The effect of poly(d,l-lactide-co-glycolide)-alendronate conjugate nanoparticles on human osteoclast precursors.

Nanoparticles (NPs) formed from polymers conjugated with bisphosphonates (BPs) allow the bone targeting of loaded drugs, such as doxorubicin, for the treatment of skeletal tumours. The additional antiosteoclastic effect of the conjugated BP could contribute to the inhibition of tumour-associated bone degradation. With this aim, we have produced NPs made of poly(d,l-lactide-co-glycolide) (PLGA) conjugated with alendronate (ALE). To show if ALE retained the antiosteoclastic properties after the conjugation with PLGA and the production of NPs, we treated human osteoclasts, derived from circulating precursors, with PLGA-ALE NPs and compared the effects on actin ring generation, apoptosis and type-I collagen degradation with those of free ALE and with NPs made of pure PLGA. PLGA-ALE NPs disrupted actin ring, induced apoptosis and inhibited collagen degradation. Unexpectedly, also NPs made of pure PLGA showed similar effects. Therefore, we cannot exclude that in addition to the observed antiosteoclastic activity dependent on ALE in PLGA-ALE NPs, there was also an effect due to pure PLGA. Still, as PLGA-ALE NPs are intended for the loading with drugs for the treatment of osteolytic bone metastases, the additional antiosteoclastic effect of PLGA-ALE NPs, and even of PLGA, may contribute to the inhibition of the disease-associated bone degradation.

Interaction of naproxen amphiphilic derivatives with biomembrane models evaluated by differential scanning calorimetry and Langmuir-Blodgett studies.

Anti-inflammatory drugs represent a potential new strategy for the treatment of Alzheimer's disease (AD). The ability to cross the blood-brain barrier and to reach brain tissues is a critical point for these drugs and is strictly related to their lipophilicity. Naproxen (NAP) is a non-steroidal anti-inflammatory drug (NSAIDs) under active investigation for AD. To improve its lipophilic character, NAP was conjugated through a diethylamine spacer (EDA) to lipoamino acids (LAA), α-amino acids containing a long alkyl side chain, to obtain the NAP-EDA-LAA10 and NAP-EDA-LAA14 prodrugs. The interaction of NAP and prodrugs with dimyristoylphosphatidylcholine phospholipids, forming either multilamellar vesicles or monolayers (at the air/water interface) and used as biomembrane models, was studied by differential scanning calorimetry and Langmuir-Blodgett techniques. Experimental data showed that NAP conjugation with LAA residues was able to enhance the drug interaction with such biomembrane models.

Evaluation of the interaction of coumarins with biomembrane models studied by differential scanning calorimetry and Langmuir-Blodgett techniques.

Three coumarins, scopoletin (1), esculetin (2), and esculin (3), were investigated by differential scanning calorimetry and Langmuir-Blodgett techniques to gain information about the interaction of these compounds with cellular membranes. Phospholipids assembled as multilamellar vesicles or monolayers (at the air-water interface) were used as biomembrane models. Differential scanning calorimetry was employed to study the interaction of these coumarins with multilamellar vesicles and to evaluate their absorption by multilamellar vesicles. These experiments indicated that 1-3 interact in this manner to different extents. The Langmuir-Blodgett technique was used to study the effect of these coumarins on the organization of phospholipids assembled as a monolayer. The data obtained were in agreement with those obtained in the calorimetric experiments.

Interaction of acyclovir and its squalenoyl-acyclovir prodrug with DMPC in monolayers at the air/water interface.

Acyclovir has been conjugated to the acyclic isoprenoid chain of squalene to form the squalenoyl-acyclovir prodrug. Its interaction with biomembrane models constituted by dimyristoylphosphatidylcholine (DMPC) monolayers has been studied by employing the Langmuir-Blodgett technique. The aim of the work was to gain information on the interaction of these compounds with phospholipid membranes. DMPC/acyclovir or squalenoyl-acyclovir prodrug mixed monolayers have been prepared at increasing molar fractions of the compound and the isotherm mean molecular area/surface pressure has been registered at 10 and 37 degrees C. Results reveal that the squalenoyl moiety enhances the affinity of acyclovir for the biomembrane model.

