Liposomal Bilayer Research Articles

Didodecyldimethylammonium Bromide Role in Anchoring Gold Nanoparticles onto Liposome Surface for Triggering the Drug Release.

Liposomes with their capacity to anchor gold nanoparticles (AuNPs) onto their surface are used in the treatment of several pathologies such as cancer. The objective of this work was the optimization of the vesicle composition by using cationic agents in order to reinforce the anchoring process of AuNPs, and for the study of the influence of local temperature and vesicle size on drug release. A Plackett-Burman design was conducted to determine the optimal composition for the anchoring of AuNPs. A comprehensive study of the influence of lipid bilayer composition on the surface charge, size, and polydispersity index (PdI) of liposomes was carried out. Afterwards, in vitro release studies by dialysis were performed and several release parameters were evaluated as a function of temperature. Cholesterol was fixed as the rigid agent and Didodecyldimethylammonium bromide (DDAB) was selected as the cationic lipid into the liposome bilayer. Photomicrographs revealed that DDAB facilitated the anchoring of AuNPs onto the liposomal surface. The anchoring of AuNPs also enhanced the amount and rate of calcein released, especially in extruded samples, at several incubating temperatures. In addition, it was observed that both the anchoring of AuNPs and the calcein release were improved by increasing the surface of the vesicles. The contributions of liposome composition (DDAB inclusion, incubation temperature, anchoring of AuNPs) and size and surface availability of the vesicles on calcein release could be used to design improved lipid nanostructures for the controlled release of anticancer drugs.

Evaluation of the effect of liposomes loaded with chlorogenic acid in treatment of 2,4,6-trinitrobenzenesulfonic acid-induced murine colitis.

Crohn's Disease (CD), one of the types of inflammatory bowel disease, poses a significant challenge to modern healthcare. This condition severely impacts patients' quality of life, and its incidence is continuously rising. Despite constant research, current treatment options are limited and largely unsuccessful and result in serious side effects, therefore new therapy alternatives are needed. Liposomal formulation provides a new hope for disease management. In our study, we characterized the anti-inflammatory activity of mesalazine (5-ASA) and chlorogenic acid (CGA) encapsulated in liposomal formulation in the animal model of CD. Liposomes were obtained by thin film hydration method and characterized in terms of suspension stability and particle size and distribution. Colitis was induced in mice by intracolonic (i.c.) administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS). The effect of treatment with liposomal suspensions of 5-ASA and CGA was evaluated macroscopically and by measuring myeloperoxidase (MPO) activity. We observed that liposome-encapsulated 5-ASA (5 mg/kg), but not CGA (20 mg/kg) attenuated colitis as evidenced by a decreased macroscopic and microscopic scores. It may be hypothesized that the composition of liposomal lipid bilayer as well as the switch in macrophage populations leading to unfavorable accumulation of anti-inflammatory agents in the cells may underly the efficiency of obtained liposomes and need to be taken into consideration in further studies on drug delivery.

Photoacoustic effect of azo derivatives modified by click reagents and parceled by liposomes

Photoacoustic (PA) imaging has been highlighted as one of the latest and the most promising biophotonic diagnostic modalities. As promising contrast agents, near-infrared (NIR) light-absorbing agents are highly in great demand. Here, the PA and photothermal (PT) effects of a series of NIR absorbing azo derivatives were characterized. Interestingly, compared with tetracyanoethylene (TCNE) and 7,7,8,8-tetracyanoquiodimethane (TCNQ), the click reagent 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquiodimethane (F4-TCNQ) caused the red shift to near infrared (NIR) region for the peaks of PA intensity of molecules. Moreover, symmetrical molecular structure of NIR dyes had better PA effect. The M2a (clicked by TCNQ) molecules were embedded into hydrophobic phospholipid bilayer of liposomes (M2a⊂L) and internalized into human breast cancer Michigan Cancer Foundation-7 (MCF-7) cell. The toxicity of the cell was low, because the cell viability reached amost 100%, which was evaluated through CCK-8 assays to determine the metabolic viability of MCF-7 cells, while the cell PA intensity was high (1.5 × 103). The systematical research of the NIR dyes not only provided a series promising NIR dyes for imaging and treatment, but also guided the design of contrast agents and PT agents which had high PA and PT effects.

