Membrane Cholesterol Research Articles

Mechanisms of lipid homeostasis in the Coxiella Containing Vacuole.

Coxiella burnetii, the causative agent of human Q fever, is an obligate intracellular bacterial pathogen that replicates in a large, membrane-bound vacuole known as the Coxiella Containing Vacuole (CCV). The CCV is a unique, phagolysosome-derived vacuole with a sterol-rich membrane containing host and bacterial proteins. The CCV membrane itself serves as a barrier to protect the bacteria from the host's innate immune response, and the lipid and protein content directly influence both the CCV luminal environment and interactions between the CCV and host trafficking pathways. CCV membrane cholesterol is critical in regulating CCV pH, while CCV phosphatidylinositol phosphate species influence CCV fusion events and membrane dynamics. C. burnetii proteins directly target host lipid metabolism to regulate CCV membrane content and generate a source of lipids that support bacterial replication or influence the innate immune response. This review provides an overview of the diverse repertoire of lipids involved in CCV formation and maintenance, highlighting the pathogen-driven strategies to modify host lipid homeostasis.

SLO co-opts host cell glycosphingolipids to access cholesterol-rich lipid rafts for enhanced pore formation and cytotoxicity.

Group A Streptococcus is a global public health concern as it causes streptococcal sore throat and less common but potentially life-threatening invasive infections. Invasive infections have been associated with bacterial strains that produce large amounts of a secreted toxin, streptolysin O (SLO), which belongs to a family of pore-forming toxins produced by a variety of bacterial species. This study reveals that SLO binds to a class of molecules known as glycosphingolipids on the surface of human cells and that this interaction promotes efficient binding of SLO to cholesterol in the cell membrane and enhances pore formation. Understanding how SLO damages human cells provides new insight into streptococcal infection and may inform new approaches to treatment and prevention.

Effects of Cholesterol on the Breast Cancer Resistance Protein: Studies through the Synthesis and Biological Evaluation of Chemical Tools.

The breast cancer resistance protein (BCRP/ABCG2) plays a major role in the multidrug resistance of cancers toward chemotherapeutic treatments. It was demonstrated that cholesterol regulates the ABCG2 activity, suggesting that lower levels of membrane cholesterol decrease the ABCG2 activity in mammalian cells. However, the precise mechanism remains unclear. To better understand the role of cholesterol in the ABCG2 activity, we studied the ABCG2-mediated efflux of different substrates in the presence of different concentrations of cholesterol. Moreover, we synthetized derivatives of cholesterol linked either to known ABCG2 inhibitors or fluorescents probes. A chalcone-cholesterol was synthetized to investigate the influence of cholesterol on ABCG2 inhibition, and a BODIPY-cholesterol was developed to track cholesterol trafficking on mammalian cells and investigate the behavior of cholesterol as an ABCG2 substrate. The obtained results with three different substrates of ABCG2 showed that cholesterol did not affect the intracellular amount of substrates nor the transport activity.

Cellular Cholesterol Loss Impairs Synaptic Vesicle Mobility via the CAMK2/Synapsin-1 Signaling Pathway.