Conjugation of squalene to acyclovir improves the affinity for biomembrane models.

Differential scanning calorimetry was used to study the interaction of acyclovir and its prodrug squalenoyl-acyclovir (obtained by conjugation of 1,1',2-tris-nor-squalene acid (squaleneCOOH) with acyclovir) with biomembrane models made of DMPC multilamellar vesicles with the aim to verify whether a stronger interaction of the prodrug with respect to the free drug can be obtained. Multilamellar vesicles were prepared in the presence of increasing molar fractions of acyclovir, squaleneCOOH or prodrug and the effect of the compounds on the thermotropic behavior of vesicles was researched, revealing no effect of acyclovir but a strong effect of squaleneCOOH and prodrug. To evaluate if acyclovir, squaleneCOOH and prodrug can be absorbed by the biomembrane model, an experiment was carried out in which the considered compounds were left in contact with the biomembrane model and their eventual uptake was evaluated analyzing the effect on the thermotropic behavior of the biomembrane model. A very small uptake was revealed for all the compounds. To check the potential use of liposomes as a delivery system for the prodrug, the biomembrane models were incubated with liposomes loaded with the compounds and the compounds transferring from the loaded liposomes to the unloaded biomembrane model was followed. The results suggest that liposomes could be used to deliver the squalenoyl-acyclovir to the biomembrane model.

Evaluation of percutaneous absorption of the repellent diethyltoluamide and the sunscreen ethylhexyl p-methoxycinnamate-loaded solid lipid nanoparticles: an in-vitro study.

OBJECTIVES: Diethyltoluamide and ethylhexyl p-methoxycinnamate (OMC) are two active ingredients in insect repellent and sunscreen products, respectively. The concurrent application of these two substances often increases their systemic absorption, compromising the safety and efficiency of the cosmetic product. In this study, diethyltoluamide and OMC were incorporated into solid lipid nanoparticles, a colloidal drug delivery system, to reduce percutaneous absorption and avoid toxic effects and also maintain the efficacy of the two active compounds on the skin surface for a long duration. METHODS: Solid lipid nanoparticles were prepared based on an ultrasonication technique and characterized by differential scanning calorimetry (DSC) analyses. In-vitro studies determined the percutaneous absorption of diethyltoluamide and OMC. KEY FINDINGS: DSC data carried out on unloaded and diethyltoluamide- and/or OMC-loaded solid lipid nanoparticles highlighted that diethyltoluamide and OMC modified the temperature and the enthalpy change associated to the calorimetric peak of solid lipid nanoparticles. The concurrent presence of the two compounds in the solid lipid nanoparticles caused a synergic effect, indicating that the lipid matrix of nanoparticles guaranteed a high encapsulation of both diethyltoluamide and OMC. Results from the in-vitro study demonstrated that the particles were able to reduce the skin permeation of the two cosmetic ingredients in comparison with an oil-in-water emulsion. CONCLUSIONS: This study has provided supplementary evidence as to the potential of lipid nanoparticles as carriers for topical administration of cosmetic active compounds.

A novel biomaterial for osteotropic drug nanocarriers: synthesis and biocompatibility evaluation of a PLGA-ALE conjugate.

