Inclusion Of Cholesterol Research Articles

How the designed processing parameters affect the liquid mixture density and viscosity of the tretinoin-loaded niosomes at different temperatures?

The current study was conducted to present novel thermophysical data on tretinoin-loaded niosomes paired with a combination of span 60 and tween 80. Measurements were carried out to analyze the liquid mixture density and viscosity of the mentioned multilayered structures for the first time, with consideration given to the diverse molecular weights of surfactants and various stabilizers at different temperatures. Through the application of equations of state, this study has the ability to set the stage for thermodynamic modeling of solutions that involve niosomes, presenting a promising avenue for further research. So, tretinoin-loaded formulations were prepared by investigating the effects of different co-surfactants, including cholesterol or dodecanol, as well as the impact of surfactant molecular weight limited to 650.525–1090.175 g mol−1. This novel investigation was conducted to assess the superior stabilizing capabilities of dodecanol in comparison to cholesterol, with a specific emphasis on optimized vesicle size, highest incorporation efficiency, and lowest zeta potential. In particulars, the response surface methodology (RSM) was applied to optimize the operative factors and the number of experiments. The experimental evidence clearly indicates that the use of dodecanol in the manufacturing process significantly improves the stability of niosomes, while the inclusion of cholesterol leads to higher liquid mixture density and viscosity in the prepared niosomes.

Insight into molecular structures and dynamical properties of niosome bilayers containing melatonin molecules: a molecular dynamics simulation approach.

Niosomes represent vesicular carriers capable of encapsulating both hydrophobic and hydrophilic drugs within their inner core or bilayer shell. They are typically composed of non-ionic synthetic surfactants such as sorbitan monostearate (Span60) with the addition of cholesterol (Chol). The physical properties and stability of niosomal vesicles strongly depend on the composition of their bilayers, which plays a significant role in determining the efficiency of drug encapsulation and release in drug delivery systems. In this study, we have explored the interactions between melatonin (Mel) molecules and the niosome bilayer, as well as their resulting physical properties. Molecular dynamics simulations were employed to investigate melatonin-inserted niosome bilayers, both with and without the inclusion of cholesterol. The simulation results revealed that cholesterol notably influences the location of melatonin molecules within the niosome bilayers. In the absence of cholesterol, melatonin tends to occupy the region around the Span60 tail groups. However, in the presence of cholesterol, melatonin is found in the vicinity of the Span60 head groups. Melatonin molecules in niosome bilayers without cholesterol exhibit a more ordered orientation when compared to those in bilayers containing 50 mol% cholesterol. The bilayer structure of the Span60/Mel and Span60/Chol/Mel systems exhibited a liquid-disordered phase (Ld). In contrast, the Span60/Chol bilayer system displays a liquid-ordered phase (Lo) with less fluidity. This study reveals that melatonin induces a disorderly bilayer structure and greater lateral expansion, whereas cholesterol induces an orderly bilayer structure and a more condensed effect. Cholesterol plays a crucial role in condensing the bilayer structure with stronger interactions between Span60 and cholesterol. The addition of 50 mol% cholesterol in the Span60 bilayers not only enhances the stability and rigidity of niosomes but also facilitates the easier release of melatonin from the bilayer membranes. This finding is particularly valuable in the context of preparing niosomes for drug delivery systems.

Development of Robust Cationic Light-Activated Thermosensitive Liposomes: Choosing the Right Lipids.

Extensive research has been conducted on cationic light-activated thermosensitive liposomes (CLTSLs) as a means for site-specific and controlled drug release; however, less attention has been given to the stability of these nanoparticles. Selecting the appropriate lipids is crucial for the development of a stable and responsive system. In this study, we investigated the impact of various lipids on the physical properties of cationic light-activated liposomes. Incorporating poly(ethylene glycol) PEG molecules resulted in uniform liposomes with low polydispersity index, while the addition of unsaturated lipid (DOTAP) resulted in extremely leaky liposomes, with almost 80% release in just 10 min of incubation at body temperature. Conversely, the inclusion of cholesterol in the formulation increased liposome stability too much and decreased their sensitivity to stimuli-responsive release, with only 14% release after 2 min of light exposure. To achieve stable and functional CLTSL, we substituted an equivalent amount of unsaturated lipid with a saturated lipid (DPTAP), resulting in stable liposomes at body temperature that were highly responsive to light, releasing 90% of their content in 10 s of light exposure. We also conducted two atomistic molecular dynamics simulations using lipid compositions with saturated and unsaturated lipids to investigate the effect of lipid composition on the dynamical properties of the liposomal lipid bilayer. Our findings suggest that the nature of lipids used to prepare liposomes significantly affects their properties, especially when the drug loading needs to be stable but triggered drug release properties are required at the same time. Selecting the appropriate lipids in the right amount is therefore essential for the preparation of liposomes with desirable properties.