Neuronal cholesterol deficiency may contribute to the synaptopathy observed in Alzheimer's disease (AD). However, the underlying mechanisms remain poorly understood. Intact synaptic vesicle (SV) mobility is crucial for normal synaptic function, whereas disrupted SV mobility can trigger the synaptopathy associated with AD. In this study, we investigated whether cellular cholesterol deficiency affects SV mobility, with the aim of identifying the mechanism that links cellular cholesterol loss to synaptopathy in AD. Lentiviruses carrying 3β-hydroxysteroid-Δ24 reductase-complementary DNA (DHCR24-cDNA), DHCR24-short hairpin RNA (DHCR24- shRNA) or empty lentiviral vectors were transfected into SHSY-5Y cells in order to construct DHCR24 knock-down and knock-in models, along with corresponding controls. Filipin III cholesterol staining was employed to visualize membrane and intracellular cholesterol in the different cell models, and fluorescence intensity was assessed using confocal microscopy. Additionally, we performed immunoblotting to quantify the expression of DHCR24, total calmodulin-dependent protein kinase 2 (CAMK-2), p-CAMK2 (T286), caveolin-1, total synapsin-1, phosphorylated synapsin-1 (p-synapsin-1; S605), and synaptophysin in each experimental group. In DHCR24-silenced cells, the loss of cellular cholesterol caused by knock-down of DCHR24 resulted in a significant decrease in the levels of phosphorylated CAMK2 (p-CAMK2) and phosphorylated synapsin-1 (p-synapsin-1) compared to control cells. The reduction in p-CAMK2 and p-synapsin-1 could disrupt SV mobility, thereby reducing replenishment of the readily releasable pool (RRP) from the reserve pool (RP). Furthermore, cells with DHCR24 knock-down showed downregulation of caveolin-1, a crucial lipid raft marker, compared to control cells. Conversely, elevated cellular cholesterol levels caused by knock-in of DHCR24 reversed the effects of cholesterol deficiency, suggesting that CAMK2-mediated synapsin-1 phosphorylation may be regulated in a lipid raft-associated manner. Additionally, we found that cellular cholesterol loss could significantly downregulate the expression of synaptophysin protein, which is vital for SV biogenesis and synaptic plasticity. These results suggest that depletion of cellular cholesterol following knock-down of DHCR24 can decrease synaptophysin protein expression and impair SV mobility by regulating the CAMK2-meditated synapsin-1 phosphorylation pathway, potentially via a lipid raft-associated mechanism. Our study indicates a critical role for cellular cholesterol deficiency in AD-related synaptopathy, thus highlighting the potential for targeting cellular cholesterol metabolism in therapeutic strategies.

Dengue Virus Fusion Peptide Promotes Hemifusion Formation by Disordering the Interfacial Region of the Membrane.

Membrane fusion is the first step in the infection process of the enveloped viruses. Enveloped viruses fuse either at the cell surface or enter the cell through endocytosis and transfer their internal genetic materials by fusing with the endosomal membrane at acidic pH. In this work, we have evaluated the effect of the Dengue virus fusion peptide (DENVFP) on the polyethylene glycol (PEG)-mediated lipid mixing of vesicles (hemifusion formation) at pH 5 and pH 7.4 with varying cholesterol concentrations. We have demonstrated that the DENVFP promotes hemifusion formation during the fusion of small unilamellar vesicles (SUVs) mainly at pH 5.0. Moreover, the fusion process demonstrates a strong correlation between fusogenicity and the amount of membrane cholesterol. We have further evaluated the partitioning ability of the peptide in three different membranes at pH 5.0 and pH 7.4. The fusogenic ability of the peptide at pH 5.0 is associated with the composition-dependent binding affinity of the peptide to the membrane. The depth-dependent fluorescence probes are used to evaluate membrane organization and dynamics utilizing steady-state and time-resolved fluorescence spectroscopic techniques. Our results show that the DENV FP promotes hemifusion formation by fluidizing the interfacial region of the membrane.

Unraveling Cholesterol-Dependent Interactions of Alkylphospholipids with Supported Lipid Bilayers.

Alkylphospholipids are single-chain lipid amphiphiles that possess clinically relevant biological activities driven by membrane-destabilizing interactions. Subtle variations in alkylphospholipid structure can lead to significant differences in their biological effects, yet corresponding membrane interactions remain underexplored. Herein, we employed the quartz crystal microbalance-dissipation (QCM-D) technique to characterize the real-time membrane interactions of three alkylphospholipids-edelfosine, miltefosine, and perifosine-on supported lipid bilayers with varying cholesterol fractions. Our findings reveal that the tested alkylphospholipids had distinct membrane-interaction profiles: (1) edelfosine exhibited irreversible binding and caused weak membrane disruption; (2) miltefosine caused major disruption by affecting membrane packing; and (3) perifosine exhibited reversible binding while triggering structural rearrangements and modest disruption. Overall, alkylphospholipid micelles showed greater activity than monomers, and higher membrane cholesterol fractions resulted in more extensive disruption, highlighting the interplay between membrane stiffness and responsiveness. These results provide biophysical evidence that different alkylphospholipids have distinct membrane-interaction behaviors that align well with reported biological activities. Our supported lipid bilayer approach offers a valuable platform for designing and assessing alkylphospholipids with tailored membrane-interaction profiles.

