Towards understanding the binding affinity of lipid drug carriers to serum albumin.

In the past several years there has been a rapid rise in the use of lipid-based drug formulations. In the case of intravenous drug administration the interaction of lipid carrier with serum albumin is crucial for the distribution of the bioactive molecules in the bloodstream and reaching the target tissue. In this work, we have explored the interaction of serum albumin with three-component lipid monolayer build of palmitoyloleoylphosphatidylcholine (POPC), sphingomyelin (SM), and cholesterol (Chol). Using wide range of lipid compositions and various concentrations of serum albumin we identified the factors governing the lipid-protein binding. Our study revealed that albumin can penetrate selectively the monolayers of POPC/SM/Chol depending on the lipid composition in the mixture. Moreover, the interaction of albumin with monolayer can be controlled by the molecular density of the film and the concentration of protein. The adsorbed albumin exists in the film on the top of lipid monolayer. This behavior may lead to the increase of the size and charge of the lipid carrier and affect the drug transport throughout the bloodstream. The results of this work provide essential physicochemical data that can be used for predicting the pharmacokinetic profile of lipid-based formulations.

The Process of Binding and Releasing of Genetic Material from Lipoplexes Based on Trimeric Surfactants and Phospholipids.

Nonviral vectors for gene therapy such as lipoplexes are characterized by low toxicity, high biocompatibility, and good transfection efficiency. Specifically, lipoplexes based on polymeric surfactants and phospholipids have great potential as gene carriers due to the increased ability to bind genetic material (multiplied positive electric charge) while lowering undesirable effects (the presence of lipids makes the system more like natural membranes). This study aimed to test the ability to bind and release genetic material by lipoplexes based on trimeric surfactants and lipid formulations of different compositions and to characterize formed complexes by circular dichroism (CD) spectroscopy and atomic force microscopy (AFM). The cytotoxicity of studied lipoplexes was tested on HeLa cells by the MTT cell viability assay and the dye exclusion test (trypan blue). The presence of lipids in the system lowered the surfactant concentration required for complexation (higher efficiency) and reduced the cytotoxicity of lipoplexes. Surfactant/lipids/DNA complexes were more stable than surfactant/DNA complexes. Surfactant molecules induced the genetic material condensation, but the presence of lipids significantly intensified this process. Systems based on trimeric surfactants and lipid formulations, particularly TRI_N and TRI_IMI systems, could be used as delivery carrier, and have proven to be highly effective, nontoxic, and universal for DNA of various lengths.

Physicochemical Characterization of Oleanolic Acid-Human Serum Albumin Complexes for Pharmaceutical and Biosensing Applications.

Among numerous compounds found in marine organisms, triterpenes have attracted considerable research interest due to a beneficial impact on health including anti-inflammatory, antitumor, antiviral, and antioxidation effects. Specifically, new functionalities of oleanolic acid (OLA) have been revealed recently, indicating possible applications in nutrition and pharmaceuticals. However, this bioactive material has limited value due to low water solubility and stability. Therefore, oleanolic acid needs a carrier that protects it and enables controlled release in the human body. Innovative drug delivery systems provide a promising strategy for overcoming these problems. However, the development of those systems requires a comprehensive understanding of the physicochemical properties of triterpenes and their carriers as well as the interactions between them. Among numerous substances, human serum albumin (HSA) has been widely studied as a drug carrier. In addition, human serum albumin is the main blood plasma protein responsible for the transport of drugs and metabolites; therefore, the interactions between that protein and other substances are of physiological and pharmaceutical importance. Moreover, sensing the HSA level in blood plasma is an important challenge that requires binding studies on a molecular scale. The aim of this study was to investigate the properties of oleanolic acid in the presence of human serum albumin in terms of thermodynamics, morphology, and viscoelasticity at the air/water interface. Moreover, the wettability, surface free energy, and topography of the films after deposition on the solid substrate were determined. The results have been discussed in terms of providing physicochemical insight into the interfacial behavior of the OLA-HSA complex, which is crucial for pharmaceutical and bioanalytical applications.

Knock Down of Plakophillin 2 Dysregulates Adhesion Pathway through Upregulation of miR200b and Alters the Mechanical Properties in Cardiac Cells.