Molecular Adsorption and Transport at Liposome Surfaces Studied by Molecular Dynamics Simulations and Second Harmonic Generation Spectroscopy.

A fundamental understanding of the factors that determine the interactions with and transport of small molecules through phospholipid membranes is crucial in developing liposome-based drug delivery systems. Here we combine time-dependent second harmonic generation (SHG) measurements with molecular dynamics simulations to elucidate the events associated with adsorption and transport of the small molecular cation, malachite green isothiocyanate (MGITC), in colloidal liposomes of different compositions. The molecular transport of MGITC through the liposome bilayer is found to be more rapid in 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPG and DOPS, respectively) liposomes, while the molecular transport is slower in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes. Interestingly, MGITC is observed to neither adsorb nor transport in trimethyl quinone-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (QPADOPE) liposomes due to shielding by the quinone group. The modified Langmuir adsorption isotherm model is used to determine the free energy of adsorption for MGITC, which is found to be less negative in DOPC than in DOPG and DOPS, caused by lower electrostatic interactions between the positively charged dye and the zwitterionic DOPC liposome surface. The results are compared to our previous investigations, which showed that malachite green (MG) adsorbs and transports in DOPG and DOPS liposomes but not in DOPC and QPADOPE liposomes. Molecular dynamics simulations are used to investigate the adsorption and transport properties of MG and MGITC in DOPC and DOPG liposomes using umbrella sampling to determine the free energy profiles and interfacial molecular orientations. Together, these time-resolved SHG studies and corresponding molecular dynamics simulations characterize the complicated chemical interactions at different lipid membranes to provide key molecular-level insights for potential drug delivery applications. The results also point toward understanding the role of chemical functional groups, in this case isothiocyanate, in controlling molecular adsorption at and transport through lipid bilayers.

Quantitative analysis of cholesterol oxidation products and desmosterol in parenteral liposomal pharmaceutical formulations.

Cholesterol is one of the major structural constituents in a liposomal bilayer. Cholesterol is susceptible to various reactions in the presence of oxygen, heat, light, certain metals, and radicals during manufacturing or storage, which may cause to generate cholesterol oxidation products (COPs). Herein, we report the development of a liquid chromatography-mass spectrometry based analytical method for screening and quantitating COPs present in liposomal parenteral pharmaceutical formulations (LPFs) from four different vendors. We detected and quantitated six COPs and desmosterol in LPFs, and desmosterol is an intermediate of cholesterol biosynthesis. 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-keto-cholesterol, and desmosterol were the major cholesterol-related impurities in LPFs. COPs were not detected in any of USP/NF grade cholesterol raw materials, implying that COPs were generated during liposome manufacturing and/or storage. This validated method presented here can be used to quantify cholesterol-related impurities present in liposomal pharmaceutical formulations to ensure the quality and the safety of liposomal pharmaceutical formulations.

Liposomal bupivacaine and novel local anesthetic formulations.

Novel preparations allowing for the extended duration of action of local anesthetics have many clinically relevant benefits. With regard to this, the development of liposomal bupivacaine has the potential to significantly impact patient care by improving perioperative pain control. The unique liposomal bilayer that encapsulates bupivacaine allows for a sustained release of local anesthetic for up to 72h after a single use and can significantly decrease postoperative opioid consumption. SABER-bupivacaine is another depot formulation that helps in sustained release of bupivacaine from an encapsulated bupivacaine in a biodegradable sucrose acetate isobutyrate biolayer. HTX-011 is an investigational extended-release local anesthetic formulation currently undergoing Phase 3 clinical trials. HTX-011 is composed of a bioerodible polymer with bupivacaine and low-dose meloxicam in which the polymer undergoes hydrolysis and allows for sustained release of bupivacaine and meloxicam for 3 days. The present investigation reviews pharmacologic considerations related to the formulation of liposomal bupivacaine, current FDA-approved indications for its use, and future extended-release local anesthetic formulations currently under investigation.

Investigation of PPIX-Lipo-MnO2 to enhance photodynamic therapy by improving tumor hypoxia.

