Characterization of lipophilic gemcitabine prodrug-liposomal membrane interaction by differential scanning calorimetry

Gemcitabine is an anticancer agent rapidly deaminated to the inactive metabolite 2',2'-difluorodeoxyuridine. Its stability as well as bioavailability can be increased by making prodrugs. A series of lipophilic prodrugs of gemcitabine were synthesized by linking the 4-amino group with valeroyl, lauroyl, and stearoyl linear acyl derivatives. We studied, by the differential scanning calorimetry technique, and compared the interaction of pure gemcitabine and its prodrugs with dimyristoylphosphatidylcholine and distearoylphosphatidylcholine vesicles with the aim of demonstrating if the gemcitabine prodrug is more able than the pure gemcitabine to interact with lipid vesicles employed both as model biomembranes and as carriers in the transport of antitumor drugs. These studies, carried out by static and kinetic calorimetric measurements, give evidence that the increase of the prodrug's lipophilic character improves the interaction with lipid bilayers, favoring the absorption through the lipid barriers and allowing the liposomes to work (when the prodrug is inserted inside the vesicles) as a lipophilic carrier which is able to deliver the drug near the cell surface. The use of different prodrugs modified in their lipophilic character, of different kinds of vesicles (multilamellar and unilamellar), and of different kinds of vesicles forming phospholipids permitted us to determine the better equilibrium between in-vesicle solubility and through-vesicle diffusion of the drug, important in the preformulative studies of antitumor carriers based on phospholipid formulations. Such studies suggest that the prodrug lipophilic tail should modulate the transport and the release of gemcitabine inside the cellular compartments, and the efficiency of the liposomal system is related to the length of the prodrug's acyl chain which has to match the phospholipid acyl chain allowing or retarding the migration through the lipid release device.     

The interaction of cortisone esters with liposomes as studied by differential scanning calorimetry

The interaction of a series of cortisone esters with dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles (MLV) has been investigated using differential scanning calorimetry (DSC). The extent of the interaction is dependent on the ester chain-length with increased interaction observed as the chain lengthens. For cortisone hexadecanoate the maximum incorporation is 11.25 mole· %. The presence of cholesterol excludes cortisone hexadecanoate from DPPC liposomes. This effect increases with increasing cholesterol concentrations.Cortisone hexadecanoate is incorporated into DPPC liposomes in such a manner that it is probable that the steroid moiety interacts strongly with the bilayer.A correlation exists between the in vitro cortisone ester release rate at 37°C from DPPC liposomes and broadening of DPPC MLV thermograms only over the range of 6–14 carbon chain-length. This indicates that the interaction of the ester chain of the steroid with the acyl chain of the lecithin are not necessarily the sole factors controlling efflux rate.     

Interaction of vinblastine sulfate with artificial phospholipid membranes. A study by differential scanning calorimetry and spin labeling

The effect of the antimitotic drug vinblastine sulfate has been studied on fully hydrated dipalmitoylphosphatidylcholine (DPPC) liposomes in the temperature range 0 degrees to 60 degrees using differential scanning calorimetry and electron spin resonance spectroscopy with two fatty acid spin labels. In the gel phase, vinblastine interacts essentially with the DPPC polar heads and induces an important disorganization of the phospholipidic bilayer. The co-operativity of the main thermal transition is decreased. In the crystal-liquid phase, the drug penetrates inside the artificial membrane and induces the formation of domains which increased thermal stability. These effects are opposite to those observed with the drug isaxonine which is used to reduce the axonal degenerating effects due to vinblastine.     

Investigation of molecular interactions between paclitaxel and DPPC by Langmuir film balance and differential scanning calorimetry

Molecular interactions between paclitaxel and dipalmitoylphosphatidyl choline (DPPC) were investigated by Langmuir film balance and differential scanning calorimetry (DSC). Both the lipid monolayer at the air-water interface and that in the lipid bilayer vesicles (liposomes) were employed as model cell membranes. Thermodynamic and kinetic analyses of the DPPC/pacltaxel monolayer system and the paclitaxel penetration into the DPPC monolayer showed that DPPC and paclitaxel can form a nonideal miscible system in the lipid monolayer over a wide range of the DPPC/paclitaxel molar ratios. Paclitaxel exerts an area-condensing effect on the DPPC monolayer at small molecular areas and an area-expanding effect at large molecular areas on the pi-A behavior of the DPPC monolayer, which can be explained by the intermolecular forces and geometric accommodation between paclitaxel and DPPC. Based on a calculation of the excess free energy of the mixed monolayer system, the most stable state of the system occurs at the monolayer composition of 5% paclitaxel. Penetration kinetics showed that the paclitaxel penetration into the DPPC monolayer increases with increasing the drug concentration in the subphase, but there is a limit of approximately 500 ng/mL. Any further increase in paclitaxel concentration had no additional significant effects on the drug penetration. Differential scanning calorimetry showed that paclitaxel caused broadening of the main phase transition. There was no significant change in the peak melting temperature of the DPPC bilayers, which demonstrated that paclitaxel was localized in the outer hydrophobic cooperative zone of the bilayer.     

