From Design to Study of Liposome-Driven Drug Release Part 1: Impact of Temperature and pH on Environment.

The marketed drug Doxorubicin (DOX) and the promising anti-cancer agent 9-(N-piperazinyl)-5-methyl-12(H)-quino[3,4-b][1,4]benzothiazinium chloride (9-PBThACl) were used to prepare and compare a range of liposomal delivery systems based on dipalmitoylphosphatidylcholine (DPPC). Liposome-assisted drug release was examined using the spectrophotometric method. In order to provide in vitro release characteristics of liposomal conjugates (LDPPC/drug vs. LDPPC/drug/drug) as well as to evaluate the impact of temperature and pH buffering on the conformation/polarity of the phospholipid bilayer, the encapsulation efficiency of the liposomes entrapping 9-PBThACl and DOX was calculated. In fact, some competition between the investigated molecules was noticed during the entrapment process because relatively high values of the encapsulation efficiency were observed only for the liposomal complexes containing one trapped drug molecule. An averaged absorbance value enabled us to indicate the pH value of the environment (pH ≈ 6.8), at which the physicochemical property profiles of the liposomal complexes were noticeably changed. Moreover, the operational factors limiting the drug release kinetics from the produced liposomes were mathematically modeled. First-order and Bhaskas models ensured satisfactory compliance with the experimental data for the liposomal complexes buffered at pH values of 5.50, 6.00, and 7.40, respectively.

The Advances and Challenges of Liposome-Assisted Drug Release in the Presence of Serum Albumin Molecules: The Influence of Surrounding pH.

The aim of this study is to prepare a liposomal delivery system for 5-methyl-12 (H)-quino[3,4-b]-1,4-benzothiazine chloride (5-MBT) and study the in vitro release characteristics. The release of 5-MBT from a liposomal complex with human serum albumin (HSA) [LDPPC/5-MBT]:HSA was examined using the spectrophotometric method and differential scanning calorimetry (DSC). Electronic paramagnetic resonance was used to assess the influence of the pH of the environment on the conformation of phospholipids, the latter determining the degree of release of the encapsulated compound. The applied mathematical models made it possible to determine the necessary analytical parameters to facilitate the process of potential drug release from liposomes. The complexes formed by liposomal 5-MBT with serum albumin (HSA) particles allowed for the description of the Fick process. The change in the polarity of the phospholipid membrane resulting from the changes in the pH of the surroundings, significantly influenced the percentage of 5-MBT entrapment in the liposomes. It also affected the release percentage.

A Comprehensive Spectroscopic Analysis of the Ibuprofen Binding with Human Serum Albumin, Part I.

Human serum albumin (HSA) plays a fundamental role in the human body. It takes part in the transport of exogenic and endogenic substances, especially drugs. Ibuprofen (IBU) is one of the most commonly used non-steroidal anti-inflammatory drugs, used for pain relief, fever relief, and for anti-inflammatory purposes. The binding of ligands with HSA is a significant factor which determines the toxicity and the therapeutic dosages of these substances. The aim of this study was to compare the degree of ibuprofen binding with human serum albumin at various temperatures and protein solution pH values. In order to evaluate conformational changes in HSA caused by interaction with ibuprofen, spectrophotometric (first and second derivatives of the UV-VIS spectrum), and spectrofluorometric analyses were performed concerning the mutual interactions of IBU-HSA. The use of fluorescent spectroscopy allowed for recording fluorescent emissive spectra of HSA (5 × 10-6 mol/dm3) without and with the presence of ibuprofen (1 × 10-5-1 × 10-4 mol/dm3) at temperatures of 308, 310, 312, and 314 K at pH values of 6.5, 6.8, 7.4, 7.8, and 8.1. System fluorescence was excited by radiation of wavelengths of λex = 275 nm and λex = 295 nm. Based on this, original and modified Stern-Volmer, Scatchard, Klotz and Hill curves were determined. The data that were obtained showed a significant effect of temperature and pH of the human serum albumin solution on the strength and type of interaction of ibuprofen with HSA.