Phytosterol-mediated glycerosomes combined with peppermint oil enhance transdermal delivery of lappaconitine by modulating the lipid composition of the stratum corneum.

Although the introduction of glycerosomes has enriched strategies for efficient transdermal drug delivery, the inclusion of cholesterol as a membrane stabilizer has limited their clinical application. The current study describes the development and optimization of a new type of glycerosome (S-glycerosome) that is formed in glycerol solution with β-sitosterol as the stabilizer. Moreover, the transdermal permeation properties of lappaconitine (LA)-loaded S-glycerosomes and peppermint oil (PO)-mediated S-glycerosomes (PO-S-glycerosomes) are evaluated, and the lipid alterations in the stratum corneum are analyzed via lipidomics. The LA-loaded S-glycerosomes prepared by the preferred formulation from the uniform design have a mean size of 145.3 ± 7.81nm and an encapsulation efficiency of 73.14 ± 0.35%. Moreover, the addition of PO positively impacts transdermal flux, peaking at 0.4% (w/v) PO. Tracing of the fluorescent probe P4 further revealed that PO-S-glycerosomes penetrate deeper into the skin than S-glycerosomes and conventional liposomes. Additionally, treatment with PO-S-glycerosomes alters the isoform type, number, and composition of sphingolipids, glycerophospholipids, glycerolipids, and fatty acids in the stratum corneum, with the most notable effect observed for ceramides, the main component of sphingolipids. Furthermore, the transdermal administration of LA-loaded PO-S-glycerosomes improved the treatment efficacy of xylene-induced inflammation in mice without skin irritation. Collectively, these findings demonstrate the feasibility of β-sitosterol as a stabilizer in glycerosomes. Additionally, the inclusion of PO improves the transdermal permeation of S-glycerosomes, potentially by altering the stratum corneum lipids.

Abstract 829: Evaluation of SK-N-DZ CLENs as a potential drug delivery system for selective targeting of neuroblastoma cells

Abstract Background: Neuroblastoma (NB) is a malignancy of the neuroendocrine system affecting infants. NB is highly resistant to drug therapy, which leads to ineffective treatments and increased refractory. Current treatments have low specificity to targeted NB cells resulting in unwanted side effects. A drug delivery system that can overcome these problems is urgently needed. Incorporating drugs in optimized lipid nanoparticles such as liposomes have proven to be an effective method for drug targeting. To achieve better cellular selectivity, lipid membrane extract (LE) is derived from NB cells using a modified Dyer and Blight lipid extraction procedure. The objective of the study was to determine an optimized cell membrane lipid-extracted nanoliposomes (CLENs) for the NB cell model while considering physicochemical properties (i.e. size and molecular charge) and composition of the preparation. Methods: The neuroblastoma cell line, SK-N-DZ, was used for drug selectivity study. Expanded culture of SK-N-DZ was employed for cellular extraction purposes. The method for lipid membrane extraction was previously reported by Alharbi and Campbell, (AAPS Open 4,5(2018)). SK-N-DZ lipid extract was the main ingredient to prepare SK-N-DZ CLENs. Phase I of the study is to prepare CLENs with various compositions of DOPC, cholesterol, DPPE-PEG5000 and SK-N-DZ lipid extract. DPPE-Rhodamine was used for fluorescence and cellular uptake studies. Phase II of the study evaluated the effect of SK-N-DZ lipid extraction inclusion on drug incorporation, cellular uptake and selectivity, and cytotoxicity. Invitrogen alamarBlue HS in vitro assay was used to determine cell viability for cytotoxicity studies. SK-N-SH (brain tissue), BT-20 (breast tissue) and G401 (kidney tissue) cell lines served as negative controls. Results: The results from the cellular uptake optimization studies revealed that the optimized composition and ratio for SK-N-DZ CLENs was DOPC/Cholesterol/LE/PEG (65/10/20/5). The average particle size and value for zeta potential was 164 nm +/- 1.2 and -7.92 +/- 1.55 mV, respectively. The inclusion of SK-N-DZ lipid membrane extract (2, 10, 20 and 40 mol%) in preparation of CLENs increased cellular uptake by SK-N-DZ cells compared to corresponding control (no LE included). No difference observed in cellular uptake of preparations containing 20 and 40 mol% of LE. The additional inclusion of cholesterol (10, 20, 40 mol%) increased overall cellular uptake of preparations compared to CLENs without the additional cholesterol content. Overall, the inclusion of PEG (5, 10, and 20 mol%) in SK-N-DZ CLENs increased their uptake by the target cell population. Conclusion: In conclusion, the inclusion of SK-N-DZ lipid membrane extract in the nanoliposome preparations increased their overall cellular uptake by SK-N-DZ target cells. Additional cellular selectively and cytotoxicity studies are currently underway. Citation Format: Kevin Bumanglag, Thi Tran, Robert Campbell, Yishan Li. Evaluation of SK-N-DZ CLENs as a potential drug delivery system for selective targeting of neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 829.

