2 phase generally offers faster drug release than an Fd3m phase, the pH responsive lipid nanoparticles developed here may find application in orally administrated formulation, where the vehicles must pass a low pH environment in the stomach before reaching neutral pH in the blood.

Application of Fluconazole-Loaded pH-Sensitive Lipid Nanoparticles for Enhanced Antifungal Therapy.

Cryptococcus neoformans is a yeast-like fungus that can cause the life-threatening disease cryptococcal meningitis. Numerous reports have shown increased resistance of this fungus against antifungal treatments, such as fluconazole (Fluc), contributing to an 80% global mortality rate. This work presents a novel approach to improve the delivery of the antifungal agent Fluc and increase the drug's targetability and availability at the infection site. Exploiting the acidic environment surrounding a C. neoformans infected site, we have developed pH-sensitive lipid nanoparticles (LNP) encapsulating Fluc to inhibit the growth of resistant C. neoformans. The LNP-Fluc delivery system consists of a neutral lipid monoolein (MO) and a novel synthetic ionizable lipid 2-morpholinoethyl oleate (O2ME). At neutral pH, because of the presence of O2ME, the nanoparticles are neutral and exhibit a liquid crystalline hexagonal nanostructure (hexosomes). At an acidic pH, they are positively charged with a cubic nanostructure (cubosomes), which facilitates the interaction with the negatively charged fungal cell wall. This interaction results in the MIC50 and MIC90 values of the LNP-Fluc being significantly lower than that of the free-Fluc control. Confocal laser scanning microscopy and scanning electron microscopy further support the MIC values, showing fungal cells exposed to LNP-Fluc at acidic pH were heavily distorted, demonstrating efflux of cytoplasmic molecules. In contrast, fungal cells exposed to Fluc alone showed cell walls mostly intact. This current study represents a significant advancement in delivering targeted antifungal therapy to combat fungal antimicrobial resistance.