Isaxonine base is a strong perturber of phospholipid bilayer order and fluidity--a differential scanning calorimetry and spin labeling study

The effects of the neurotropic drug isaxonine on fully hydrated dipalmitoyl-phosphatidyl-choline (DPPC) bilayers has been studied in the temperature range 0 degree-60 degrees, using differential scanning calorimetry and electron spin resonance spectroscopy, with two stearic acid spin labels. At low concentration (1% mol/mol), isaxonine is trapped in the polar interface and enhances the phospholipid multibilayers organization in the gel state. In contrast, at high concentration (30% mol/mol), the drug disorganizes the phospholipidic structures and may induce domain formation by phase separation. The strong interactions of isaxonine at the lipid-water interface change the ionization state of the stearic acid spin labels which become totally ionized. Then isaxonine acts as a modifier of the surface pH of the bilayer. The strong membrane effects of isaxonine may explain in part its pharmacological properties in vivo.     

PAMAM dendrimers and model membranes: differential scanning calorimetry studies

Dendrimers attract much attention as potential drug and gene carriers for intracellular delivery. From this point of view, it is crucial to extend our knowledge about their interactions with membranes. The influence of polyamidoamine (PAMAM) dendrimers on the thermotropic behavior of DPPC multilamellar vesicles and DMPC small unilamellar vesicles was examined by differential scanning calorimetry. We used three types of PAMAM dendrimers to determine how a dendrimer structure determines interactions with liposomes. We show that the strength of interactions depends on both the dendrimers' structure and degree of hydrophobicity. A model for the interaction of each type of dendrimer with liposomes was proposed.     

Characterization of latex-antineoplastic drug complexes by differential scanning calorimetry and microphotography

Choline kinase inhibitors have recently been identified as potentially useful antitumoral agents. Here we determine the best conditions for obtaining drug-polymer complexes with 5-fluorouracil (5-FU), and JCR791B, a new drug representing a significant advance in the development of new molecules to inhibit tumour proliferation. As polymers we used the cellulose derivatives Aquacoat and Aquateric. The variables in the adsorption process measured were time to adsorbent-adsorbate equilibrium, pH and concentration. The drug-polymer complexes were characterized by differential scanning calorimetry and microphotography. Our results show that adsorption of 5-FU and JCR was similar with both polymers although slightly greater with Aquacoat. The chemical structure of the drug and its solubility in water and oil are fundamental characteristics that determine the performance of polymers as drug carriers able to provide controlled release.     

Interaction of cortisol-21-palmitate with liposomes examined by differential scanning calorimetry.

Liposomes have been suggested as carriers for corticosteroids in the local treatment of arthritis by intra-articular injection. The long chain 21-esters of cortisol such as the palmitate or octanoate are taken up and retained by liposomes in higher concentration than cortisol itself. Differential scanning calorimetry has been used to show that the cortisol ester is anchored in the liposome phospholipid bilayer by the acyl side chain. In addition, the limiting concentration of cortisol-21-palmitate which can be incorporated into dipalmitolyl phosphatidylcholine liposomes has been measured by observing changes in the DSC spectrum at different steroid concentrations. Steroid in excess of this concentration limit forms a separate phase which can be identified by nuclear magnetic resonance. For optimum effect, the treatment of arthritis with liposomes must be carried out with liposomes containing steroid below the limiting concentration.     

Further investigations into the use of high sensitivity differential scanning calorimetry as a means of predicting drug-excipient interactions

The early prediction of drug-excipient incompatibility is vital in the pharmaceutical industry to avoid costly material wastage and time delays. We report here on the use of high sensitivity differential scanning calorimetry (HSDSC) to examine the compatibility between an experimental drug (Drug A) and common pharmaceutical excipients. Short-term HSDSC experiments (up to 25h) indicated that Drug A was stable in the presence of moisture and was compatible with both lactose monohydrate and magnesium stearate in the dry state, but showed degradation in the presence of magnesium stearate and water in combination. These results agreed with conventional stability studies, in which extensive degradation was observed in the Drug A-magnesium stearate system after storage at 40 degrees C/75% RH for 4 weeks but not under other conditions. These results indicate that HSDSC may be used to examine the compatibility of experimental drugs with conventional excipients and, in particular, illustrate the importance of incorporating humidity as an experimental variable in order to fully establish the stability profile of the material under test.     