Cholesterol content regulates silica-induced lysosomal membrane permeability.

Inhalation of crystalline silica has been well documented to cause pulmonary inflammation and lung disease such as silicosis. Respirable silica particles deposit in the lungs and are phagocytosed by alveolar macrophages. Subsequently, phagocytosed silica remains undegraded within lysosomes causing lysosomal damage known as phagolysosomal membrane permeability (LMP). LMP can trigger the assembly of the NLRP3 inflammasome resulting in release of inflammatory cytokines that contribute to disease. In order to better understand the mechanisms of LMP this study used murine bone marrow derived macrophages (BMdM) as a cellular model to investigate the mechanism of silica-induced LMP. Reduction of lysosomal cholesterol in bone marrow derived macrophages with 18:1 phosphatidylglycerol (DOPG) liposome treatment increased silica-induced LMP and IL-1β release. Conversely, increasing lysosomal and cellular cholesterol with U18666A reduced IL-1β release. Co-treatment of bone marrow derived macrophages with 18:1 phosphatidylglycerol and U18666A resulted in a significant reduction of the effects of U18666A on lysosomal cholesterol. Phosphatidylcholine 100-nm liposome model systems were used to examine the effects of silica particles on lipid membrane order. Time-resolved fluorescence anisotropy of the membrane probe, Di-4-ANEPPDHQ, was used to determine changes to membrane order. Silica increased lipid order that was attenuated by inclusion of cholesterol in the phosphatidylcholine liposomes. These results demonstrate that increased cholesterol can attenuate silica-induced membrane changes in liposomes and cell models, while decreasing cholesterol exacerbates silica-induced membrane changes. Selective manipulation of lysosomal cholesterol may be a way of attenuating lysosomal disruption and preventing silica-induced chronic inflammatory disease progression.

IMMU-41. VERSATILE MRNA-NANOPARTICLE PLATFORM TO REPROGRAM THE BRAIN TUMOR MICROENVIRONMENT AND PREDICT TREATMENT OUTCOME

Abstract RNA vaccines have shown great promise as activators of immune responses against viral pathogens but their efficacy in cancer is unclear. Here, we describe a versatile, personalized mRNA-nanoparticle (RNA-NP) platform that can be customized to produce powerful anti-tumor immune activation, reprogram the brain tumor microenvironment, and predict vaccine efficacy just two days after treatment. Lipid mixtures (i.e. DOTAP, Cholesterol) with or without iron oxide nanoparticle-cores were complexed with mRNA encoding tumor antigens. Intravenous administration of RNA-NPs induced robust activation of innate immune cells resulting in prolonged survival in murine models of subcutaneous and intracranial melanoma. Inclusion of cholesterol in the lipid backbone enabled delivery of nucleic acids across the blood brain barrier and into tumor-associated myeloid cells in intracranial GL261 and KR158b tumors. These cholesterol-bearing liposomes not only activated innate immune cells in the tumor microenvironment (i.e. increased expression of CD80 and MHCII), but also enabled further manipulation of this compartment. Use of RNA-NPs to deliver siRNA targeting PD-L1 resulted in significant reduction in PD-L1 expression among tumor associated myeloid cells, leading to 37% long term survivorship in combination with systemic checkpoint blockade in an otherwise fatal model of GL261. We have also shown that IONPs incorporated into the cores of these particles enable non-invasive tracking of dendritic cells by MRI, enabling creation of a theranostic approach to: 1) enhance immunologic effects; 2) facilitate translocation across the blood-brain barrier; and 3) enable non-invasive imaging to predict response to RNA-NPs.

Determination of the boundary lipids of sticholysins using tryptophan quenching

Sticholysins are α-pore-forming toxins produced by the sea-anemone Stichodactyla helianthus. These toxins exert their activity by forming pores on sphingomyelin-containing membranes. Recognition of sphingomyelin by sticholysins is required to start the process of pore formation. Sphingomyelin recognition is coupled with membrane binding and followed by membrane penetration and oligomerization. Many features of these processes are known. However, the extent of contact with each of the different kinds of lipids present in the membrane has received little attention. To delve into this question, we have used a phosphatidylcholine analogue labeled at one of its acyl chains with a doxyl moiety, a known quencher of tryptophan emission. Here we present evidence for the contact of sticholysins with phosphatidylcholine lipids in the sticholysin oligomer, and for how each sticholysin isotoxin is affected differently by the inclusion of cholesterol in the membrane. Furthermore, using phosphatidylcholine analogs that were labeled at different positions of their structure (acyl chains and headgroup) in combination with a variety of sticholysin mutants, we also investigated the depth of the tryptophan residues of sticholysins in the bilayer. Our results indicate that the position of the tryptophan residues relative to the membrane normal is deeper when cholesterol is absent from the membrane.