Background: Mutations in genes encoding intercalated disk/desmosome proteins, such as plakophilin 2 (PKP2), cause arrhythmogenic cardiomyopathy (ACM). Desmosomes are responsible for myocyte-myocyte attachment and maintaining mechanical integrity of the myocardium. Methods: We knocked down Pkp2 in HL-1 mouse atrial cardiomyocytes (HL-1Pkp2-shRNA) and characterized their biomechanical properties. Gene expression was analyzed by RNA-Sequencing, microarray, and qPCR. Immunofluorescence was used to detect changes in cytoskeleton and focal adhesion. Antagomirs were used to knock down expression of selected microRNA (miR) in the rescue experiments. Results: Knockdown of Pkp2 was associated with decreased cardiomyocyte stiffness and work of detachment, and increased plasticity index. Altered mechanical properties were associated with impaired actin cytoskeleton in HL-1Pkp2-shRNA cells. Analysis of differentially expressed genes identified focal adhesion and actin cytoskeleton amongst the most dysregulated pathways, and miR200 family (a, b, and 429) as the most upregulated miRs in HL-1Pkp2-shRNA cells. Knockdown of miR-200b but not miR-200a, miR-429, by sequence-specific shRNAs partially rescued integrin-α1 (Itga1) levels, actin organization, cell adhesion (on collagen), and stiffness. Conclusions: PKP2 deficiency alters cardiomyocytes adhesion through a mechanism that involves upregulation of miR-200b and suppression of Itga1 expression. These findings provide new insights into the molecular basis of altered mechanosensing in ACM.

Langmuir-Blodgett films of membrane lipid in the presence of hybrid silsesquioxane, a promising component of biomaterials.

Functionalized polyhedral oligomeric silsesquioxanes (POSS) derivatives have great potential in biomedical applications such as tissue engineering, drug delivery, biosensors, dental composites and biomedical devices. Having the above in mind, in this paper, the study of the surface characteristics of binary Langmuir-Blodgett films consisting of an open cage silsesquioxane POSS-poly (ethylene glycol) (POSS-PEG) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), as a representative of phospholipid was conducted based on contact angle measurements of three liquids. The measured values of the contact angle (with water, formamide and diiodomethane as the wetting liquids) allowed to calculate surface free energy of the films from van Oss et al. approach. The film structure of the deposited layers was evaluated using an atomic force microscope. Analysis of the obtained results led to the conclusion, that the pure DMPE molecules create agglomerates onto a solid substrate, whereas the POSS-PEG molecules form a homogenous monolayer. After an addition of POSS-PEG to lipid film, changes in the surface properties are visible. The wettability as well as surface free energy depend on the molar ratio of both components. The AFM images shed more light on the changes of the DMPE monolayer topography caused by the POSS-PEG addition.

How Complex Is the Concanavalin A-Carboxypeptidase Y Interaction?

Lectin-carbohydrate interactions can be exploited in ultrasensitive biochemical recognition or medical diagnosis. For this purpose, besides the high specificity of the interactions, an appropriate methodology for their quantitative and detailed characterization is demanded. In this work, we determine the unbinding properties of the concanavalin A-carboxypeptidase Y complex, which is important for characterization of glycoproteins on the surface of biological cells. To achieve the goal, we have developed a methodology based on dynamic force spectroscopy measurements and two advanced theoretical models of force-induced unbinding. Our final results allowed excluding both, rebinding processes and the multibarrier character of the interaction potential, as possible explanations of the concanavalin A-carboxypeptidase Y unbinding mechanisms. Such characteristics as the position and height of the activation barrier and the force-free dissociation rate were determined. We hope our paper contributes to a better understanding of the unbinding processes in receptor-ligand complexes.

Temperature, pH, and Molecular Packing Effects on the Penetration of Oleic Acid Monolayer by α-Lactalbumin.

Recently, we reported on the interfacial behavior of mixed oleic acid (OA)-α-lactalbumin monolayer and its relevance in the formation of tumoricidal HAMLET (human α-lactalbumin made lethal to tumor cells)-like complex. This complex is probably formed in the gastrointestinal tract, but it has not been proved so far. The molecular base and the underlying physicochemical forces leading to such complexation remain to be known as well. There are also several other issues related with the complex stoichiometry that need to be fully explained. This study provides insight into the mechanism of temperature, pH, and physical state of monolayer-dependent binding of OA by the milk protein- apo-α-lactalbumin. Using the Langmuir and Langmuir-Blodgett approaches, we investigated the interactions between the OA monolayer and the apo-bovine α-lactalbumin (BLA III) at different pH, temperatures, and molecular packing. We found that the most favorable conditions for the formation of mixed OA-BLA III film are relevant to the gastric environment. The stabilization of OA-BLA III at the interface is associated with the conformational changes of protein in the presence of fatty acids induced by low pH and high temperature in the expanded monolayer. Our approach helps to understand the molecular mechanism of HAMLET/bovine α-lactalbumin made lethal to tumor cells formation in vivo.

Bioengineering the spider silk sequence to modify its affinity for drugs.