Characterization of lipid nanoparticles by differential scanning calorimetry, X-ray and neutron scattering

Differential scanning calorimetry and X-ray diffraction play a prominent role in the characterization of lipid nanoparticle (LNP) dispersions. This review shortly outlines the measurement principles of these two techniques and summarizes their applications in the field of nanodispersions of solid lipids. These methods are particularly useful for the characterization of the matrix state, polymorphism and phase behavior of the nanoparticles which may be affected by, for example, the small particle size and the composition of the dispersions. The basics of small angle X-ray and neutron scattering which are also very promising methods for the characterization of LNPs are explained in some more detail. Examples for their use in the area of solid LNPs regarding the evaluation of particle size effects and the formation of superstructures in the nanoparticle dispersions are given. Some technical questions concerning the use of the different characterization techniques in the field of LNP research are also addressed.     

Characterization of dimethyldiacyloxysilanes by differential scanning calorimetry, Raman scattering and X-ray diffraction

The phase behaviour of diacyloxydimethylsilanes (DMS Cn; n = 10, 12, 14, 16, 18, 20, 22) was investigated by differential scanning calorimetry, X-ray diffraction and Raman spectroscopy. All DMS Cn melt from a crystalline phase to an isotropic liquid with a single sharp transition. On cooling, the homologous DMS C16 up to DMS C22 show a characteristic monotropic phase (L beta'H). In contrast to the calorimetrical investigations, it was not possible to analyse the monotropic phase of DMS C16 by X-ray diffraction. This behaviour is due to a two-phase region (gel phase--crystalline phase). The Raman spectra of all DMS are very similar. Only in the low frequency range we find different bands of the longitudinal acoustic modes. The Raman measurements demonstrate undoubtedly that in the solid state the alkyl chains are in all-trans conformation. The factor group splitting of the CH2 scissoring Raman mode show that the DMS Cn are arranged in a subcell packing with two molecules per unit cell. The highly ordered all-trans structure of the alkyl chains is present up to the melting transition. On melting there are changes in different regions of the Raman spectra: C-H stretching, CH2 scissoring mode, C-C skeletal stretching, CH3 rocking and longitudinal acoustic modes. On cooling DMS C18 and DMS C20 from the melt to the crystalline state, the gel phase is also proved by Raman scattering. Based on the results of the Raman and X-ray data the gel phase is characterized by a hexagonal subcell packing and by an ordered structure of the alkyl chain residues.     

Quantification of the leaching of triethyl citrate/polysorbate 80 mixtures from Eudragit RS films by differential scanning calorimetry.

The influence of triethyl citrate and polysorbate 80 (Tween 80) on the glass transition temperature (T(G)) of Eudragit RS membranes was investigated using differential scanning calorimetry (DSC). The T(G)-decreasing effect of TEC and Tween 80 displayed an almost identical performance in extent at a linear relationship between weight proportion and T(G) resulting in a specific T(G)-decrease (T(G,spec.)) of -1.98(K/%TEC) and -1.86(K/%Tween), respectively. Thus, the proportion of each adjuvant could be summarized as the plasticizer complex weight proportion (PC) with T(G,spec.)=1.96(K/%PC). Vice versa this linear relationship could be used to determine the proportion of plasticizer complex within the polymer membrane after swelling and diffusion processes, i.e. plasticizer leaching. For membranes containing 20% (w/w) TEC and 8% (w/w) Tween 80 as plasticizer complex a fast leaching resulted during the dissolution test reaching an equilibrium at 6.08% (+/-0.5) PC after 30 min in demineralised water. The DSC method proved to be a simple method to determine plasticizer leaching via T(G), however, without respect on the film forming properties of the two different excipients. Plasticizing with TEC or TEC/Tween 80 mixtures led to smooth and continuous films, while plasticizing with Tween 80 only resulted in mosaic like fissured films.     