Lipid-peptide nanocomplexes for mRNA delivery in vitro and in vivo.

Despite recent advances in the field of mRNA therapy, the lack of safe and efficacious delivery vehicles with pharmaceutically developable properties remains a major limitation. Here, we describe the systematic optimisation of lipid-peptide nanocomplexes for the delivery of mRNA in two murine cancer cell types, B16-F10 melanoma and CT26 colon carcinoma as well as NCI-H358 human lung bronchoalveolar cells. Different combinations of lipids and peptides were screened from an original lipid-peptide nanocomplex formulation for improved luciferase mRNA transfection in vitro by a multi-factorial screening approach. This led to the identification of key structural elements within the nanocomplex associated with substantial improvements in mRNA transfection efficiency included alkyl tail length of the cationic lipid, the fusogenic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. The peptide component (K16GACYGLPHKFCG) was further improved by the inclusion of a linker, RVRR, that is cleavable by the endosomal enzymes cathepsin B and furin, and a hydrophobic motif (X-S-X) between the mRNA packaging (K16) and receptor targeting domains (CYGLPHKFCG). Nanocomplex transfections of a murine B16-F10 melanoma tumour supported the inclusion of cholesterol for optimal transfection in vivo as well as in vitro. In vitro transfections were also performed with mRNA encoding interleukin-15 as a potential immunotherapy agent and again, the optimised formulation with the key structural elements demonstrated significantly higher expression than the original formulation. Physicochemical characterisation of the nanocomplexes over time indicated that the optimal formulation retained biophysical properties such as size, charge and mRNA complexation efficiency for 14days upon storage at 4°C without the need for additional stabilising agents. In summary, we have developed an efficacious lipid-peptide nanocomplex with promising pharmaceutical development properties for the delivery of therapeutic mRNA.

Lactobacilli spp. and Zataria multiflora essence as antibiotic substituent on broiler health and performance parameters

This experiment was conducted to evaluate the effects of commercial probiotics (Lactobacillus spp.) (LS) with Zataria multiflora essential oil (ZEO) and an antibiotic growth promoter (AGP) on broiler health and performance. In experiment 1, 300 one-day-old chicks (Ross 308) were assigned to one of 4 treatments, comprising five replicates per treatment in a completely randomised design. Dietary treatments included: basal diet (control), 0.25 and 0.50 g/kg LS, or 0.50 g/kg Flavomycine added to basal diet. In experiment 2, 225 one-day-old chicks, were assigned to one of 3 treatments, comprising five replicates per treatment in a completely randomised design Dietary treatments included: basal diet (control), 0.50 g/kg LS, or 1 g/kg ZEO added to the basal diet. The inclusion of LS increased body weight gain significantly (P ≤ .05) at 42 days of age. Both AGP and LS reduced feed intake and feed conversion ratio compared to the control diet (P ≤ .05). The lowest triglyceride, cholesterol, LDL and ALT were observed in chicks administered by LS compare to control and AGP (P ≤ .05). The inclusion of LSdecreased triglyceride, cholesterol, LDL and ALT, and increased immune titre against IBD and IB compare to control and AGP (P ≤ .05), and Intestinal health index were increased in LS group compared to control and AGP. The addition of ZEO, decreased LDL and ALT, and increased the immune titre against AI, ND and IBD compared to LS significantly (P ≤ .001). The results showed that usage of LS (0.5 g/kg) population of salmonella and coliform bacteria were greatly reduced, while in ZEO fed birds, E. coli population of caeca was reduced considerably (P ≤ .001). These results confirmed that LS and ZEO have effective on broiler chicken and can be considered a potent antibiotic replacer. HIGHLIGHTS Use of antibiotic growth promoters (AGP) in poultry diets, has been banned worldwide. To control and eliminate of intestinal pathogens, and increase body weight and improve FCR, alternative procedures as adding phytogenic compounds and probiotics in the poultry diet. Lactobacilli spores and Zataria multiflora essence can decrease the microbial population of the caecum, and increase the intestinal health index.