The efficacy of photodynamic therapy (PDT) is reduced in the context of hypoxic environments. This is problematic, considering that hypoxia is exhibited in the vast majority of malignant tumors. Thus, increasing the concentration of oxygen in malignant tumors improves PDT treatment outcomes. Studies show that MnO2 nanoparticles can produce oxygen when it reacts with endogenous H2O2. Herein, we encapsulated Protoporphyrin IX (PPIX) in the liposome bilayer (PPIX-Lipo), which was then coated with MnO2 nanoparticles to construct PPIX-Lipo-MnO2 (PPIX-Lipo-M) in order to enhance PDT efficacy under tumor hypoxia. The PDT results show that PPIX-Lipo-M was more cytotoxic to breast cancer cells than PPIX-Lipo while under hypoxic conditions, indicating that the production of oxygen gas in hypoxic conditions improved treatment outcomes. Upon encapsulating PPIX into the liposome, the aqueous solubility of PPIX significantly improved. Consequently, the cellular uptake of both PPIX-Lipo and PPIX-Lipo-M also increased significantly compared to that of bare PPIX. Overall, PPIX-Lipo-M has the capacity to act as a therapeutic agent that relieves hypoxia and hence improve PDT efficacy.

MODEL BIOMEMBRANES AS TEST OBJECTS FOR THE DETERMINATION OF CONCENTRATION RANGES OF HARMFUL CHEMICAL SUBSTANCES IN BIOLOGICAL MEDIUMS AND OBJECTS OF EXTERNAL ENVIRONMENT

The paper presents data on changes in model biomembranes (liposomes, erythrocyte shadows, erythrocytes) used as test objects to determine those ranges of concentrations of biologically active substances in which there is no violation of the structure or function of experimental objects. Melaphene, plant growth regulator used in small doses in seed pre-treatment, and antioxidant phenosan derivatives, phenoxane and IHFANs, have been used as biologically active substances. It was shown by DSC that phenosan derivatives at concentrations equal to 10-5 M and higher destroy the microdomain organization in the bilayers of phospholipid multilamellar liposomes and reshape protein microdomains in the shadows of red blood cells. Melaphene in small and large concentrations changes polymodal the microdomain organization in the bilayers of phospholipid multilamellar liposomes without destroying the structure and does not affect the protein microdomains in the shadows. An increase in the membrane permeability in isolated intact erythrocytes in the presence of melaphene in large and small concentrations has been revealed by means of spectral anaslysis. The method of small-angle diffraction scattering showed the absence of the effect of melaphene in a wide range of concentrations on the thickness of phospholipid bilayers and the order of their packaging in multilamellar liposomes.

Studies on the Interactions of 2-Hydroxyoleic Acid with Monolayers and Bilayers Containing Cationic Lipid: Searching for the Formulations for More Efficient Drug Delivery to Cancer Cells.

Drug delivery in cationic liposomes seems to be a promising therapeutic approach in cancer treatment. The rational design of the positively charged lipid vesicles as anticancer drug carriers should be supported by a detailed analysis of the interactions of the carrier components with anticancer drugs. In the present work, 2-hydroxyoleic acid (2OHOA; Minerval), a membrane lipid therapy drug, was incorporated into positively charged mono- and bilayer membranes containing 1-palmitoyl-2-oleoyl- sn-glycero-3-ethylphosphocholine (EPOPC), the synthetic cationic lipid, and 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC). The intermolecular interactions, fluidity, and miscibility of the studied monolayers were analyzed by utilizing Langmuir balance experiments. The morphology of two-dimensional films was inspected using a Brewster angle microscopy technique. The properties of the liposomes were investigated by dynamic light scattering (DLS) and zeta potential measurements, steady-state fluorescence anisotropy experiments, and the spectrofluorimetric titration of calcein-encapsulated vesicles with a lysis-inducing agent. According to the collected results, 2OHOA intercalation into films of pure phospholipids or a binary EPOPC/DOPC film is thermodynamically favorable. Surprisingly, no significant effect of the presence of unsaturated 2OHOA chains on the EPOPC/DOPC monolayer order was observed. The experiments carried out for 2OHOA-inserted cationic EPOPC/DOPC (1:4) liposomes indicate effective incorporation of the drug into the liposome bilayer and the formation of stable vesicles without affecting their properties markedly. On the basis of the obtained results, EPOPC/DOPC/2OHOA cationic liposomes with 15% 2OHOA content in the phospholipid bilayer seem to be the most suitable for potential biomedical applications.

