Poly(methoxypolyethyleneglycol cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to diffuse through the blood-brain barrier after intravenous administration. However, the mechanism of transport of these nanoparticles into brain has not yet been clearly elucidated. The development of a model of rat brain endothelial cells (RBEC) in culture has allowed investigations into this mechanism. A study of the intracellular trafficking of nanoparticles by cell fractionation and confocal microscopy showed that nanoparticles are internalized by the endocytic pathway. Inhibition of the caveolae-mediated pathway by preincubation with filipin and nystatin did not modify the cellular uptake of the nanoparticles. In contrast, chlorpromazine and NaN(3) pretreatment, which interferes with clathrin and energy-dependent endocytosis, caused a significant decrease of nanoparticle internalization. Furthermore, cellular uptake experiments with nanoparticles preincubated with apolipoprotein E and blocking of low-density lipoprotein receptors (LDLR) clearly suggested that the LDLR-mediated pathway was involved in the endocytosis of PEGPHDCA nanoparticles by RBEC.

In the last ten years folate targeting has emerged as one of the most promising approach in specific drug targeting towards solid tumors. Despite this growing interest, some of the cellular models generally used to investigate folate-targeted drug carriers lack accurate characterization. Furthermore, because of the variability of the folate receptor expression in vivo, it should be important to evaluate the carriers in a situation of increasing expression levels. The aim of this note is to present an in vitro model, consisting of three different cell lines (HeLa, KB 3–1 and MCF7 cells), in which the conditions leading to a reliable and constant over-expression of the folate receptor have been determined and the quantity of receptor present at the surface of the cells quantified. The selected cell lines express various amounts of the protein, from a non-detectable level in MCF7 cells to a high level of over-expression rate in KB cells, resulting in an interesting range of conditions useful for the investigation of folate-targeted vectors.

The aim of this work was to assess the influence of various formulation parameters on the incorporation of a poorly water-soluble crystalline drug into nanoparticles. For this purpose, the influence of the polymer (polylactic acid, polysebacic acid terminated with lithocholic acid, and polysebacic acid-co-lithocholic acid) as well as the effect of the dispersion medium (aqueous phases at different temperatures, saline medium and ethanol) on the encapsulation was investigated. 3H-labelled drug was used in order to determine the loading efficiency by liquid scintillation counting. The solubility of the drug in the various polymer materials was assessed by differential scanning calorimetry (DSC). The solubility of the drug in the different dispersion media was then determined by gas chromatographic-mass spectrometric measurements. The highest loading ratios were obtained using poly (lactic acid) (PLA). However, the drug solubility in the polymers, determined by DSC analysis, cannot be considered as predictive for encapsulation efficiency. The study of the influence of the liquid outer phase showed that the encapsulation efficiency increased when the drug solubility in the dispersion medium (before acetone evaporation) decreased. These experiments made it possible to propose a mechanism to account for the leakage of the crystalline drug during the nanoprecipitation process. So, when acetone is eliminated by evaporation, the drug solubility in the dispersion medium decreases, leading to the formation of crystals. During nanoparticles storage, the crystals continue to grow, the nanoparticles serving as drug reservoirs. These findings highlight the importance of using a polymer with a specific affinity for the drug, and a dispersion medium with the lowest drug solubility to achieve an efficient encapsulation of a crystalline drug.