Versatile mRNA-Nanoparticle Platform for Treatment of Glioblastoma

Purpose/Objective(s)Glioblastoma (GBM) is difficult to treat due to isolation behind the blood brain barrier and a notoriously immune-suppressive tumor microenvironment. RNA vaccines have recently shown great promise as activators of immune responses against viral pathogens but their efficacy in cancer is not well understood. Here, we describe a versatile, personalized mRNA-nanoparticle (RNA-NP) platform that can be customized to produce powerful anti-tumor immune activation, reprogram the brain tumor microenvironment, and enable tracking of particle localization to predict vaccine efficacy just two days after treatment.Materials/MethodsLipid mixtures (i.e., DOTAP, cholesterol) were complexed with mRNA encoding tumor antigens and evaluated for their ability to activate anti-tumor immune responses in vitro and in vivo. Particle localization after intravenous injection was assessed to identify constructs that enabled delivery of nucleic acids to intracranial tumors. Iron oxide nanoparticles (IONPs) were then incorporated into the cores of liposomes to enable particle tracking with MRI.ResultsIntravenous administration of RNA-NPs composed of DOTAP and RNA induced robust activation of innate immune cells resulting in prolonged survival in murine models of subcutaneous and intracranial melanoma. Inclusion of cholesterol in the lipid backbone enabled delivery of nucleic acids across the blood brain barrier and into tumor-associated myeloid cells in intracranial GL261 and KR158b tumors. These cholesterol-bearing liposomes not only activated innate immune cells in the tumor microenvironment (i.e., increased expression of CD80 and MHCII), but also enabled further manipulation of this compartment. Use of RNA-NPs to deliver siRNA targeting PD-L1 resulted in significant reduction in PD-L1 expression among tumor associated myeloid cells, leading to 37% long term survivorship in combination with systemic checkpoint blockade in an otherwise fatal model of GL261. IONPs were also incorporated into the cores of these particles to enable non-invasive tracking of particle localization with MRI. Preliminary experiments utilizing dendritic cells treated with iron-oxide-loaded RNA-NPs ex vivo demonstrate that a single dose of this therapy produces immunologic rejection of a murine melanoma model in which a dendritic cell vaccine currently in clinical trials yields no benefit. Furthermore, quantification of particle accumulation in lymph nodes with MRI correlates strongly with survival outcome (R =, indicating that tracking particle localization may be used to reliably differentiate responders from non-responders just two days after treatment.ConclusionRNA-NPs are a versatile platform for RNA delivery to immune cells. These particles are currently under investigation in canine trials with human clinical trials of our first-generation therapy beginning this year. Glioblastoma (GBM) is difficult to treat due to isolation behind the blood brain barrier and a notoriously immune-suppressive tumor microenvironment. RNA vaccines have recently shown great promise as activators of immune responses against viral pathogens but their efficacy in cancer is not well understood. Here, we describe a versatile, personalized mRNA-nanoparticle (RNA-NP) platform that can be customized to produce powerful anti-tumor immune activation, reprogram the brain tumor microenvironment, and enable tracking of particle localization to predict vaccine efficacy just two days after treatment. Lipid mixtures (i.e., DOTAP, cholesterol) were complexed with mRNA encoding tumor antigens and evaluated for their ability to activate anti-tumor immune responses in vitro and in vivo. Particle localization after intravenous injection was assessed to identify constructs that enabled delivery of nucleic acids to intracranial tumors. Iron oxide nanoparticles (IONPs) were then incorporated into the cores of liposomes to enable particle tracking with MRI. Intravenous administration of RNA-NPs composed of DOTAP and RNA induced robust activation of innate immune cells resulting in prolonged survival in murine models of subcutaneous and intracranial melanoma. Inclusion of cholesterol in the lipid backbone enabled delivery of nucleic acids across the blood brain barrier and into tumor-associated myeloid cells in intracranial GL261 and KR158b tumors. These cholesterol-bearing liposomes not only activated innate immune cells in the tumor microenvironment (i.e., increased expression of CD80 and MHCII), but also enabled further manipulation of this compartment. Use of RNA-NPs to deliver siRNA targeting PD-L1 resulted in significant reduction in PD-L1 expression among tumor associated myeloid cells, leading to 37% long term survivorship in combination with systemic checkpoint blockade in an otherwise fatal model of GL261. IONPs were also incorporated into the cores of these particles to enable non-invasive tracking of particle localization with MRI. Preliminary experiments utilizing dendritic cells treated with iron-oxide-loaded RNA-NPs ex vivo demonstrate that a single dose of this therapy produces immunologic rejection of a murine melanoma model in which a dendritic cell vaccine currently in clinical trials yields no benefit. Furthermore, quantification of particle accumulation in lymph nodes with MRI correlates strongly with survival outcome (R =, indicating that tracking particle localization may be used to reliably differentiate responders from non-responders just two days after treatment. RNA-NPs are a versatile platform for RNA delivery to immune cells. These particles are currently under investigation in canine trials with human clinical trials of our first-generation therapy beginning this year.

Preparation of Asymmetric Vesicles with Trapped CsCl Avoids Osmotic Imbalance, Non-Physiological External Solutions, and Minimizes Leakage.