BACKGROUND: Silk is a biocompatible and biodegradable material, able to self-assemble into different morphological structures. Silk structures may be used for many biomedical applications, including carriers for drug delivery. The authors designed a new bioengineered spider silk protein, EMS2, and examined its property as a carrier of chemotherapeutics. MATERIALS AND METHODS: To obtain EMS protein, the MS2 silk monomer (that was based on the MaSp2 spidroin of Nephila clavipes) was modified by the addition of a glutamic acid residue. Both bioengineered silks were produced in an Escherichia coli expression system and purified by thermal method. The silk spheres were produced by mixing with potassium phosphate buffer. The physical properties of the particles were characterized using scanning electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, and zeta potential measurements. The MTT assay was used to examine the cytotoxicity of spheres. The loading and release profiles of drugs were studied spectrophotometrically. RESULTS: The bioengineered silk variant, EMS2, was constructed, produced, and purified. The EMS2 silk retained the self-assembly property and formed spheres. The spheres made of EMS2 and MS2 silks were not cytotoxic and had a similar secondary structure content but differed in morphology and zeta potential values; EMS2 particles were more negatively charged than MS2 particles. Independently of the loading method (pre- or post-loading), the loading of drugs into EMS2 spheres was more efficient than the loading into MS2 spheres. The advantageous loading efficiency and release rate made EMS2 spheres a good choice to deliver neutral etoposide (ETP). Despite the high loading efficiency of positively charged mitoxantrone (MTX) into EMS2 particles, the fast release rate made EMS2 unsuitable for the delivery of this drug. A faster release rate from EMS2 particles compared to MS2 particles was observed for positively charged doxorubicin (DOX). CONCLUSION: By modifying its sequence, silk affinity for drugs can be controlled.

Lipid nanoparticles assessment by flow cytometry.

BACKGROUND: Liposomes are promising carriers for drugs and bioactive compounds. Size and structure are their crucial parameters. Thus, it is essential to assess individual vesicles as prepared. Currently available techniques fail to measure liposome's size and structure simultaneously, with a high throughput. To solve this problem, we have developed a novel, flow cytometric method quantifying liposomes. METHODS: Firstly, the following fluorescent staining combinations were tested: DiD/TO, Rh123/DiD, Syto9/DiD. Further, chosen fluorochromes were used to compare three populations of vesicles: raw (R), obtained by thin film hydration and extruded ones (populations E10 and E21). Dynamic light scattering (DLS) was used for determination of average diameter and size distribution of nanocarriers. Structural differences between the raw and the extruded liposomes, as well as additional information concerning vesicles size were acquired employing atomic force microscopy (AFM). RESULTS: DLS analysis indicated that, three distinct populations of vesicles were obtained. Liposomes were characterized by mean diameter of 323nm, 220nm and 170nm for population R, E10 and E21 respectively. All the populations were stable and revealed zeta potential of -29mV. AFM confirmed that raw and extruded liposomes were differed in structure. CONCLUSIONS AND GENERAL SIGNIFICANCE: DiD/TO was the optimal fluorochrome combination that enabled to resolve distinctly the sub-populations of liposomes. Results obtained by flow cytometry were in a good agreement with those from DLS and AFM. It was proved that, flow cytometry, when proper fluorescent dyes are used, is an adequate method for liposomes assessment. The proposed method enables fast and reliable analysis of liposomes in their native environment.

Gemini Surfactants Based on Bis-Imidazolium Alkoxy Derivatives as Effective Agents for Delivery of Nucleic Acids: A Structural and Spectroscopic Study.

The success rate of gene therapy depends on the efficient transfection of genetic material into cells. The golden mean between harmlessness and high effectiveness can be provided by synthetic lipid-like molecules that are similar to the components of biological membranes. Cationic gemini surfactants are one such moiety and because of their favourable physicochemical properties (double positive electric charge, reduced toxicity, low values of critical micelle concentration), they show great potential as delivery system components for genetic material in gene therapy. The aim of this study was to investigate the process of the complexation of cationic gemini surfactants with nucleic acids: double-stranded DNA of different sizes (21 bp, ~185 bp, ~20 kbp) and siRNA (21 bp). The tested series of dicationic surfactants consists of bis-imidazolium quaternary salts with varying lengths of hydrophobic side chains (m = 5, 6, 7, 8, 9, 11, 12, 14, 16). On the basis of the data obtained by circular dichroism spectroscopy and electrophoresis, we concluded that the studied gemini surfactants with long side chains effectively bind nucleic acids at low concentrations, which leads to the formation of stable lipoplexes. Images obtained by atomic force microscopy also confirmed the formation of vesicular structures, i.e., complexes between DNA and surfactants. The cytotoxicity of selected surfactants was also tested on HeLa cells. The surfactant toxicity significantly depends on surfactant geometry (the length of hydrophobic chain).