Cholesterol- and ssDNA-binding fusion protein-mediated DNA tethering on the plasma membrane.

DNA modification of the plasma membrane is an excellent approach for controlling membrane-protein interactions, modulating cell-cell/cell-biomolecule interactions, and extending the biosensing field. The hydrophobic insertion of DNA conjugated with hydrophobic anchoring molecules is utilized for tethering DNA on the cell membrane. In this study, we developed an alternative approach to tether DNA on the plasma membrane based on ssDNA- and cholesterol-binding proteins. We designed a fusion protein (Rep-ALOD4) composed of domain 4 of anthrolysin O (ALOD4), which binds to cholesterol in the plasma membrane, and a replication initiator protein derived from porcine circovirus type 2 (Rep), which forms covalent bonds with single-stranded DNA (ssDNA) with a Rep recognition sequence. Rep-ALOD4 conjugates ssDNA to Rep and binds to the plasma membrane via cholesterol, thus tethering ssDNA to the cells. Quartz crystal microbalance measurements showed that membrane cholesterol binding of Rep-ALOD4 to the lipid bilayer containing cholesterol was accelerated above 20% (w/w) cholesterol in the lipid bilayer. Rep-ALOD4 was conjugated to fluorescein-labeled ssDNA (S-FITC-Rep-ALOD4) and used to treat human cervical tumor HeLa cells. The green signal assigned to S-FITC-Rep-ALOD4 was detected along HeLa cells, whereas diminished by cholesterol removal with methyl β-cyclodextrins. Moreover, ssDNA-conjugated Rep-ALOD4 tethered ssDNA-conjugated functional proteins on the HeLa cell plasma membrane via complementary base pairing. Collectively, Rep-ALOD4 has the potential as an ssDNA-tethering material via plasma membrane cholesterol to extend cell surface engineering.

Near-infrared light therapy normalizes amyloid load, neuronal lipid membrane order, rafts and cholesterol level in Alzheimer's disease.

Cholesterol dysregulation, disorder of neuronal membrane lipid packing, and lipid rafts lead to the synthesis and accumulation of toxic amyloid-β (Aβ), contributing to the development of Alzheimer's disease (AD). Our study shows that near-infrared (NIR) transcranial photobiomodulation therapy (tPBMT) can reduce Aβ load and restore the properties of neuronal plasma membrane, including Aβ production, bilayer order, rafts, lipid content, and Ca2+ channels during AD. Mice in the experiments were exposed to 808-nm LED for 1h daily over 3months. In the APOE transgenic model with cholesterol dysregulation, the cholesterol levels increased by 22 times, causing healthy neurons to produce toxic Aβ three times faster, increasing its load by five times. Consequently, Aβ disrupts the membrane bilayer and prompts the formation of lipid rafts and pores. NIR-tPBMT can nearly half attenuate Aβ load, restore membrane lipid order and rigidity, reduce the number of lipid rafts, modulate cholesterol synthesis, normalize Ca2+ influx by activated endocytosis, and reduce neuronal death. The Ca2+ influx induced by light does not cause excitotoxicity but modulates Ca2+/calmodulin signaling involved in AD mechanisms and cell viability. The transcriptome analysis of the brain cortex and hippocampus shows that light can downregulate Ca2+/calmodulin-dependent AD-risk genes BACE, PSEN, and APP, and normalize cholesterol homeostasis via the HMGCR, DHCR7, and INSIG1 genes. Additionally, light enhances neuron resistance to the endoplasmic reticulum stress via activating transcription factors of the unfolded protein response. Thus, red/NIR light could be promising in combating AD, restoring synaptic plasticity in degenerating neurons and reducing Aβ load.