Polarity of Hydrated Phosphatidylcholine Headgroups.

The headgroup (H) stratum (sometimes called the polar region) of membrane bilayers is a relevant yet poorly understood solvation phase for small molecules and macromolecules interacting with the membranes. Solvation of compounds in bilayer strata is characterized experimentally by wide- and small-angle X-ray scattering, neutron diffraction, and various NMR techniques. The quantification is tedious and only available for a limited set of small molecules. Our recently published model of liposome partitioning of small molecules shows that solvation of compounds in the H-stratum of fluid phosphatidylcholine (PC) bilayers correlates well with their solvation in hydrated diacetyl phosphatidylcholine (DAcPC), and solvation in the core (C) depends in a similar way on that in n-hexadecane. These two correlations became a basis for a model describing the location of compounds in the H- and C-strata and at the connecting interface as a nonlinear function of the fragment solvation characteristics of the compounds. In this study, refractivity of hydrated DAcPC phases with varying water contents was measured and polarity was determined using the steady-state fluorescence of indole and Nile Red. The results were compared with the published data obtained by other techniques for PC bilayers in liposomes or on solid supports. The demonstrated qualitative agreement, as well as the polarity and refractivity dependencies on the DAcPC concentration, supports the suitability of hydrated DAcPC as the H-stratum surrogate. Interestingly, depending on hydrations typical for the H-strata of fluid PC bilayers, the dielectric constant could decrease significantly from 31.0 to 7.3 for 16 and 8 water molecules per headgroup, respectively. Although additional experiments are needed for confirmation, this observation could help set proper dielectric constant magnitudes in continuum-based computational models of accumulation and crossing of the PC bilayers with varying hydration levels thanks to the temperature or the structure of fatty acid chains.

Liposomal integration method for assessing antioxidative activity of water insoluble compounds towards biologically relevant free radicals: example of avarol

The liposomal integration method, in conjunction with electron paramagnetic resonance (EPR) spectroscopy, has been presented for the investigation of antioxidant activity of selected water-insoluble compound towards biologically relevant free radicals. This method was applied to avarol, a sesquiterpenoid hydroquinone isolated from the marine sponge Dysidea avara. The antioxidant activity of water-insoluble avarol towards •OH, O2•− and NO• radicals was attained by its incorporation into the DPPC liposomes bilayer, and towards ascorbyl radicals in the organic solvent. Avarol’s activity towards •OH, O2•−, NO• and ascorbyl radicals was 86.2%, 50.9%, 23.6% and 61.8%, respectively, showing its significant radical scavenging potential.

Influence of Cholesterol and Bilayer Curvature on the Interaction of PPO-PEO Block Copolymers with Liposomes.

Interactions of nonionic poly(ethylene oxide)- b-poly(propylene oxide) (PEO-PPO) block copolymers, known as Pluronics or poloxamers, with cell membranes have been widely studied for a host of biomedical applications. Herein, we report how cholesterol within phosphatidylcholine (POPC) lipid bilayer liposomes and bilayer curvature affects the binding of several PPO-PEO-PPO triblocks with varying PPO content and a tPPO-PEO diblock, where t refers to a tert-butyl end group. Pulsed-field-gradient NMR was employed to quantify the extent of copolymer associated with liposomes prepared with cholesterol concentrations ranging from 0 to 30 mol % relative to the total content of POPC and cholesterol and vesicle extrusion radii of 25, 50, or 100 nm. The fraction of polymer bound to the liposomes was extracted from NMR data on the basis of the very different mobilities of the bound and free polymers in aqueous solution. Cholesterol concentration was manipulated by varying the molar percentage of this sterol in the POPC bilayer preparation. The membrane curvature was varied by adjusting the liposome size through a conventional pore extrusion technique. Although the PPO content significantly influences the overall amount of block copolymer adsorbed to the liposome, we found that polymer binding decreases with increasing cholesterol concentration in a universal fashion, with the fraction of bound polymer dropping 10-fold between 0 and 30 mol % cholesterol relative to the total content of POPC and cholesterol. Increasing the bilayer curvature (decreasing the radius of the liposome) in the absence of cholesterol increases polymer binding between 2- and 4-fold over the range of liposome sizes studied. These results demonstrate that cholesterol plays a dominant role, and bilayer curvature has a less significant impact as the curvature decreases, on polymer-membrane association.