BACKGROUND & AIMS: Osteotropic drug-delivery systems have been proposed as a means to provide drugs with affinity to bone tissues. Drugs or proteins have been linked chemically to bone-seeking agents, such as bisphosphonates (BPs); alternatively, drug-loaded nanoparticles have been used to target specific tissues, such as tumor areas. In our current research, these approaches were merged by synthesizing a novel bone-seeking polymer conjugate, from which targetable nanoparticles can be produced. MATERIALS & METHODS: An amino-BP, alendronate (ALE) was bound covalently to a biodegradable polymer, poly(lactic-co-glycolide) (PLGA), containing a free end carboxylic group. Blood compatibility and cytotoxicity were assessed in vitro. RESULTS & DISCUSSION: By a classical solvent-evaporation method, nanoparticles with a mean size of 200-300 nm were prepared from the conjugate; sterilization was achieved by gamma-irradiation, confirming their potential as injectable drug nanocarriers. Owing to the presence of the BP residue, PLGA-ALE nanoparticles were adsorbed onto hydroxyapatite to a higher extent than pure PLGA nanoparticles. The PLGA-ALE conjugate did not induce either hemolysis or alterations of the plasmatic phase of coagulation, or cytotoxic effects on endothelial cells and trabecular osteoblasts. CONCLUSION: The prepared conjugate represents a novel biomaterial that is able to provide nanoparticles, which can be further loaded with drugs, such as anticancer agents, and addressed to osteolytic or other bone diseases.

Absorption of nitro-polycyclic aromatic hydrocarbons by biomembrane models: effect of the medium lipophilicity.

To demonstrate the relationship between the structure of nitro-polycyclic aromatic hydrocarbons and their effect on biomembranes, we have investigated the influence of three structurally different nitro-polycyclic aromatic hydrocarbons, 2-nitrofluorene, 2,7-dinitrofluorene and 3-nitrofluoranthene, on the thermotropic behavior of dimyristoylphosphatidylcholine multilamellar vesicles, used as biomembrane models, by means of differential scanning calorimetry. The obtained results indicate that the studied nitro-polycyclic aromatic hydrocarbons affected the thermotropic behavior of multilamellar vesicles to various extents, modifying the pretransition and the main phase transition peaks and shifting them to lower temperatures. The effect of the aqueous and lipophilic medium on the absorption process of these compounds by the biomembrane models has been also investigated revealing that the process is hindered by the aqueous medium but strongly allowed by the lipophilic medium.

Differential scanning calorimetry study on drug release from an inulin-based hydrogel and its interaction with a biomembrane model: pH and loading effect.

Inulin has been derivatized with methacrylic anhydride (MA) and succinic anhydride (SA) to obtain a methacrylated/succinilated derivative (INU-MA-SA) able to produce a pH sensitive hydrogel after UV irradiation. The hydrogel was characterized and loaded with diflunisal (10.4, 17 and 24%, w/w) chosen as a model drug. The drug release from INU-MA-SA-based hydrogel to a biomembrane model made by unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) was investigated at pH 4.0 and 7.4 by differential scanning calorimetry (DSC) that appears to be a suitable technique to follow the transfer kinetics of a drug from a controlled release system to a biomembrane model. The drug release from the hydrogel was compared with the dissolution of drug solid form by examining the effects exerted on the thermotropic behaviour of the DMPC unilamellar vesicles. The transferred drug and the release rate were affected by the drug loading as well as by the pH of the external medium. In particular the release was not linearly related to the drug loading but an intermediate loading allowed a better release at both investigated pHs, with a faster and more complete release observed at pH 7.4.

Biocompatibility of poly(D,L-lactide-co-glycolide) nanoparticles conjugated with alendronate.

Nanoparticles made of a conjugate of poly(D,L-lactide-co-glycolide) with alendronate (PLGA-ALE NPs), were prepared by emulsion/solvent evaporation technique. The conjugation yield, determined by MALDI TOF analysis, was 30-35%. PLGA-ALE NPs size, evaluated by photon correlation spectroscopy, was 198.7+/-0.2 nm. Haemocompatibility studies using different concentrations of PLGA-ALE NPs did not show any significant effect on haemolysis, leukocyte number, platelet activation, APTT and complement consumption, in comparison with blood incubated with phosphate buffered saline (PBS). A significant reduction of the prothrombin activity was demonstrated after incubation with 560 microg/ml of PLGA-ALE NPs; a significant increase was observed at the highest dilutions. The viability of human umbilical vein endothelial cells and bone marrow stromal cells (BMSC), evaluated through the neutral red test, was not affected by PLGA-ALE NPs. There were no significant differences in cell-associated alkaline phosphatase between BMSC incubated with PLGA-ALE NP- and PBS-added media. These results demonstrated that PLGA-ALE NPs had an acceptable degree of blood compatibility and were not cytotoxic; therefore, they may be considered suitable for intravenous administration.