Methods for Visualizing and Quantifying Cholesterol Distribution in Mammalian Cells Using Filipin and D4 Probes.

Cholesterol is a key component of biological membranes and, like many cellular lipids, is unevenly distributed among organelles. Disruptions in cholesterol trafficking are associated with various pathologies, including lysosomal lipid storage disorders, often characterized by intracellular cholesterol accumulation. A significant challenge in studying cholesterol trafficking is the lack of easy methods to trace this molecule in situ. Fluorescent probes that specifically bind cholesterol have enabled the visualization and imaging of cholesterol distribution within cells. This chapter details optimized methods for visualizing and quantifying free cholesterol at the plasma membrane and intracellulaly, both inindividual cellsand in large cell populations. These methods use two fluorescent probes: the D4 fragment of perfringolysin O fused to monomeric EGFP (mEGFP-D4 and the more sensitive mutant mEGFP-D4H) and the polyene macrolide filipin. We describe robust methods for quantifying plasma membrane cholesterol by flow cytometry and to visualize intracellular cholesterol poolsby light microscopy. Furthermore, we introduce a refined filipin staining protocol that enhances intracellular cholesterol detection. For precise quantification, we developed an automated image analysis pipeline. This chapter provides a comprehensive guide for staining and quantifying cellular cholesterol, offering valuable tools for studying cholesterol dynamics in mammalian cells.

Monitoring Accessible Cholesterol Levels in Immune Cells.

Cholesterol is a critical lipid that is present at high concentrations in the plasma membranes of animal cells. Most of the membrane cholesterol is sequestered by other membrane lipids and the transmembrane domains of proteins. Cholesterol in excess of such sequestration forms a pool that is referred to as "accessible cholesterol." This pool of cholesterol plays a crucial role in maintaining lipid homeostasis and in controlling cell growth. The accessible cholesterol pool can also be exploited by bacteria and viruses to promote infection and host immune responses rapidly lower levels of this pool to confer protection. We had previously developed a bacterial toxin sensor called ALOD4 to monitor and quantify accessible cholesterol in cultured cells. Here, we report the characterization of a modified version of ALOD4 that is specialized to detect and monitor accessible cholesterol levels in primary immune cells by flow cytometry analysis.

Opposite Roles of Cholesterol and Lanosterol in Lipid Membrane on Amyloid-Beta 42 Peptide Nucleation and Fibril Formation.

Molecular self-assembly of amyloid-beta peptides to form fibrillar aggregates is a known cause of Alzheimer's disease. Although homogeneous nucleation of amyloid-beta is unfavorable, heterogeneous nucleation of amyloid-beta in cell membranes plays a key role in fibril formation. We observed these opposite roles in the effects of cholesterol and lanosterol, the precursor of cholesterol in the brain, on nucleation. As previously reported, cholesterol accelerated nucleation, whereas lanosterol decelerated it when mixed with dioleoyl-phosphatidylcholine at 20%. The observed opposite effects of cholesterol and lanosterol on nucleation do not correlate with the differences in the mechanical and thermodynamic nature of mixed membranes. However, the affinity of amyloid-beta to the inner membrane seems to be related to the opposite effects on nucleation kinetics. Cholesterol reduced the insertion of amyloid-beta into the lipid membrane, whereas lanosterol promoted the insertion of amyloid-beta into the membrane, which would make amyloid-beta more tightly bound by lipid molecules and reduce its diffusivity in the membrane and consequently inhibit nucleation. Our study provides insights into the effects of sterol compounds other than the well-investigated cholesterol on the self-assembly of amyloid-beta to clarify the molecular basis underlying Alzheimer's disease pathology and to develop targeted therapeutic strategies.