Novel Class of Ultrasound-Triggerable Drug Delivery Systems for the Improved Treatment of Tumors.

The controlled release of anticancer drugs at the tumor site is a central challenge in treating cancer. To achieve this goal, our strategy was based on tumor-specific targeting and ultrasound-triggered release of an anticancer agent from liposomal nanocarriers. To enhance the ultrasound-triggered drug release, we incorporated a lipophilic sonosensitizer, chlorin e6 (Ce6) ester, into the lipid bilayer of liposomes. Additionally, asparagine-glycine-arginine (NGR) that binds to CD13, which is overexpressed in tumor cells, was introduced into these liposomes. Under the navigation effects of the NGR, the novel ultrasound-triggerable NGR-modified liposomal nanocarrier (NGR/UT-L) accumulates in tumor sites. Once irradiated by ultrasound in tumor tissues, the sonodynamic effect produced by Ce6 could create more efficient disruptions of the lipid bilayer of the liposomal nanocarriers. After encapsulating doxorubicin (DOX) as the model drug, the ultrasound triggered lipid bilayer breakdown can spring the immediate release of DOX, making it possible for ultrasound-responsive chemotherapy with great selectivity. By combining tumor-specific targeting and stimuli-responsive controlled release into one system, NGR/UT-L demonstrated a perfect antitumor effect. Moreover, this report provides an example of controlled-release by means of a novel class of ultrasound triggering systems.

One-Step Assembly of TiO2-Liposomes Based on Interfacial Sol-Gel Process within Lipid Bilayer.

There is a growing interest in the use of hybrid liposomes for various biochemical and biomedical applications. In this study, we report the first preparation and characterization of a class of TiO2-reinforced liposomes by a one-step assembly approach. The amphiphilic natural structure of lipids is exploited to localize a hydrophobic molecule, namely, precursor tetrabutyl titanate (TBOT), in the mid-plane of the liposomal bilayer assemblies in the aqueous phase. In situ TiO2 nanoshell formation is driven by subsequent interfacial hydrolysis of TBOT and the ensuing condensation within the hydrophobic interstices of the lipid bilayer. The core-shell structure, like cell and wall, is demonstrated by means of scanning electron microscopy and transmission electron microscopy images, and the formation of the TiO2 shell is confirmed using energy-dispersive X-ray spectroscopy, thermogravimetric analysis, and differential scanning calorimetry. To study the structural evolution of the hybrid liposomes during titania formation, fluorescence probe technique and surface pressure versus molecular area (π- A) isotherms are designed. The results demonstrate that the incorporation of TBOT into the mid-membrane of the lipid and titania in the core of the membrane strengthened the assembly of the lipid bilayer. We further demonstrate that titania shell improved the stability and release property of liposomes. We expect that the reported new TiO2-coated liposomes by co-assembly will be valuable in designing hybrid liposomes, exhibiting integrative capacity for drug encapsulation, compartment reaction, and photocatalysis.

Reversible Aggregation of Chlorophyll Derivative Induced by Phase Transition of Lipid.

Controlling the supramolecular organization of pigment molecules will provide innovative materials that exhibit variable optical properties. In nature, photosynthetic systems employ chlorophyllous supramolecules in which each pigment molecule is suitably organized in proteins, and their properties are adequately optimized by changing the structures of the surrounding amino acid residues. Here, we report a strategy for varying the aggregation behavior of a chlorophyll derivative by using a phase-transition phenomenon of lipid bilayers. Methyl pyropheophorbide a (MPP) was employed as a chlorophyllous pigment in our artificial system, and synthetic phosphatidylcholines with saturated acyl chain(s) were also used. The MPP molecules successfully accumulated within the lipid bilayer of liposomes without changing the vesicular structure. When the lipid bilayer was in a gel form (under the phase-transition temperature, Tm), the embedded MPP aggregated to yield a dimeric form showing red-shifted absorption bands and circular dichroism signals. When the solutions of MPP-containing liposomes were heated to higher temperatures than their Tm, MPP disaggregated to monomeric form as the absorption spectrum changed into its original fashion in dichloromethane. The reversible thermochromic (dis)aggregation of the MPP molecules had good cyclability. Additional careful examination of the phase transition in the MPP-lipid co-assemblies clarified that the critical temperatures of the MPP (dis)aggregation were in good agreement with the phase-transition temperatures of the pigment-containing bilayers. The reversible MPP aggregation in the lipid bilayers occurred in a wide range of temperatures (around 10-55 °C) by changing the length of the diacyl side chains of phospholipids. The reversible thermochromism of the chlorophyllous system was established by varying the nature of the surrounding lipid bilayer. This study can provide a useful strategy for making variable tetrapyrrolic aggregate systems induced by mild extrinsic stimuli.