Assessment of fluidity of different invasomes by electron spin resonance and differential scanning calorimetry

The aim of this study was to investigate the influence of membrane-softening components (terpenes/terpene mixtures, ethanol) on fluidity of phospholipid membranes in invasomes, which contain besides phosphatidylcholine and water, also ethanol and terpenes. Also mTHPC was incorporated into invasomes in order to study its molecular interaction with phospholipids in vesicular membranes. Fluidity of bilayers was investigated by electron spin resonance (ESR) using spin labels 5- and 16-doxyl stearic acid and by differential scanning calorimetry (DSC). Addition of 1% of a single terpene/terpene mixture led to significant fluidity increase around the C16 atom of phospholipid acyl chains comprising the vesicles. However, it was not possible to differentiate between the influences of single terpenes or terpene mixtures. Incorporation of mTHPC into the bilayer of vesicles decreased fluidity near the C16 atom of acyl chains, indicating its localization in the inner hydrophobic zone of bilayers. These results are in agreement with DSC measurements, which showed that terpenes increased fluidity of bilayers, while mTHPC decreased fluidity. Thus, invasomes represent vesicles with very high membrane fluidity. However, no direct correlation between fluidity of invasomes and their penetration enhancing ability was found, indicating that besides fluidity also other phenomena might be responsible for improved skin delivery of mTHPC.     

Determination of the free/included piroxicam ratio in cyclodextrin complexes: comparison between UV spectrophotometry and differential scanning calorimetry.

Few analytical techniques allow to evaluate the inclusion yield of cyclodextrin-drug complexes, because most manufacturing processes give amorphous products. In this study, we have developed an alternative method to differential scanning calorimetry, to accurately determine the free/complexed piroxicam ratio by UV spectroscopy. This method is based on the differential solubility of the piroxicam-beta-cyclodextrin 1:2.5 mol/mol complex in water-acetonitrile (1:1, v/v) (Solvent A) or in anhydrous acetonitrile (Solvent B), both containing 0.05 M HCl. In anhydrous acetonitrile, beta-cyclodextrin is insoluble and the included drug remains entrapped, allowing the free piroxicam determination, while with 50% of water, the complex is totally dissolved, allowing the determination of the total guest content. This method was validated for linearity, precision and accuracy. The presence of cyclodextrin does not influence the assays, but more than 0.5% of water in Solvent B significantly affects the determination of the free piroxicam content. In comparison with differential scanning calorimetry, both detectability and precision were improved. It is now possible to analyse complexes with an inclusion purity greater than 99%.     

Interaction of calcium and neomycin with anionic phospholipid-lecithin liposomes. A differential scanning calorimetry study

The interactions of calcium and neomycin with liposomes of various anionic phospholipids plus lecithin were studied by differential scanning calorimetry. Phosphatidylinositol bisphosphate differed from other acidic phospholipids in its interactions with both calcium and neomycin. Calcium, at concentrations as low as 1 mM, induced the appearance of a second transition peak in phosphatidylinositol bisphosphate-enriched liposomes only. Neomycin acted antagonistically and precluded this phase separation. In addition, neomycin lowered the phase transition temperature of phosphatidylinositol bisphosphate-lecithin liposomes while it raised the transition temperature of all other anionic phospholipid-lecithin liposomes tested. This fluidizing effect of neomycin and the antagonism to calcium may induce critical alterations of properties of biological membranes. The study supports and extends our previous findings and conclusions that phosphatidylinositol bisphosphate may play a crucial role in the expression of aminoglycoside toxicity.     

Molecular mechanisms of photosensitization XIII: a combined differential scanning calorimetry and DNA photosensitization study in non steroidal antiinflammatory drugs-DNA interaction.

A combined differential scanning calorimetry (DSC) and photosensitization study has been carried out on the interaction of several NSAID on DNA, both from calf thymus and pBR 322 plasmid. The investigated compounds were both non-steroidal anti-inflammatory drugs as well as compounds related to NSAIDs for structural similar properties, to find evidence for their ability to interact with DNA as a function of steric hindrance and polarity of the chemical structures. The considered NSAIDs were diflunisal (DFN, a salicylic derivative), naproxen (NAP), ketoprofen (KPF), suprofen (SPF) and tiaprofenic acid (TIA, arylpropionic acids). The structural criterion used was related to three different aromatic groups, biphenyl, naphthalene and benzophenone (BZP). In fact drug-DNA interaction can be revealed by variations of the enthalpies and temperatures of unfolding of DNA obtained by comparison of calorimetric peaks, where a decrease of the enthalpy is associated with the drug-DNA interaction, by engaging electrostatic bonds. Testing their ability in inducing DNA cleavage when UVA irradiated can evidence the photosensitizing properties of the drug. A good correlation was found between calorimetric and photosensitization studies. From the results obtained it can be reasonably supposed that the photocleavage depends only on the drug molecules bound to DNA. Copyright