Diet therapy abates mutant APC and KRas effects by reshaping plasma membrane cholesterol nanodomains.

Cholesterol-enriched plasma membrane domains are known to serve as signaling platforms in a diverse array of cellular processes. However, the link between cholesterol homeostasis and mutant APC-KRas-associated colorectal tumorigenesis remains to be established. Thus, we investigated the impact of Apc-Kras on (i) colonocyte plasma membrane cholesterol homeostasis, order, and receptor nanoclustering, (ii) colonocyte cell proliferation, and (iii) whether these effects are modulated by select membrane active dietaries (MADs). We observed that oncogenic APC-KRas increased membrane order by perturbing cholesterol homeostasis when cell proliferation is upregulated, in part by altering the expression of genes associated with cholesterol influx, export and de novo synthesis in mouse colorectal cancer (CRC) models and CRC patients. Additionally, oncogene-induced loss of cholesterol homeostasis altered Fzd7, LRP6 and KRas cluster structure/organization. Notably, we show that the combination of chemo-protective MADs, i.e., n-3 PUFAs and curcumin, reduced colonic membrane free cholesterol, order, receptor cluster size, cell proliferation and the number of dysplastic foci in mutant APC-KRas models. This work highlights the dynamic shaping of plasma membrane organization during colon tumorigenesis and the utility of membrane-targeted cancer therapy.

Chemical Warfare in the Environment-Secondary Metabolites From Starfish (Asterias amurensis) Induce Teratogenicity in Medaka Embryos (Oryzias melastigma).

Starfish saponins, known for their role as feeding deterrents against predators like crabs and fish, have been extensively studied for their antifeedant and cytolytic effects. Recent research suggests that starfish secondary metabolites possess antifouling and antifeedant properties and play a role in biological interactions and various ecological functions. Previous research demonstrated that saponins from starfish exert their toxic effects on fish by interacting with cholesterol in the blood cell membrane. This study investigated the hypothesis that secondary metabolites, other than saponins, from the starfish Asterias amurensis may be harmful to fish eggs, particularly marine medaka (Oryzias melastigma) eggs, which share starfish habitat. We systematically separated the secondary metabolites of A. amurensis by a variety of chromatographic methods. Two oligoglycosides related to embryonic teratogenicity were identified, including one previously undescribed tetrasaccharide (1) and one known pyrrole oligoglycoside (2). Their structures were established mainly on the basis of detailed analysis of the nuclear magnetic resonance spectroscopy (NMR) and mass spectroscopic data. Compounds 1 and 2 exhibited strong lethality and significantly reduced voluntary movements in medaka embryos, with compound 2 showing more pronounced effects on teratogenicity and heart rates. The main morphological abnormalities observed included delayed head development (DHD), tiny spines (TS), incomplete absorption of oil balls (OB), cardiac abnormalities (CA), and shrunken yolk sacs (SYS). Our findings imply that starfish secondary metabolites may have broader ecological effects, influencing habitat-sharing species in subtle but crucial ways.

Chicken genome-wide CRISPR library screen identifies potential candidates associated with Avian influenza virus infection.

The avian influenza virus (AIV) poses a significant threat to both the poultry industry and public health. Systematic identification of host factors involved in AIV infection in chicken is critical. In this study, we developed a comprehensive chicken genome-wide sgRNA library containing 76,350 sgRNAs, with 4-6 sgRNAs designed per gene. Then, we constructed a genome-wide CRISPR/Cas9 knockout chicken fibroblasts cells (DF-1 cells) library, covering 99.9 % of the total sgRNAs. Following multiple rounds of survival selection during AIV infection, 706 potential genes were identified, including 107 genes previously associated with AIV infection. These candidate genes were primarily involved in ubiquitin-related pathways, RNA transport, endocytosis, and other cellular processes. Among these, 18 novel hits were selected and confirmed to contribute to AIV-induced cell death, with eight genes specifically implicated in AIV proliferation. Notably, RNF2 was found to negatively regulate interferon-stimulated genes (ISGs), DCP1A was suggested to influence gene expression linked to AIV proliferation, and CREB3L3 may regulate membrane cholesterol levels during AIV invasion, further validating the screening results. This study identified 599 potential chicken genes involved in AIV infection, providing a foundation for a deeper understanding of the mechanisms underlying AIV infection in avian cells.