Derivatives of Graphene Oxide as Potential Drug Carriers.

Chemically functionalized graphene oxides could be used as novel drug carriers. Covalent alterations of graphene oxides lead to surface changes via formation of chemical bonding while non-covalent ones involve van der Waals forces, hydrogen bonding, and π-π stacking interactions. Covalent modifications appear to be superior as they can yield compounds with desired properties and carriers prepared by other methods are less stable. Synthesis of graphene oxide-iminodiacetic acid and graphene oxide-glycine involves nucleophilic substitution of graphene oxide nanoparticles with iminodiacetic acid or glycine. As the first step, iminodiacetic acid or glycine were transformed into iminodiacetic acid or glycine methyl ester hydrochlorides, respectively, for C-terminus protection. The obtained product, activated in situ, was then used to form amide bonds between graphene oxide and iminodiacetic acid or glycine.

The Influence of Fatty Acids on Metoprolol - Human Serum Albumin Interaction in Low Affinity Binding Sites: A Multifactorial NMR Approach.

BACKGROUND: Metoprolol (MTP) is a cardio-selective ß1-blocker used in hypertension, angina pectoris and chronic heart failure therapies. Serum albumin is the most frequently occurring protein in blood plasma. The binding of ligands to human serum albumin (HSA) has an important effect on pharmacokinetics and final clinical effects. OBJECTIVE: The objectives of this study included a detailed analysis of metoprolol - serum albumin interactions in low affinity binding sites, on the surface or within the hydrophobic subdomain of a macromolecule, as well as an analysis of the competition between MTP and fatty acids in binding with protein. METHODS: The analysis of the drug-albumin interaction was based on the observed chemical shifts in combination with correlation Times (T1 -1 = τ) [1/s], 2D NOESY 1H NMR spectra and association constants Ka [M-1]. For the determination of chemical shifts σ [ppm], relaxation times T1 [s] and for the NOESY experiment, the final concentrations of MTP and albumins (in the presence (HSA) and absence of fatty acids (dHSA)) were 5 x 10-3 M and 2 x 10-5 M - 4.55 x 10-4 M, respectively. In order to calculate the association constants, the final concentrations of MTP and both HSA and dHSA were 2.75 x 10-3 M - 6.25 x 10-2 M and 2.5 x 10-4 M, respectively. For the analysis, the MTP proton resonances of aliphatic H17, aromatic (H2/H6 and H3/H5) and the methoxy group H14 were chosen. RESULTS: Changes in the values of the 1H NMR chemical shift [ppm] are evidence of interaction between MTP, fatted (HSA) and defatted (dHSA) human serum albumin. With an increase of albumin concentration, changes in the chemical shift values were observed for the aromatic protons H2/H6 (Δσ = 0.013 ppm and 0.018 ppm) and H3/H5 (Δσ = 0.015 ppm and 0.019 ppm), the aliphatic proton H17 (Δσ = 0.018 ppm and 0.022 ppm) and the aliphatic protons of the methoxy group H14 (Δσ = 0.019 ppm and 0.022 ppm) for dHSA and HSA, respectively. Greater changes in chemical shifts in the presence of fatty acids (FA) were observed. Changes in the correlation times of MTP aromatic H2/H6 (Δτc = 0.224 1/s and 0.189 1/s) and H3/H5 (Δτc = 0.269 1/s and 0.210 1/s), aliphatic from the methoxy group H14 (Δτc = 0.472 1/s and 0.271 1/s) and aliphatic H17 protons (Δτc = 0.178 1/s and 0.137 1/s) for dHSA and HSA systems, respectively. It confirms the interaction between the drug and albumin are evidence for the dynamics of the process. In the presence of FA the relaxation time of all analyzed MTP proton resonance signals significantly increases (due to the decrease of correlation time). This phenomenon is due to the increase of electron density in the MTP protons' surroundings. Association constants for the MTP-dHSA complex in the low affinity site range between 0.29 x 102 M-1 and 0.47 x 102 M-1. The presence of FA results in a two to three-fold increase of the Ka values of protons from aromatic (H2/H6 and H3/H5), aliphatic H17 and methoxy (H14) groups. In 2D NOESY spectra proton magnetization transfer was observed between cysteine (Cys-34) and aromatic H3/H5 and H2/H6 protons. Cross-peaks were also observed between cysteine and aliphatic protons from the methoxy group. CONCLUSION: The selective changes in σ [ppm] and τc [1/s] values indicated the unequal participation of chemical groups of MTP in the interaction with HSA and dHSA. The data obtained suggest that the presence of fatty acids increases the accessibility of low affinity sites of serum albumin to MTP, which results in the higher affinity of albumin towards the drug. The results showed that the main binding site of MTP and fatty acid is probably a low affinity site in subdomain IB, where Cys-34 can be located.

