Permeability of PEGylated immunoarsonoliposomes through in vitro blood brain barrier-medulloblastoma co-culture models for brain tumor therapy.

PURPOSE: Owing to restricted access of pharmacological agents into the brain due to blood brain barrier (BBB) there is a need: 1. to develop a more representative 3-D-co-culture model of tumor-BBB interaction to investigate drug and nanoparticle transport into the brain for diagnostic and therapeutic evaluation. 2. to address the lack of new alternative methods to animal testing according to replacement-reduction-refinement principles. In this work, in vitro BBB-medulloblastoma 3-D-co-culture models were established using immortalized human primary brain endothelial cells (hCMEC/D3). METHODS: hCMEC/D3 cells were cultured in presence and in absence of two human medulloblastoma cell lines on Transwell membranes. In vitro models were characterized for BBB formation, zonula occludens-1 expression and permeability to dextran. Transferrin receptors (Tfr) expressed on hCMEC/D3 were exploited to facilitate arsonoliposome (ARL) permeability through the BBB to the tumor by covalently attaching an antibody specific to human Tfr. The effect of anticancer ARLs on hCMEC/D3 was assessed. RESULTS: In vitro BBB and BBB-tumor co-culture models were established successfully. BBB permeability was affected by the presence of tumor aggregates as suggested by increased permeability of ARLs. There was a 6-fold and 8-fold increase in anti-Tfr-ARL uptake into VC312R and BBB-DAOY co-culture models, respectively, compared to plain ARLs. CONCLUSION: The three-dimensional models might be appropriate models to study the transport of various drugs and nanocarriers (liposomes and immunoarsonoliposomes) through the healthy and diseased BBB. The immunoarsonoliposomes can be potentially used as anticancer agents due to good tolerance of the in vitro BBB model to their toxic effect.

Development of poly(glycerol adipate) nanoparticles loaded with non-steroidal anti-inflammatory drugs.

The aim of this study was to assess acylated and non-acylated poly(glycerol adipate) polymers (PGA) as suitable nanoparticulate systems for encapsulation and release of ibuprofen, ibuprofen sodium salt (IBU-Na) and ketoprofen as model drugs. Drug encapsulated nanoparticles were prepared using the interfacial deposition method in the absence of surfactants. Physicochemical characterisation studies of the produced loaded nanoparticles showed that drug-polymer interactions depend on the characteristics of the actual active substance. IBU-Na showed strong interactions with the polymers and it was found to be molecularly dispersed within the polymer matrix while ibuprofen and ketoprofen retained their crystalline state. The drug release profiles showed stepwise patterns which involve an initial burst release effect, diffusion of the drug from the polymer matrix and eventually drug release possibly via a combined mechanism. PGA polymers can be effectively used as drug delivery carriers for various active substances.

The preparation of magnetically guided lipid based nanoemulsions using self-emulsifying technology.

This paper reports an easy and highly reproducible preparation route, using self-emulsifying technology, for an orally administered high quality magnetically responsive drug delivery system. Hydrophobic iron oxide nanoparticles of about 5 nm in diameter were prepared and incorporated into the lipid core of the produced oil droplets of a self-nanoemulsifying drug delivery system (MagC(18)/SNEDDS). The produced nanoemulsion exhibits colloidal stability at high ionic strengths and temperatures. The observed value of the saturation magnetization at 2 K is approximately 4.1 emu g(-1). The nanoemulsion displayed the magnetic properties of a non-interacting assembly of superparamagnetic particles and a low blocking temperature. Moreover the effect of MagC(18)/SNEDDS on biological systems in vitro was investigated in rodent fibroblasts (3T3 cells). The cytotoxicity studies show that none of the formulations tested affected cell activity significantly over the 24 h incubation. Such systems might have a potential use for oral delivery of poorly soluble compounds by extending the residence time of the formulation in the small intestine resulting in increased drug absorption values.

Arsonoliposomes for the potential treatment of medulloblastoma.