Enhancement of gemcitabine affinity for biomembranes by conjugation with squalene: differential scanning calorimetry and Langmuir-Blodgett studies using biomembrane models.

Molecular interactions between gemcitabine, alone or conjugated with squalene to form the gem-squalene prodrug, with dimyristoylphosphatidylcholine have been investigated by differential scanning calorimetry and Langmuir film balance techniques to gain information about the interaction of gemcitabine and its prodrug with mammalian cell membranes and to evaluate the potential of liposomes as a delivery system for gemcitabine prodrugs. Phospholipids assembled as multilamellar vesicles or monolayers (at the air water interface) have been used as biomembrane models. Different interactions of gemcitabine, its prodrug, and squalene with the lipid were detected by dispersing the compounds in the MLV and were compared with kinetic experiments carried out to consider the ability of the examined compounds to dissolve in an aqueous medium, to migrate through it, and to be captured by multilamellar vesicles. Their ability to be released from drug-loaded liposomes and be taken up by empty vesicles mimicking biomembranes was also considered. Analysis of the differential scanning calorimetry curves reveals that gemcitabine has very little interaction with multilamellar vesicles whereas the gem-squalene prodrug strongly interacts with multilamellar vesicles. The kinetic experiments suggest that an aqueous medium does not permit the prodrug uptake by the biomembrane models, whereas it is allowed when gem-squalene is gradually released by the liposomes. The molecular area/surface pressure isotherms of the gemcitabine/lipid, gem-squalene/lipid, and pure compound monolayers, in agreement with the calorimetric results, indicate that gem-squalene interacts with the phospholipid monolayer with the squalene moiety in contact with the phospholipid chains and gemcitabine protruding in the aqueous medium.

Interaction of four monoterpenes contained in essential oils with model membranes: implications for their antibacterial activity.

The present article reports the antimicrobial efficacy of four monoterpenes (thymol, carvacrol, p-cymene, and gamma-terpinene) against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli. For a better understanding of their mechanism of action, the damage caused by these four monoterpenes on biomembranes was evaluated by monitoring the release, following exposure to the compounds under study, of the water-soluble fluorescent marker carboxyfluorescein (CF) from large unilamellar vesicles (LUVs) with different lipidic composition (phosphatidylcholine, PC, phosphatidylcholine/phosphatidylserine, PC/PS, 9:1; phosphatidylcholine/stearylamine, PC/SA, 9:1). Furthermore, the interaction of these terpenes with dimyristoylphosphatidylcholine multilamellar vesicles as model membranes was monitored by means of differential scanning calorimetry (DSC) technique. Finally, the results were related also with the relative lipophilicity and water solubility of the compounds examined. We observed that thymol is considerably more toxic against S. aureus than the other three terpenes, while carvacrol and p-cymene are the most inhibitory against E. coli. Thymol and carvacrol, but not gamma-terpinene and p-cymene, caused a concentration-dependent CF leakage from all kinds of LUVs employed; in particular, thymol was more effective on PC and PC/SA LUVS than on PC/PS vesicles, while carvacrol challenge evoked a CF leakage from PC/PS LUVs similar to that induced from PC/SA LUVs, and lower than that measured with PC vesicles. Concerning DSC experiments, these four terpenes caused a decrease in Tm and (especially carvacrol and p-cymene) DeltaH values, very likely acting as substitutional impurities. Taken together, our findings lead us to speculate that the antimicrobial effect of thymol, carvacrol, p-cymene, and gamma-terpinene may result, partially at least, from a gross perturbation of the lipidic fraction of the plasmic membrane of the microorganism. In addition to being related to the physicochemical characteristics of the compounds (such as lipophilicity and water solubility), this effect seems to be dependent on the lipidic composition and net surface charge of the microbic membranes. Furthermore, the compounds might cross the cell membranes, thus penetrating into the interior of the cell and interacting with intracellular sites critical for antibacterial activity.