In Vitro Investigation of the Interaction of Tolbutamide and Losartan with Human Serum Albumin in Hyperglycemia States.

Serum albumin is exposed to numerous structural modifications which affect its stability and activity. Glycation is one of the processes leading to the loss of the original properties of the albumin and physiological function disorder. In terms of long lasting states of the hyperglycemia, Advanced Glycation End-products (AGEs) are formed. AGEs are responsible for cellular and tissue structure damage that cause the appearance of a number of health consequences and premature aging. The aim of the present study was to analyze the conformational changes of serum albumin by glycation-"fructation"-using multiple spectroscopic techniques, such as absorption (UV-Vis), fluorescence (SFM), circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy and evaluate of possible alteration of binding and competition between tolbutamide (TB, a first-generation sulfonylurea oral hypoglycemic drug) and losartan (LOS, an angiotensin II receptor (AT1) blocker used in hypertension (1st line with a coexisting diabetes)) in binding to non-glycated (HSA) and glycated (gHSAFRC) human serum albumin in high-affinity binding sites. The studies allowed us to indicate the structural alterations of human serum albumin as a result of fructose glycation. Changes in binding parameters, such as association ( K a ) or Stern-Volmer ( K S V ) constants suggest that glycation increases the affinity of TB and LOS towards albumin and affects interactions between them. The process of albumin glycation influences the pharmacokinetics of drugs, thus monitored pharmacotherapy is reasonable in the case of diabetes and hypertension polypharmacy. This information may lead to the development of more effective drug treatments based on personalized medicine for patients with diabetes. Our studies suggest the validity of monitored polypharmacy of diabetes and coexisting diseases.

Effect of Temperature on Tolbutamide Binding to Glycated Serum Albumin.

Glycation process occurs in protein and becomes more pronounced in diabetes when an increased amount of reducing sugar is present in bloodstream. Glycation of protein may cause conformational changes resulting in the alterations of its binding properties even though they occur at a distance from the binding sites. The changes in protein properties could be related to several pathological consequences such as diabetic and nondiabetic cardiovascular diseases, cataract, renal dysfunction and Alzheimer's disease. The experiment was designed to test the impact of glycation process on sulfonylurea drug tolbutamide-albumin binding under physiological (T = 309 K) and inflammatory (T = 311 K and T = 313 K) states using fluorescence and UV-VIS spectroscopies. It was found in fluorescence analysis experiments that the modification of serum albumin in tryptophanyl and tyrosyl residues environment may affect the tolbutamide (TB) binding to albumin in subdomain IIA and/or IIIA (Sudlow's site I and/or II), and also in subdomains IB and IIB. We estimated the binding of tolbutamide to albumin described by a mixed nature of interaction (specific and nonspecific). The association constants Ka (L∙mol-1) for tolbutamide at its high affinity sites on non-glycated albumin were in the range of 1.98-7.88 × 104 L∙mol-1 (λex = 275 nm), 1.20-1.64 × 104 L∙mol-1 (λex = 295 nm) and decreased to 1.24-0.42 × 104 L∙mol-1 at λex = 275 nm (T = 309 K and T = 311 K) and increased to 2.79 × 104 L∙mol-1 at λex = 275 nm (T = 313 K) and to 4.43-6.61 × 104 L∙mol-1 at λex = 295 nm due to the glycation process. Temperature dependence suggests the important role of van der Waals forces and hydrogen bonding in hydrophobic interactions between tolbutamide and both glycated and non-glycated albumin. We concluded that the changes in the environment of TB binding of albumin in subdomain IIA and/or IIIA as well as in subdomains IB and IIB influence on therapeutic effect and therefore the studies of the binding of tolbutamide (in diabetes) to transporting protein under glycation that refers to the modification of a protein are of great importance in pharmacology and biochemistry. This information may lead to the development of more effective drug therapy in people with diabetes.