PURPOSE: To investigate the arsonoliposome effect on medulloblastoma cells (VC312Rs) related to uptake, endocytotic mechanism and cell viability. METHODS: VC312R viability in presence of either arsonoliposomes or stealth liposomes was studied using MTT assay for 1-4 days. Fibroblasts (3T3) were used as control. Apoptosis was studied for 2 h, 5 h and 24 h. Bodipy-labelled arsonoliposome uptake (time- and dose-dependent) was estimated using FACS analysis. The endocytotic mechanism was investigated using inhibitors of clathrin- (chlorpromazine) and caveolae-mediated endocytosis (filipin). RESULTS: Arsonoliposomes affected significantly the VC312R viability compared to 3T3 cells and induced apoptosis to VC312Rs after 2 h of incubation. Apoptosis was not observed for 3T3 cells. Liposome uptake versus time showed a bimodal pattern. Clathrin-mediated endocytosis was the main endocytotic mechanism at low lipid concentrations and caveolae at higher ones; thus, dose-dependent uptake did not show a plateau at increased lipid concentrations. CONCLUSIONS: Arsonoliposomes showed "selective" toxicity towards medulloblastoma cells inducing apoptosis after 2 hs of incubation. Therefore, arsonoliposomes are promising anticancer vehicles for brain tumour treatment.

Dexamethasone incorporating liposomes: an in vitro study of their applicability as a slow releasing delivery system of dexamethasone from covered metallic stents.

AIM: To investigate the possibility of covering PET-covered commercially available metallic stents, with liposomal dexamethasone that will act as a slow releasing drug-depot at the site of interest. METHODS: Large multilamellar (MLV), sonicated (SUV) and dried reconstituted (DRV) liposomes entrapping dexamethasone were prepared by thin film hydration, sonication and the DRV method, respectively, and applied on stents using a simple evaporation technique. Drug encapsulation and retention in liposomes were measured by HPLC. The presence of liposomes on the stent surface was confirmed by scanning electron microscopy, while the release of dexamethasone and lipid from the liposome-covered stent was evaluated under different conditions (flow rate, presence of plasma proteins), in an in vitro assembly that was developed to simulate in vivo conditions. RESULTS: The release of dexamethasone from liposome-covered stents ranged from 25% to 50% after 48 h of incubation in buffer, depending on the type of liposome. The release was highest from stents covered with DRV liposomes. When increasing the flow rate from 2 to 6 ml/min a slight increase in release of drug was observed, while a higher release was measured when stents were incubated in plasma proteins. Liposome size does not affect liposome placement on stents. CONCLUSION: The basic characteristics that should be considered when preparing liposomes to cover stents should be their drug loading capacity and their stability under the conditions prevailing at the site of interest. By preparing the appropriate formulation, it is possible that liposomal drugs may be used to cover stents and serve as drug releasing depots at the site of interest. Further in vitro and in vivo studies are needed in order to exploit the possible applications of this methodology.

Cholesterol level affects surface charge of lipid membranes in saline solution.

Cholesterol is an important component of all biological membranes as well as drug delivery liposomes. We show here that increasing the level of cholesterol in a phospholipid membrane decreases surface charge in the physiological environment. Through molecular dynamics simulation we have shown that increasing the level of cholesterol decreases Na+ ion binding. Complementary experimental ζ--potential measurements have shown a decreased ζ--potential with increasing cholesterol content, indicative of reduced surface charge. Both experiments and simulations have been carried out on both saturated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. This result is particularly important because membrane surface charge plays an important role in the interactions of biomembranes with peripheral membrane proteins and drug delivery liposomes with the immune system.

Drug incorporation and release of water soluble drugs from novel functionalized poly(glycerol adipate) nanoparticles.