OBJECTIVES:The aim of this study was to evaluate the toxicity of a new lipid complex formulation of amphotericin B (LC-AmB) produced by a simple process.METHODS:Toxicity was evaluated after daily administration for 21 consecutive days in female CD1 mice. Doses of LC-AmB up to 20 mg/kg were used, and compared with Fungizone at 0.5 mg/kg and Abelcet at 10 mg/kg. Acute toxicity after a single bolus injection was also determined, as well as the haemolytic activity and toxicity to mouse macrophages in vitro.RESULTS:LC-AmB reduced both the haemolytic activity of amphotericin B and its toxicity towards mouse peritoneal macrophages. Its acute toxicity (LD50 > 200 mg/kg in CD1 mice) was similar to that in the literature for the least toxic lipid formulations of amphotericin B. The relative liver weight increased slightly in mice treated daily with a dose of 20 mg/kg LC-AmB, as did the kidney weight in this group and the group treated with Fungizone. There was also a dose-dependent decrease in the haematocrit with all formulations. All treatments caused significant increases in transaminase levels. Total hepatic CYP 450 was slightly but not significantly increased in the groups treated with 20 mg/kg LC-AmB, Abelcet and Fungizone. However, expression of some isoforms of CYP 450 was reduced, the most marked being the hepatic CYP 3A1 after treatment with 20 mg/kg LC-AmB, Abelcet and Fungizone. The effects on hepatic function are probably related to accumulation in organs rich in phagocytic cells.CONCLUSION:LC-AmB did not induce any new toxicity compared with Abelcet and Fungizone.

Tamoxifen represents the endocrine treatment of choice for hormone-dependent breast cancer. However, undesirable side effects appeared after long-term therapy. Therefore, the concept of a drug delivery system at the tumour site is an interesting alternative. The aim of this work was to evaluate the potential of an original in situ forming gel to allow sustained delivery of tamoxifen. The gel forms spontaneously when two aqueous polymeric solutions are put in contact: a cyclodextrin (CD) polymer and dextran grafted with alkyl side chains. Some hydrophobic chains form inclusion complexes with CDs, leaving also free CDs available for the inclusion of drug molecules. Phase solubility studies showed a dramatically increased apparent solubility of tamoxifen in water (80-fold) in the presence of the CD polymer. Moreover, this drug was successfully entrapped in the gels, with loading efficiencies around 90%. Tamoxifen was released following a zero-order release profile during four days, followed by gel dissolution. The estimated amount of gel to be formed in situ to achieve in vivo anti-proliferative effects was found to be compatible with an injection. Moreover, this new concept opens a large domain of perspectives for other types of pathologies, where soft matrices allowing local drug sustained release are required.

In this article, microspheres able to induce the controlled release of oligonucleotide/polyethylenimine complexes are proposed. A model oligonucleotide (the oligothymidilate pdT16) was encapsulated within poly(lactide-co-glycolide) microspheres alone or associated with polyethylenimine (PEI) at different nitrogen/phospate (N/P) ratios. Microspheres were prepared by the multiple emulsion-solvent evaporation technique and characterized for morphology, diameter, encapsulation efficiency, and release kinetics. The introduction of PEI in the internal aqueous phase resulted in the formation of a soluble complex with pdT16 and in a strong increase of the oligonucleotide encapsulation efficiency. PEI affected microsphere morphology inducing the formation of very porous particles yielding to an accelerated release of pdT16. When incubated with HeLa cells, microspheres encapsulating pdT16/PEI complexes allowed both a reduction of the complex toxicity and an improvement of the intracellular penetration of the released oligonucleotide. We conclude that biodegradable microspheres encapsulating oligonucleotides/PEI complexes have a great potential as controlled release system because they allow the sustained release of an oligonucleotide carrier that crosses biological membranes and locates in nucleus.

When dealing with solid tumors in vivo, pegylated long-circulating carrier systems show, after intravenous administration, an attractive extravasation profile with an enhanced localization in the tumoral interstitium. These systems could be of help for the delivery of cancer fighting drugs, such as Tamoxifen, a well known antiestrogen used in breast cancer therapy that possesses an extended biodistribution in vivo. This work aimed at encapsulating Tamoxifen in long-circulating poly(MePEGcyanoacrylate-co-hexadecylcyanoacrylate) 1:4 nanospheres. Tamoxifen-loaded poly(MePEGcyanoacrylate-co-hexadecylcyanoacrylate) nanospheres were successfully synthesized and characterized in terms of hydrophilicity/hydrophobicity by a model made up from near infrared spectra using principal component analysis. Zeta potential, drug loading, encapsulation efficiency, as well as biological effect, in vitro release and nanospheres integrity were also investigated. Even though near infrared spectroscopy could not detect Tamoxifen, it revealed that Pluronic F68 was associated with the pegylated nanospheres. HPLC measurements demonstrated that Tamoxifen was encapsulated in the pegylated nanospheres following a partition equilibrium between the polymeric and the aqueous phases. The Tamoxifen encapsulated in the nanospheres still showed a transcription inhibitory activity in ex vivo experiments. However, zeta potential and in vitro release suggested that Tamoxifen was essentially localized at the nanoparticles surface, resulting in an important and immediate drug release.

