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.

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.

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.

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.

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.

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.

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.

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.