The natural asymmetry of cellular membranes influences their properties. In recent years, methodologies for preparing asymmetric vesicles have been developed that rely on cyclodextrin-catalyzed exchange of lipids between donor lipid multilamellar vesicles and acceptor lipid unilamellar vesicles, and the subsequent separation of the, now asymmetric, acceptor vesicles from the donors. Isolation is often accomplished by preloading acceptor vesicles with a high concentration of sucrose, typically 25% (w/w), and separating from donor and cyclodextrin by sucrose gradient centrifugation. We found that when the asymmetric vesicles prepared using methyl-α-cyclodextrin exchange were dispersed under hypotonic conditions using physiological salt solutions, there was enhanced leakage of an entrapped probe, 6-carboxyfluorescein. Studies with symmetric vesicles showed this was due to osmotic pressure and was specific to hypotonic solutions. Inclusion of cholesterol partly reduced leakage but did not completely eliminate it. To avoid having to use hypotonic conditions or to suspend vesicles at nonphysiological solute concentrations to minimize leakage, a method for preparing asymmetric vesicles using acceptor vesicle-entrapped CsCl at a physiological ion concentration (100 mM) was developed. Asymmetric vesicles prepared with the entrapped CsCl protocol were highly resistant to 6-carboxyfluorescein leakage out of the vesicles.

Abstract 1322: Evaluating intracellular accumulation profiles of chemotherapeutic agents using CLENs for the treatment of Y79 retinoblastoma

Abstract Retinoblastoma is the most common intraocular malignancy in children, with approximately 200-300 new cases in the United States each year. Currently, conventional chemotherapy agents (carboplatin, cisplatin, doxorubicin, vincristine, and cyclophosphamide) used to treat retinoblastoma, are rapidly eliminated from circulation. Novel lipid nanoparticles (LNP) have been shown to improve delivery of encapsulated chemotherapeutic agents to Y79 retinoblastoma cells, and therefore the agents are less toxic to healthy cells. Past studies have supported the use of LNP delivery systems to enhance chemotherapeutic drug action against Y79 retinoblastoma cells as well. The experimental plan will now evaluate the use of cellular membrane lipid-extracted nanoliposomes (CLENs) to examine targeting using cellular models of retinoblastoma. Y79 cells were cultured in RPMI growth medium supplemented by 20% FBS. CLENs and lipid extracts (LE) were prepared according to previously published protocols. Following the development of Y79-LE nano-preparations consisting of DOPC, Y79-LE, and cholesterol at different ratios, preparations were evaluated in terms of physicochemical properties and uptake by Y79 cells. The additional inclusion of cationic lipid DOTAP in Y79-LE preparations was evaluated for select preparations. Particle sizes and zeta potential values were determined for each preparation type by ZetaPALS. CLENs exhibited an average particle size between 400 to 600 nm. Experiments with Y79-CLENs prepared in the absence and presence of DOTAP, showed they were taken up by the target cell population. In general, the inclusion of Y79-LE increased cellular uptake. The inclusion of cholesterol in combination with DOTAP showed enhanced liposomal uptake in Y79 retinoblastoma cells. Evidence from previous studies suggest a benefit of using lipid nanoparticles compared to more conventional treatment options. Preliminary findings suggest a positive correlation between the particle size and degree of cellular uptake. Additional optimization studies are currently underway. Citation Format: Nan Luo, Virali Patel, Minesh Patel, Eric Conte, Robert Campbell. Evaluating intracellular accumulation profiles of chemotherapeutic agents using CLENs for the treatment of Y79 retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1322.

Simulation Study of Breast Cancer Lipid Changes Affecting Membrane Oxygen Permeability: Effects of Chain Length and Cholesterol.

Tumor radiotherapy relies on intracellular oxygen (O2) to generate reactive species that trigger cell death, yet hypoxia is common in cancers of the breast. De novo lipid synthesis in tumors supports cell proliferation but also may lead to unusually high levels of the 16:1 palmitoleoyl (Y) phospholipid tail, which is two carbons shorter than the 18:1 oleoyl (O) tail abundant in normal breast tissue. Here, we use atomic resolution molecular dynamics simulations to test two hypotheses: (1) the shorter, 16:1 Y, tail of the de novo lipid biosynthesis product 1-palmitoyl,2-palmitoleoyl-phosphatidylcholine (PYPC) promotes lower membrane permeability relative to the more common lipid 1-palmitoyl,2-oleoylphosphatidylcholine (POPC), by reducing oxygen solubility in the interleaflet region, and (2) cholesterol further lessens the permeability of PYPC by reducing overall O2 solubility and promoting PYPC tail order adjacent to the rigid cholesterol ring system. The simulationsconducted here indicate that PYPC has a permeability of 14±1cm/s at 37°C, comparable to 15.4±0.4cm/s for POPC. Inclusion of cholesterol in a 1:1 ratio with phospholipid intensifies the effect of chain length, giving permeabilities of 10.2±0.2cm/s for PYPC/cholesterol and 11.0±0.6cm/s for POPC/cholesterol. These findings indicate that PYPC maynot substantiallyinfluence membrane-level oxygen flux and is unlikely to hinder breast tissue oxygenation.