We have previously demonstrated the ability of poly(glycerol adipate) backbone (PGA) and PGA polymer backbone substituted with varying amounts of pendant C(18) chain length acyl groups to yield Dexamethasone phosphate DXMP loaded nanoparticles. The aim of this study was to obtain a deeper understanding of the underlying principles responsible for good drug incorporation and controlled release of drugs from poly (glycerol adipate) (PGA) nanoparticles. We compared the incorporation of the water soluble drugs DXMP and Cytosine arabinoside (CYT-ARA) in both unmodified and substituted PGA polymers. We investigated the effect of change in acyl group chain length and the degree of substitution on the physicochemical properties, drug loading and release of DXMP and CYT-ARA. Nanoparticles were prepared by the interfacial deposition technique and the simultaneous emulsification method. Amongst the nanoparticles prepared using acylated polymers with varying chain lengths (C(2) to C(10)) for DXMP incorporation, polymers with acyl group chain lengths containing 8 carbon atoms (C(8)) showed maximum drug incorporation. Amongst the C(8) series, polymers with 100% acylation provided both good drug incorporation and a controlled release for DXMP while for CYT-ARA it was the unsubstituted polymer backbone that had maximum drug loading and slower release. A number of inter-related factors are responsible for producing particles with particular size, zeta potential, drug loading and release characteristics. Drug loading and release from nanoparticles are primarily influenced by the nature of interactions between the drug and polymers which in turn depend upon the type of drug used and the physical chemistry of the polymer.

Novel functionalized biodegradable polymers for nanoparticle drug delivery systems.

We have prepared and screened a library of novel functionalized polymers for development of nanoparticle drug delivery systems. The polymer backbone consisting of two ester-linked, nontoxic, biological monomers, glycerol and adipic acid, was prepared using a hydrolytic enzyme. The specificity of the chosen enzyme yields a linear polymer with one free pendant hydroxyl group per repeat unit, which can be further functionalized. This protocol gives control over the backbone polymer molecular weight, together with the ability to incorporate various amounts of different fatty acyl substituents. These functionalized polymers are able to self-assemble into well-defined small particles of high homogeneity with a very low toxicity. They are able to incorporate a water soluble drug, dexamethasone phosphate, with a high efficiency and drug loading which varies with the polymer specification. The above characteristics strongly suggest that these polymers could be developed into useful nanoparticulate drug delivery systems.

Arsenic trioxide liposomes: encapsulation efficiency and in vitro stability.

The use of arsenic-containing compounds in cancer therapy is currently being re-considered, after the recent approval of arsenic trioxide (Trisenox) for the treatment of relapsed promyelocytic leukemia (PML). In an attempt to prepare a carrier system to minimize the toxicity of this drug, the aim of this study is to prepare and characterize liposomes encapsulating arsenic trioxide (ATO). For this, we prepared different types of liposomes entrapping ATO: large multilamellar (MLV), sonicated (SUV) and dried reconstituted vesicles (DRV). The techniques used were: thin film hydration, sonication and the DRV method, respectively. Two lipid compositions were studied for each liposome type, EggPC/Chol (1:1) and DSPC/Chol (1:1). After liposome preparation, drug encapsulation was evaluated by measuring arsenic in liposomes. For this, energy-dispersive X-ray fluorescence spectroscopy or atomic absorption was used. In addition, the retention of the drug in the liposomes was evaluated after incubating the liposomes in buffer at 37 degrees C. The experimental results reveal that encapsulation of ATO in liposomes ranges between 0.003 and 0.506 mol/ mol of lipid, and is highest in the DRV vesicles and lowest in the small unilamellar vesicles, as anticipated. Considering the in vitro stability of ATO-encapsulating liposomes: 1) For the PC/Chol liposomes (DRV and MLV), after 24 hours of incubation, more than 70% (or 90% in some cases) of the initially encapsulated amount of ATO was released. 2) The liposomes composed of DSPC/Chol could retain substantially higher amounts of ATO, especially the DRV liposomes (54% retained after 24 h). 3) In the case of PC/Chol, temperature of incubation has no effect on the ATO release after 24 hours, but affects the rate of ATO release in the MLV liposomes, while for the DSPC/Chol liposomes there is a slight increase (statistically insignificant) of ATO release at higher temperature.