The role of nanoparticles in the albumin-cytarabine and albumin-methotrexate interactions.

Understanding the interactions which occur between nanomaterials and biomolecules is one of the most important issues in nanotechnology. Determining the properties of nanoparticles obtained through the use of novel methods and defining the scope of their application as drug carriers has important practical significance. Nanoparticles containing methotrexate and cytarabine obtained by a modified reverse-phase evaporation method (mREV) were characterized through the use of the UV/Vis and NMR methods. Obtained results confirmed high degree of analysed drugs encapsulation. The encapsulation efficiencies of cytarabine (AraC) and methotrexate (MTX) in LDPPC/AraC/MTX were found to be 86.30% (AraC) and 86.00% (MTX). The increased permeability of the phospholipid membranes, resulting from physico-chemical properties and the location of the drug, as well as from the physico-chemical properties of the phospholipids themselves, has been confirmed by increase in the length of the T1 relaxation time of protons in the N+(CH3)3 group. The study of analysed drugs release process from the liposomes has been made for bovine serum albumin, both in the absence (dBSA) and in the presence of fatty acid (BSA). Moreover two types of kinetic models (Bhaskar equation and Rigter-Peppas equation) have been used. Based on the study it has been concluded that mathematical modelling of drug release can be very helpful in speeding up product development and in better understanding the mechanisms controlling drug release from advanced delivery systems.

Methotrexate and Cytarabine-Loaded Nanocarriers for Multidrug Cancer Therapy. Spectroscopic Study.

Determining the properties of nanoparticles obtained by novel methods and defining the scope of their application as drug carriers has important practical significance. This article presents the pioneering studies concerning high degree incorporation of cytarabine (AraC) and methotrexate (MTX) into liposome vesicles. The main focus of this study were cytarabine-methotrexate-dipalmitoylphosphatidylcholine (DPPC) interactions observed in the gel and fluid phases of DPPC bilayers. The proposed new method of use the Transmittance2919/2850 ratio presented in our research is sensitive to subtle changes in conformational order resulting from rotations, kinks and bends of the lipid chains. The transition temperatures characterized by Fourier Transform Infrared Spectroscopy (FT-IR) were consistent with the results obtained by Differential Scanning Calorimetry (DSC). Transmission Electron Microscopy (TEM) was used in order to determine the size and shape of the liposomes obtained. The mutual interactions occurring between the drugs studied and the phospholipids were analyzed using the Nuclear Magnetic Resonance (NMR).

In vitro spectroscopic study of piperine-encapsulated nanosize liposomes.