Evaluating a clinical tool (FAMCAT) for identifying familial hypercholesterolaemia in primary care: a retrospective cohort study.

BackgroundFamilial hypercholesterolaemia (FH) is an inherited lipid disorder causing premature heart disease, which is severely underdiagnosed. Improving the identification of people with FH in primary care settings would help to reduce avoidable heart attacks and early deaths.AimTo evaluate the accuracy of the familial hypercholesterolaemia case ascertainment identifcation tool (FAMCAT) for identifying FH in primary care.Design & settingA retrospective cohort study of 1 030 183 patients was undertaken. Data were extracted from the UK Royal College of General Practitioners (RCGP) Research and Surveillance Centre (RSC) database. Patient were aged >16 years.MethodThe FAMCAT algorithm was compared with methods of FH detection recommended by national guidelines (Simon Broome diagnostic criteria, Dutch Lipid Clinic Network [DLCN] Score, and cholesterol levels >99th centile). Discrimination and calibration were assessed by area under the receiver operating curve (AUC) and by comparing observed versus predicted cases.ResultsA total of 1707 patients had a diagnosis of FH. FAMCAT showed a high level of discrimination (AUC = 0.844, 95% confidence interval [CI] = 0.834 to 0.854), performing significantly better than Simon Broome criteria (AUC = 0.730, 95% CI = 0.719 to 0.741), DLCN Score (AUC = 0.766, 95% CI = 0.755 to 0.778), and screening cholesterols >99 th centile (AUC = 0.579, 95% CI = 0.571 to 0.588). Inclusion of premature myocardial infarction (MI) and fitting cholesterol as a continuous variable improved the accuracy of FAMCAT (AUC = 0.894, 95% CI = 0.885 to 0.903).ConclusionBetter performance of the FAMCAT algorithm, compared with other approaches for case finding of FH in primary care, such as Simon Broome criteria, DLCN criteria or very high cholesterol levels, has been confirmed in a large population cohort.

Abstract 1151: Inclusion of cholesterol and A549 lipid extract in CLENs improved cellular uptake in adenocarcinoma non-small cell lung cancer target cells

Abstract Purpose:The purpose of this investigation was to determine the optimal amount of cholesterol and A549-derived lipid extract (LE) included in the initial design and development of A-549-cell membrane lipid extracted nanoliposomes (A549-CLENs) for cell targeting studies. Methods and Materials:A549 (ATCC® CCL-185™), used as a positive control and NCI-H1703 (ATCC® CRL-5889™), used as a negative control, and respective growth media were purchased from ATCC (Manassas, VA). A549-GFP (green fluorescent protein) cell line was purchased from AntiCancer, Inc. DOPC, DOTAP, cholesterol, and DSPE-PEG5000 were obtained from Avanti Polar Lipids (Alabastar, AL). Cellular membrane lipids of A549 cells were extracted by modified Bligh & Dyer method. CLENs were fluorescently labeled with rhodamine-B. Particle sizes and zeta potentials were performed using ZetaPALS (Brookhaven Instruments Corporation, Holtsville, NY, USA). For cellular uptake studies cells were seeded at 2.0 x 104 cells in a 48-well plate followed by incubation for 24 hours. A fluorescence microplate reader and/or fluorescence microscopy were used to evaluate cellular uptake as a function of relative amount of cholesterol and A549-lipid extract inclusion in CLENs. Results:In general, an increase in the inclusion of cholesterol reduced the particle size of CLENs. Amount of 10 mol% of cholesterol content in CLENs demonstrated the smallest mean size diameters and a zeta potential value recorded at 243 ± 4.4 nm and 1 ± 2.61 mV, respectively. The finding correlated also with a relatively high degree of cellular uptake of the preparation type for part I of the physicochemical characterization studies. We next maintained 10 mol% cholesterol content consistent in all future preparations and increased the total A-549 lipid extract material at the expense of DOPC. In general, the mean diameter and zeta potential for A549-CLENs were 246 ± 3.8 nm and 20.4 ± 0.84 mV, respectively. Approximately 10-15 mol% of A-549 lipid extract inclusion resulted in a consistently high degree of overall cellular uptake by GFP-A549 and A549 target cells compared to control and the non-target cell population, NCI-H1703. Conclusion: Preparation of A549-CLENs consisting of DOPC/ DOTAP/ Chol/ LE/PEG (65/10/10/10/5) established the greatest increase in cellular uptake and overall selectivity. Future studies will consider antibody conjugation and development of tumor-specific biomarkers for serodiagnostic detection and lung cancer treatment. Citation Format: Maxime Langlois, Guo-Yun Kao, Diana Lasker. Inclusion of cholesterol and A549 lipid extract in CLENs improved cellular uptake in adenocarcinoma non-small cell lung cancer target cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1151.