ABSTRACT The present study compared the abilities of different lipid carriers of amphotericin B (AMB) to activate murine peritoneal macrophages, as assessed by their capacities to produce nitric oxide (NO) and tumor necrosis factor alpha (TNF-α). Although AMB alone did not induce NO production, synergy was observed with gamma interferon but not with lipopolysaccharide. This synergy could not be explained by the mobilization of the nuclear activation factor NF-κB by AMB. On the other hand, AMB induced TNF-α production without a costimulator and no synergy was observed. Anti-TNF-α antibodies did not influence NO production, and an inhibitor of NO synthase did not affect TNF-α production, indicating that the production of one of these effector molecules was independent of that of the other. The incorporation of AMB into lipid carriers reduced NO and TNF-α production with all formulations but more so with liposomes than with lipid complexes. NO production was correlated with the induction of NO synthase II, revealed by Western blotting. The extent of association of AMB with macrophages depended on the formulation, especially on the AMB/lipids ratio: the higher the ratio was, the greater the AMB association with macrophages. However, there was no clear correlation between AMB association with macrophages, whether internalized or bound to the membrane, and immunostimulating effects. These results may explain the reduced toxicities of lipid-based formulations of AMB.

The interactions of naked and surface-modified poly(D,L-lactic acid) (PLA) nanocapsules (NC), where polyethyleneglycol (PEG) was adsorbed or covalently attached, have been studied with a macrophage-like cell line. The fluorescent oil marker, DiD, was successfully encapsulated in NCs in order to follow their interactions with cells. The cell-associated fluorescence obtained with PEG-PLA NC was about 3- to 13-fold lower than that obtained with naked-PLA NC. The effects of PEG chain length, its content as a percentage of total polymer and NC concentration in the culture medium were evaluated. PEG-PLA NC showed dramatically reduced fluorescence association with cells during an 18 h incubation compared with naked-PLA NC, showing that covalent attachment of PEG is important for the persistence of low uptake. The best results in reducing cell-associated fluorescence were obtained with a surface-modified PEG-PLA NC bearing a chain with 20000 MW. Increasing the percentage of PEG produced a reduction in marker association for a given PEG chain length. Moreover, when the PEG-containing poloxamer was simply adsorbed, marker association was dependent on the extent of dilution and the type of serum in the culture medium. Serum proteins, especially immunoglobulins, increased cell-associated fluorescence for PEG-adsorbed NC, but had very little effect on PEG-PLA NC. Marker association was only partially inhibited in the presence of cytochalasin B. The mechanisms of cell-NC interaction depended on the characteristics of the NC surface in each formulation. When the NC was physically separated from cells no diffusion of fluorescent marker in aqueous medium occurred. Nevertheless, collision-mediated transfer of DiD from NC to J774 cells was a non-negligible route of marker transfer, mainly for naked NC. However, this collision-mediated transfer was reduced for the PEG-PLA NC probably due to the restricted contact between NC and cells afforded by PEG steric hindrance at the surface.