Critical process parameters in manufacturing of liposomal formulations of amphotericin B

Identifying the critical process parameters (CPPs) of a complex drug product manufacture and the associated impact on critical quality attributes (CQAs) is essential to the development and quality control of both new and generic drugs. AmBisome, a liposomal amphotericin B (AMB) macrolide antibiotic widely adopted as an important antifungal drug product, was used as a model complex drug product in the current study. This study investigated how multi-step production approaches and related manufacturing conditions may affect essential physico-chemical and toxicological properties of the final drug product. A key challenge in the manufacture and analysis of liposomal AMB was the drug substance’s propensity to aggregate, with associated poor solubility in water and organic solvents. This study identified three key CPPs in a four step manufacturing process: (i) proper acidification during formation of the drug-lipid complexes (Step 1), (ii) liposome heat curing following liposomal particle sizing (Step 3), and (iii) flash-freezing at the initial stages of the lyophilization cycle (Step 4). Over-acidification led to rapid degradation of the drug, whereas under-acidification hampered full solubilization and formation of the soluble drug-lipid complexes. Extended heat treatment of the formed liposomes at 65 °C, just above the lipid phase transition temperature, brought dramatic changes in the aggregated state and/or packing of the drug in the liposomal bilayer, as followed by the complex changes in the UV/Vis spectra. Such thermal conditioning resulted in a five- to ten-fold reduction in the in-vitro toxicity of the drug product, bringing it close to the values for AmBisome used as control and measured by the RBC assay. Finally, flash-freezing conditions during lyophilization was critical to prevent aggregation and maintaining the 80–120 nm liposome size when reconstituted. Our research found that changes in the amphotericin’s UV/Vis spectra were a sensitive CQA measure and provided a set of quantitative parameters for a facile non-destructive process monitoring in-situ, as well as for comparison of the quality of final formulations.

Layer-by-layer nanoparticles for novel delivery of cisplatin and PARP inhibitors for platinum-based drug resistance therapy in ovarian cancer.

Advanced staged high‐grade serous ovarian cancer (HGSOC) is the leading cause of gynecological cancer death in the developed world, with 5‐year survival rates of only 25–30% due to late‐stage diagnosis and the shortcomings of platinum‐based therapies. A Phase I clinical trial of a combination of free cisplatin and poly(ADP‐ribose) polymerase inhibitors (PARPis) showed therapeutic benefit for HGSOC. In this study, we address the challenge of resistance to platinum‐based therapy by developing a targeted delivery approach. Novel electrostatic layer‐by‐layer (LbL) liposomal nanoparticles (NPs) with a terminal hyaluronic acid layer that facilitates CD44 receptor targeting are designed for selective targeting of HGSOC cells; the liposomes can be formulated to contain both cisplatin and the PARPi drug within the liposomal core and bilayer. The therapeutic effectiveness of LbL NP‐encapsulated cisplatin and PARPi alone and in combination was compared with the corresponding free drugs in luciferase and CD44‐expressing OVCAR8 orthotopic xenografts in female nude mice. The NPs exhibited prolonged blood circulation half‐life, mechanistic staged drug release and targeted codelivery of the therapeutic agents to HGSOC cells. Moreover, compared to the free drugs, the NPs resulted in significantly reduced tumor metastasis, extended survival, and moderated systemic toxicity.

Effect of the Incorporation of Functionalized Cyclodextrins in the Liposomal Bilayer.