Prevalence of multiple chronic conditions by U.S. state and territory, 2017.

Having multiple (two or more) chronic conditions (MCC) is associated with an increased risk of mortality and functional decline, health resource utilization, and healthcare expenditures. As a result, understanding the prevalence of MCC is increasingly being recognized as a public health imperative. This research describes the prevalence and distribution of adults with MCC across the United States using 2017 data from the Behavioral Risk Factors Surveillance System (BRFSS). Prevalence of MCC was calculated for each U.S. state and territory overall, by sex and by age. Additionally, the most common condition dyads (two condition combinations) and triads (three condition combinations) were assessed for each state. Prevalence of MCC ranged from 37.9% in the District of Columbia to 64.4% in West Virginia. Females had a higher prevalence than males in 47 of 53 states and territories, and MCC prevalence increased with age in every state and territory. Overall prevalence estimates were higher than estimates using data from the National Health Interview Survey (NHIS), especially in the younger population (aged 18–44), due partly to the inclusion of high cholesterol, obesity, and depression as chronic conditions. Analysis of the most prevalent dyads and triads revealed the greatest state-by-state variability in the 18-44-year-old population. Multiple states’ most prevalent dyads and triads for this population included obesity and depression. These findings build an accurate picture of the prevalence of multiple chronic conditions across the United States and will aid public health officials in creating programs targeted to their region.

Niosomes: a promising drug delivery system

Targeting is a phenomenon in which the distribution of a drug in the body occurs in such a way that the main part of it interacts with the target tissue at the cellular or subcellular level to achieve the desired pharmacological effect on the selected site without undesirable interactions in other organs. This can be achieved using a drug delivery system such as niosomes, which are non-ionic vesicles of surfactants obtained by hydrating synthetic non-ionic surfactants with the inclusion of cholesterol. They are vestibular systems similar to liposomes that can be used as carriers of amphiphilic or lipophilic drugs. Niosomes are a promising drug delivery tool, and it has been widely evaluated as a possibility of controlled release and targeted delivery of the active substance for the treatment of cancer, autoimmune diseases, viral and other infectious diseases. It can be assumed that encapsulation of the drug in the vesicular system prolongs its presence in the systemic circulation and increases the possibility of penetration into the target tissue, possibly reducing toxicity if selective absorption can be achieved.

Hierarchy of interactions dictating the thermodynamics of real cell membranes: Following the insulin secretory granules paradigm up to fifteen-components vesicles

A fifteen-components model membrane that reflected the 80 % of phospholipids present in Insulin Secretory Granules was obtained and thermodynamic exploitation was performed, through micro-DSC, in order to assess the synergic contributions to the stability of a mixed complex system very close to real membranes. Simpler systems were also stepwise investigated, to complete a previous preliminary study and to highlight a hierarchy of interactions that can be now summarized as phospholipid tail unsaturation > phospholipid tail length > phospholipid headgroup > membrane curvature. In particular, Small Unilamellar Vesicles (SUVs) that consisted in phospholipids with different headgroups (choline, ethanolamine and serine), was step by step considered, following inclusion of sphingomyelins and lysophosphatidylcholines together with a more complete fatty acids distribution characterizing the phospholipid bilayer of the Insulin Secretory Granules. The inclusion of cholesterol was finally considered and the influence of three FFAs (stearic, oleic and elaidic acids) was investigated in comparison with simpler systems, highlighting the magnitude of the effects on such a detailed membrane in the frame of Type 2 Diabetes Mellitus alterations.

Do Cholesterol and Sphingomyelin Change the Mechanism of Aβ25-35 Peptide Binding to Zwitterionic Bilayer?

Using replica exchange with solute tempering all-atom molecular dynamics, we studied the equilibrium binding of Aβ25-35 peptide to the ternary bilayer composed of an equimolar mixture of dimyristoylphosphatidylcholine (DMPC), N-palmitoylsphingomyelin (PSM), and cholesterol. Binding of the same peptide to the pure DMPC bilayer served as a control. Due to significant C-terminal hydrophobic moment, binding to the ternary and DMPC bilayers promotes helical structure in the peptide. For both bilayers a polarized binding profile is observed, in which the N-terminus anchors to the bilayer surface, whereas the C-terminus alternates between unbound and inserted states. Both ternary and DMPC bilayers feature two Aβ25-35 bound states, surface bound, S, and inserted, I, separated by modest free energy barriers. Experimental data are in agreement with our results but indicate that cholesterol impact is Aβ fragment dependent. For Aβ25-35, we predict that its binding mechanism is independent of the inclusion of PSM and cholesterol into the bilayer.