Energetics of cholesterol perception and translocation in the human Smoothened receptor.

Smoothened (SMO), a member of the G Protein-Coupled Receptor superfamily, mediates Hedgehog signaling and is linked to cancer and birth defects. SMO responds to accessible cholesterol in the ciliary membrane, translocating it via a longitudinal tunnel to its extracellular domain. Reaching a complete mechanistic understanding of the cholesterol translocation process would help in the development of cancer therapies. Competing hypotheses based on available structures support entry of cholesterol from outer and inner membrane leaflets, but the exact mechanism of translocation remains unclear. Using atomistic molecular dynamics simulations (∼2 millisecond simulations) and biochemical assays of SMO mutants, we assess the energetic feasibilities of proposed hypotheses. We show that the energetic barriers for cholesterol translocation from either leaflets are comparable. Mutagenesis experiments and complementary simulations of SMO mutants validate the role of critical amino acid residues along the translocation pathways. Our data suggests that cholesterol can take either pathway to enter SMO, thus explaining contradictory experimental observations in literature. Thus, our results illuminate the energetics and provide a first molecular description of cholesterol translocation in SMO.

Cholic-Acid Derived, Guanidine-Functionalized Polymers as Broad-Spectrum Antimicrobial Agents.

In this study, we report the design of a new guanylated, cholic-acid-based monomer (GM) to combat antimicrobial resistance. The microbial activity stems from the interfacial amphiphilicity of cholic acid, while guanidine shows a strong association with phosphate, which promotes binding to membrane phospholipids. The monomer showed strong antimicrobial activity; however, surprisingly, homopolymers synthesized by photoiniferter reversible addition-fragmentation chain-transfer (RAFT) polymerization of GM completely lost their activity likely due to the conformation of the polymer. In contrast, the design of GM copolymers with poly(ethylene glycol) methyl ether methacrylate (PEGMA) or 2-hydroxyethyl methacrylate (HEMA) allowed recovery of their antimicrobial activity. Due to the existence of cholesterol in cell membranes, hemolysis was highly dependent on the content of GM incorporated. This study highlights the unique and intriguing properties of this novel amphiphilic monomer and its polymers, providing valuable insights into the development of more potent antimicrobial materials.

A Mechanical Immune Checkpoint Inhibitor Stiffens Tumor Cells to Potentiate Antitumor Immunity.

Tumor progression is associated with tumor-cell softening. Improving the stiffness of the tumor cells can make them more vulnerable to lymphocyte-mediated attack. Tumor cell membranes typically exhibit higher cholesterol levels than normal cells, making tumor cells soft. Herein, we demonstrate a mechanical immune checkpoint inhibitor (MICI) formulated by cyclodextrin (CD) lipids and fusogenic lipids. Through fusing CD lipids into the tumor cell membrane using a fusogenic liposome formulation, the cholesterol in the plasma membrane is reduced due to the specific host-guest interactions between CD lipid and cholesterol. As a result, tumor cells are stiffened, and the activation of lymphocytes (including NK and cytotoxic effector T cells) is improved when contacting the stiffened tumor cells, characterized by robust degranulation and effector cytokine production. Notably, this treatment has negligible influence on the infiltration and proliferation of lymphocytes in tumor tissues, confirming that the enhanced antitumor efficacy should result from activating a specific number of lymphocytes caused by direct regulation of the tumor cell stiffness. The combination of MICIs and clinical immunotherapies enhances the lymphocyte-mediated antitumor effects in two tumor mouse models, including breast cancer and melanoma. Our research also reveals an unappreciated mechanical dimension to lymphocyte activation.