Interaction of resveratrol and its trimethyl and triacetyl derivatives with biomembrane models studied by differential scanning calorimetry.

The interaction of resveratrol (trans-3,5,4'-trihydroxystilbene) and two of its derivatives (3,5,4'-tri-O-methylresveratrol and 3,5,4'-tri-O-triacetylresveratrol) with biomembrane models, represented by dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV), has been studied by differential scanning calorimetry (DSC). The analysis of MLV prepared in the presence of increasing molar fraction of such compounds has been carried out to reveal their maximum interaction with biomembrane models. The results from these studies have been compared with kinetic experiments results, in order to detect the entity and rate of compound absorption by the biomembrane models. The findings indicate that the compounds affected the thermotropic properties of DMPC MLV by suppressing the pretransition peak and broadening the DMPC main phase transition calorimetric peak and shifting it to lower temperatures. The order of effectiveness found was resveratrol > trimethylresveratrol > triacetylresveratrol. The kinetic experiments reveal that in an aqueous medium the absorption of resveratrol by the biomembranes models is allowed, whereas the absorption of its derivatives is hindered; in contrast when a lipophilic medium is employed, all three compounds are easily absorbed.

Differential scanning calorimetry evidence of the enhancement of beta-sitosterol absorption across biological membranes mediated by beta-cyclodextrins.

beta-Sitosterol is a plant sterol that has received much attention because of its effectiveness in reducing the absorption of dietary cholesterol, as well as in offering protection from cardiovascular diseases and cancer development. Thus, the knowledge of the interaction of beta-sitosterol with biological membranes can help in understanding its mechanism of action. In the present paper, the differential scanning calorimetry technique has been used to study the interaction of beta-sitosterol with a biomembrane model constituted by dimyristoylphosphatidylcholine multilamellar vesicles. Furthermore, kinetic experiments have been carried out to follow the uptake of beta-sitosterol by biomembranes and the effect of beta-cyclodextrins on such a process. Our results indicate that opportune concentrations of beta-cyclodextrins improve the uptake of beta-sitosterol by phospholipid membranes.

Biomimetic approach to biomembrane models studies: medium influence on the interaction kinetics of some phenylurea derivatives herbicides.

The ability of herbicides to interact with cell membranes outer lipid layer and subsequently to penetrate inside cells can be a prerequisite for exhibiting a toxic activity for both the directly exposed workers and the end consumers as the herbicides are present in the soil and water. The effect exerted by fenuron, chlorotoluron, metobromuron, monolinuron, and chlorbromuron, five structurally similar phenylurea herbicides, on the thermotropic behavior of model membranes, represented by dimyristoylphosphatidylcholine (DMPC) vesicles, was investigated by differential scanning calorimetry. The examined compounds, when dispersed in liposomes during their preparation, exerted a different action on the gel-to-liquid crystal phase transition of DMPC multilamellar vesicles. The ability of phenylurea herbicides, as a finely powdered solid, to migrate through an aqueous medium and interact with biomembrane models was also studied. This transfer process was compared with these compounds intermembrane transfer from herbicide-loaded liposomes to empty ones. These processes can mimic absorption kinetics mediated by hydrophilic or lipophilic media. Different rate and entity of interaction occurred between model membranes and solid phenylurea herbicides. Different behavior was observed by considering the time-dependent studies carried out by contacting, for increasing times, equivalent amounts of empty DMPC vesicles with phenylurea herbicide-loaded ones; all compounds were able to migrate from loaded to empty DMPC vesicles. Thus, phenylurea herbicides are able to reach and penetrate biological membranes when dispersed in a lipophilic or hydrophilic medium; these processes are related to the substituents present on the compounds backbone. The obtained experimental results seem to validate the employed strategy to study the ability of bioactive compounds to both interact with biological membranes and be adsorbed inside a membrane mimicking a biological cell when dispersed in a lipophilic or hydrophilic medium.