The aim of the present work was to investigate the biodistribution characteristics of PEG-coated polycyanoacrylate nanoparticles prepared by the nanoprecipitation/solvent diffusion method using the previously synthesized poly(MePEGcyanoacrylate-hexadecylcyanoacrylate) copolymer. It was observed that [14C]-radiolabeled PEGylated nanoparticles remained for a longer time in the blood circulation after intravenous administration to mice, compared to the non-PEGylated poly(hexadecylcyanoacrylate) (PHDCA) nanoparticles. Furthermore, hepatic accumulation was dramatically reduced, whereas a highly increased spleen uptake was shown. The PEGylation degree of the polymer seemed not to affect the in vivo behavior of the nanoparticles, whereas previously obtained in vitro data have shown a modification of plasma protein adsorption depending on the density of PEG at the surface of the particles. Moreover, the study of the in vitro cytotoxicity of the nanoparticles revealed that the PEGylation of the cyanoacrylate polymer reduced its toxicity. These results open up interesting perspectives for the targeting of drugs to other tissues than the liver.

Polyalkylcyanoacrylate (PACA) nanoparticles loaded with doxorubicin allowed multidrug resistance to be overcome in vitro. However, increased cytotoxicity is not always correlated with an increased level of intracellular drug. Although we have previously shown that PACA nanoparticles are not endocytosed by tumour cells, we report here that a direct interaction between nanoparticles and cells is a necessary requirement for overcoming resistance. In addition, the results showed that the degradation products of PACA (mainly polycyanoacrylic acid) in the presence of doxorubicin are able to increase both accumulation and cytotoxicity, thus suggesting the formation of a doxorubicin-polycyanoacrylic acid ion pair. It is therefore concluded that resistance is overcome as a result of both the adsorption of nanoparticles to the cell surface and increased doxorubicin diffusion by the accumulation of an ion pair at the plasma membrane.

The in-vitro and in-vivo toxicity and activity of a new emulsion-based delivery system for amphotericin B (AmB-E) and of deoxycholate-amphotericin B (Fungizone) were studied. In vitro, Candida albicans and human red blood cells (RBCs) were treated with either product and dose-response curves for various cellular effects (changes in potassium cell content, haemoglobin leakage from RBCs and colony-forming ability of fungal cells) were obtained. AmB-E was less toxic than Fungizone against human RBCs and equally active against C. albicans cells. In-vivo studies showed that the LD50 of AmB-E and Fungizone in noninfected OF1 mice were 7.24 and 3.46 mg/kg, respectively. The therapeutic efficacy of AmB-E was assessed in murine candidiasis. Firstly, the efficacy of equal doses (0.8 mg/kg) of AmB-E and Fungizone was evaluated in infected mice. Both formulations increased the survival time compared to the control and were equally effective in reducing the cfu counts in the kidney. In the same model of infection, the maximum tolerated doses (MTD) of Fungizone and AmB-E were determined in order to study the efficacies of Fungizone and AmB-E at their respective MTD. AmB-E significantly increased the number of long-term survivors compared with Fungizone (MTD:2 and 1 mg/kg, respectively). Thus, AmB-E was more effective than Fungizone for treatment of systemic mycoses at the MTD.

The present study evaluates the ability of a new drug carrier: nanocapsules of poly(D,L-lactide) containing muramyldipeptide-L-alanyl-cholesterol (MTP-Chol NC) to induce activation of mouse macrophage cell lines. MTP-Chol NC stimulated nitric oxide (NO) expression and tumor necrosis factor-α (TNF-α) production, these are two important mediators of macrophage-mediated cytotoxicity. The encapsulated form was more effective than free muramyldipeptide, at low immunomodulator concentrations. The dose-response curves were completely different for NO and TNF-α, implying different regulatory mechanisms. In RAW 264.7 cells, the addition of anti-TNF-α antibodies during the activation period did not affect the level of nitrite induced by MTP-Chol NC and lipopolysaccharide. Therefore, autocrine stimulation by TNF-α did not contribute to NO production. On the other hand, the presence of an NO synthase inhibitor led to an increase in TNF-α secretion. In J774.A1 cells, which were activated by MTP-Chol NC and interferon-γ, TNF-α production seemed to act as a second messenger. Thus, under certain conditions, NO can play a role in modulating the cytotoxic activities of mouse macrophages.