A Mechanical Immune Checkpoint Inhibitor Stiffens Tumor Cells to Potentiate Antitumor Immunity

AbstractTumor progression is associated with tumor‐cell softening. Improving the stiffness of the tumor cells can make them more vulnerable to lymphocyte‐mediated attack. Tumor cell membranes typically exhibit higher cholesterol levels than normal cells, making tumor cells soft. Herein, we demonstrate a mechanical immune checkpoint inhibitor (MICI) formulated by cyclodextrin (CD) lipids and fusogenic lipids. Through fusing CD lipids into the tumor cell membrane using a fusogenic liposome formulation, the cholesterol in the plasma membrane is reduced due to the specific host–guest interactions between CD lipid and cholesterol. As a result, tumor cells are stiffened, and the activation of lymphocytes (including NK and cytotoxic effector T cells) is improved when contacting the stiffened tumor cells, characterized by robust degranulation and effector cytokine production. Notably, this treatment has negligible influence on the infiltration and proliferation of lymphocytes in tumor tissues, confirming that the enhanced antitumor efficacy should result from activating a specific number of lymphocytes caused by direct regulation of the tumor cell stiffness. The combination of MICIs and clinical immunotherapies enhances the lymphocyte‐mediated antitumor effects in two tumor mouse models, including breast cancer and melanoma. Our research also reveals an unappreciated mechanical dimension to lymphocyte activation.

Cell membrane cholesterol and regulation of cellular processes: new and the same old thing

Membranes of living cells, or biological membranes, are unique molecular systems in which the functioning of all molecules is interdependent and coordinated, and disruption of this coordination can be fatal for the cell. One example of such coordination and mutual regulation is the functioning of membrane proteins, whose activity depends on their interaction with membrane lipids. This review summarizes the facts about the importance of the cholesterol component of cell membranes for the normal functioning of membrane proteins and the whole cell. This lipid component provides fine regulation of a variety of cellular functions and provides clues to understanding changes in the activity of a number of proteins under various physiologic and pathologic conditions. This review provides examples of cholesterol-dependent membrane proteins and cellular processes and discusses their role in several pathologies. Understanding the mechanisms of cholesterol-protein interactions represents a significant resource for the development of drugs that affect the cholesterol-protein interface.

Methyl-β-cyclodextrin Enhances Tumor Cellular Uptake and Accumulation of α-Linolenic Acid-Paclitaxel Conjugate Nanoparticles.

Improving nanomedicine uptake by tumor cells is key to achieving intracellular drug delivery. In this study, we found that methyl-β-cyclodextrin (MβCD) can significantly promote the intracellular accumulation of nanoparticulated α-linolenic acid-paclitaxel conjugates (ALA-PTX NPs) via enhanced clathrin-mediated endocytosis and limited degradation in lysosomes. Our in vitro results indicated that MβCD not only reduced the plasma membrane cholesterol content and increased plasma membrane fluidity, leading to ALA-PTX NPs being more easily incorporated into the plasma membrane, further enhancing membrane fluidity and making the plasma membrane more susceptible to tensile deformation, forming intracellular vesicles to enhance ALA-PTX NP cellular uptake, but also destroyed lysosomes and then limited ALA-PTX NPs' degradation in lysosomes. In HepG2 tumor-bearing mice, MβCD was also able to enhance the antitumor activity of ALA-PTX NPs in vivo. Moreover, we found that MβCD specifically promoted PUFA-paclitaxel conjugate NP cellular uptake. The cellular uptake of PTX liposome which shares an endocytosis pathway with ALA-PTX NPs could be enhanced by MβCD combined with ALA or ALA-PTX NPs. Therefore, we suggested that MβCD combined with polyunsaturated fatty acid-conjugation would be an effective strategy for improving intracellular delivery of nanoparticulated chemotherapeutic drugs used for combination administration to enhance antitumor efficiency.