Black pepper is a source of effective antioxidants. It contains several powerful antioxidants and is thus one of the most important spices for preventing and curtailing oxidative stress. There is considerable interest in the development of a drug-delivery systems that would result in the selective delivery of antioxidants to tissues in sufficient concentrations to ameliorate oxidant-induced tissue injuries. Liposomes are biocompatible, biodegradable and nontoxic artificial phospholipid vesicles that offer the possibility of carrying hydrophilic, hydrophobic and amphiphilic molecules. This article focuses on the use of liposomes for the delivery of antioxidants in the prevention or treatment of pathological conditions related to oxidative stress. Liposome formulations of piperine were analyzed with various spectroscopic methods. The formulation with the highest entrapment efficiency (90.5%) was formulated with an L-α-phosphatidylcholine dipalmitoyl (DPPC):piperine, 30:1 molar ratio, and total lipid count of 19.47 mg/ml in the final liposomal preparation. The liposome formulation was found to be stable after storage at 4 °C, protected from light, for a minimum of 3 weeks. The incremental process of piperine penetration through the phospholipid membrane was analyzed using the FT-IR, UV-Vis and NMR methods. Temperature stability studies carried out at 37 °C showed the highest percentage of piperine release in the first 3 h of incubation.

Physicochemical properties of liposomes as potential anticancer drugs carriers. Interaction of etoposide and cytarabine with the membrane: spectroscopic studies.

The interactions between etoposide, cytarabine and 1,2-dihexadecanoyl-sn-glycerol-3-phosphocholine bilayers were studied using differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR). These techniques have proven to be a very powerful tool in studying the structure and dynamics of phospholipid bilayers. In particular, DSC can provide information on the phase transition temperature and cooperativity of the lipid molecules in the absence and presence of the drug. Vibrational spectroscopy is well suited to the study of drug-lipid interactions, since it allows for an investigation of the conformation of phospholipid molecules at different levels in lipid bilayers and follows structural changes that occur during the gel to liquid-crystalline phase transition. NMR supported the determination of the main phase transition temperatures (TC) of 1,2-dihexadecanoyl-sn-glycerol-3-phosphocholine (DPPC). The main phase transition temperature (TC) determined by (1)H NMR is comparable with values obtained by DSC for all studied liposomes. The location of cytarabine and etoposide in liposomes was also determined by NMR. Atomic force microscopy (AFM) images, acquired immediately after sample deposition on a mica surface, revealed the spherical shape of lipid vesicles.

Diaquadichloridobis(1H-imidazole)manganese(II) at 100 K.

The mononuclear title complex, [MnCl(2)(C(3)H(4)N(2))(2)(H(2)O)(2)], is located on a crystallographic inversion center. The Mn(II) ion is coordinated by two imidazole ligands [Mn-N = 2.2080 (9) A], two Cl atoms [Mn-Cl = 2.5747 (3) A] and two water molecules [Mn-O = 2.2064 (8) A]. These six monodentate ligands define an octahedron with almost ideal angles: the adjacent N-Mn-O, N-Mn-Cl and O-Mn-Cl angles are 90.56 (3), 92.04 (2) and 90.21 (2) degrees , respectively. Hydrogen bonds between the coordinated water molecules and Cl atoms form a two-dimensional network parallel to (100) involving R(4)(2)(8) rings. The two-dimensional networks link into a three-dimensional framework through weaker N-H...Cl interactions. Thermogravimetric analysis results are in accordance with the water-coordinated character of the substance and its dehydration in two successive steps.

Liposomes formed in sintered glass pores.

The method for preparation of vesicles, by evaporation of hydrophobic solvent from double emulsion (w/o/w) formed in the properly designed device is described. These method leads to multiple increase of encapsulation efficiency of aqueous solutions of drug in liposomes in comparison with other method. The w/o/w was passed through the glass sinter with the use of negative pressure to disrupt w/o/w drops into smaller ones. At low pressure and at heigher temperature, the hydrophobic solvent from oil phase evaporated off and the lipids that were diluted in oil phase had created bilayer. When the relatively small quantity of lipids was used, the final encapsulation efficiency (ee) was about 50% and the uppermost encapsulation volume (ev) was 160 mL/g of lipids. Similar ee was noted for a 4-amino-10-methylfolic acid (MTX), Patent Blue V (PB) and bovine serum albumin (BSA). Liposomes loaded with drug at high concentration may be easily separated from suspension with the use of simple centrifugation.