Liposomes loaded with drug–cyclodextrin complexes are widely used as drug delivery systems, especially for species with low aqueous solubility and stability. Investigation of the intimate interactions of macrocycles with liposomes are essential for formulation of efficient and stable drug-in-cyclodextrin-in-liposome carriers. In this work, we reported the preparation of unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) embedded with native β-cyclodextrin and two synthetic derivatives: heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TMCD) and heptakis(2,3-di-O-acetyl)-β-cyclodextrin (DACD). We then studied the effect of these macrocycles on the liposomal size, membrane viscosity, and liposomal stability at different temperatures and concentrations. We observed that TMCD and DACD affected vesicle size and the change of size was related to CD concentration. Irrespective of its nature, the macrocycle established interactions with the phospholipidic head groups, preventing cyclodextrins to diffuse into the lipid bilayer, as confirmed by molecular dynamics simulations. Such supramolecular structuring improves liposome stability making these colloid systems promising carriers for biologically active compounds.

Implication of Cholesterol in Regulating the Pore‐formation Mechanism of Vibrio Cholerae Cytolysin, a β‐barrel Pore‐forming toxin

Vibrio cholerae cytolysin (VCC) is a β‐barrel pore‐forming toxin with potent cell‐killing cytolytic/cytotoxic activity. To exert it's cytolytic effect, VCC binds to the target cell membranes and forms transmembrane oligomeric β‐barrel pores. Earlier studies have indicated that cholesterol might play a critical role in regulating the mode of action of VCC. However, exact mechanistic basis of the process still remains unknown. In the present study, we have explored and examined the roles of cholesterol in the different steps of VCC mode of action. Employing the membrane lipid bilayer of lipid vesicles or liposomes containing varying amount of cholesterol, we have shown that cholesterol governs the efficient interaction of the toxin with the membrane lipid bilayer that in turn facilitates subsequent oligomeric pore‐formation process. We have also studied the physiological relevance of membrane cholesterol in the context of the biomembranes of the eukaryotic cells. Our results have shown that the depletion of membrane cholesterol from erythrocytes compromises the pore‐forming activity of VCC. In the biomembrane of erythrocytes, where accessory interactions are available, binding of VCC is not affected to any significant extent. However, upon depletion of membrane cholesterol in the erythrocytes membranes, membrane‐bound fraction of VCC remains trapped in the form of abortive oligomeric assembly, and does not form the functional pores. Our study has also revealed that VCC shows a marked tendency to associate with the cholesterol‐rich membrane micro‐domains on erythrocytes that, in turn, appears to facilitate cholesterol‐dependent pore‐formation mechanism of the toxin. In sum, our results suggest that the presence of cholesterol in the target membranes play critical role for the pore‐formation mechanism of VCC.Support or Funding InformationWe acknowledge the support through funding from the Department of Biotechnology (DBT), Govt. of India [DBT grant no. BT/HRD/NBA/37/01/2014 (x)] and also the support through funding under the Centre of Excellence (COE) in Frontier Areas of Science and Technology (FAST) program of the Ministry of Human Resource Development, Govt. of India, in the area of protein science, design, and engineering (to K.C.). We also thank the Indian Institute of Science Education and Research Mohali for the support.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dual-Sensitive On-Off Switch in Liposome Bilayer for Controllable Drug Release.

To improve the controllability of drug release from liposome, a series of pH- and photosensitive block copolymers C7H15-AZO- b-PDPAn- b-mPEG were designed and served as an on-off switch in the liposome bilayer. The functional properties of liposomes were studied by dynamic light scattering, fluorophotometry, UV-vis spectroscopy, and fluorescence spectrophotometry. The liposomes with or without copolymer were relatively uniform in size. Their membrane stability was improved obviously after inserting copolymer under pH 7.4, but it decreased in an acidic environment and caused a large amount of drug release. Meanwhile, UV irradiation could also result in more drug release because of the photoisomerization of the azobenzene (AZO) group. Furthermore, intermittent drug-release experiments showed that the PDPA blocks could reversibly get in and out of the liposome bilayer and ultimately realized the complete drug release. All results suggested that the designed copolymers could be inserted into liposome bilayer through self-assembly and could act as a switch for controllable drug release.