Conference Abstract| November 01 1996 INDUCTION OF NO-SYNTHASE ACTIVITY AND CYTOKINE PRODUCTION IN MACROPHAGES BY A CARRIER-ASSOCIATED MURAMYL PEPTIDE G. Barratt; G. Barratt 1URA CNRS 1218, Université Paris -Sud, 92296 CHATENAY-MALABRY, France Search for other works by this author on: This Site PubMed Google Scholar I. Seyler; I. Seyler 1URA CNRS 1218, Université Paris -Sud, 92296 CHATENAY-MALABRY, France Search for other works by this author on: This Site PubMed Google Scholar M; Appel; M; Appel 1URA CNRS 1218, Université Paris -Sud, 92296 CHATENAY-MALABRY, France Search for other works by this author on: This Site PubMed Google Scholar P. Legrand; P. Legrand 1URA CNRS 1218, Université Paris -Sud, 92296 CHATENAY-MALABRY, France Search for other works by this author on: This Site PubMed Google Scholar F. Puisieux; F. Puisieux 1URA CNRS 1218, Université Paris -Sud, 92296 CHATENAY-MALABRY, France Search for other works by this author on: This Site PubMed Google Scholar J.P. Devissaguet J.P. Devissaguet 1URA CNRS 1218, Université Paris -Sud, 92296 CHATENAY-MALABRY, France Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1996) 24 (4): 527S. https://doi.org/10.1042/bst024527s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation G. Barratt, I. Seyler, M; Appel, P. Legrand, F. Puisieux, J.P. Devissaguet; INDUCTION OF NO-SYNTHASE ACTIVITY AND CYTOKINE PRODUCTION IN MACROPHAGES BY A CARRIER-ASSOCIATED MURAMYL PEPTIDE. Biochem Soc Trans 1 November 1996; 24 (4): 527S. doi: https://doi.org/10.1042/bst024527s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1996 Biochemical Society1996 Article PDF first page preview Close Modal You do not currently have access to this content.

The structure of an amphotericin B emulsion-based delivery system (AmB-E) was investigated using spectroscopic methods (electronic absorption and circular dichroism (CD)), photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) at various amphotericin B (AmB) concentrations, in comparison with AmB-deoxycholate micelles, Fungizone® (Fungi-SS) and free AmB suspension (AmB-SS). Our results show that AmB-E absorption and CD spectra, mean particle size determination by PCS and morphological examination are only weakly concentration-dependent, between 5.10−5 and 5.10−7 M, as compared with Fungizone® and free AmB suspension. The only difference observed was at high dilution (5.10−8 M), where AmB dissociation appeared in spectroscopic studies, together with emulsion droplet fusion observed in PCS. These data seem to indicate that AmB could be located in the droplets of the emulsion, preferentially in the phospholipid layer, because AmB is not soluble in the oil inner phase. All these results suggest that AmB-E is a system which has potential applications and merits further evaluation.

The use of amphotericin B for the treatment of disseminated mycoses in AIDS and transplanted patients is limited by the toxicity of the commercial injectable form (Fungizone(R)). It has been demonstrated that an amphotericin B emulsion with Intralipid(R) is able to decrease both in vivo and in vitro toxicity. We propose here the application of two techniques of emulsion formation for the design of an amphotericin B injectable formulation. The first one is based on the spontaneous emulsification process, and the second uses the submerged jet homogenization process, the Microfluidizer(R). The physical parameters and the stability of the emulsion were evaluated. For the first method, the nature of the surfactants, their relative concentrations as well as that of oil, were important factors for the stability of the emulsion. The emulsion with 5 % (w/w) of oil and 1.2 % (w/w) of phospholipids gave the best results in stability. For the second technique, a pressure 5.5.10(4) Pa and four cycles makes the most stable emulsions. The absorption and circular dichroism spectra of amphotericin B emulsions at different concentrations were analyzed in order to determine the molecular state of the drug in these preparations.