Targeting liposomes with protein drugs to the blood-brain barrier in vitro.

In this study, we aim to target pegylated liposomes loaded with horseradish peroxidase (HRP) and tagged with transferrin (Tf) to the BBB in vitro. Liposomes were prepared with the post-insertion technique: micelles of polyethylene glycol (PEG) and PEG-Tf were inserted into pre-formed liposomes containing HRP. Tf was measured indirectly by measuring iron via atomic absorption spectroscopy. All liposomes were around 100 nm in diameter, contained 5-13 microg HRP per mumol phospholipid and 63-74 Tf molecules per liposome (lipo Tf) or no Tf (lipo C). Brain capillary endothelial cells (BCEC) were incubated with liposomes at 4 degrees C (to determine binding) or at 37 degrees C (to determine association, i.e. binding+endocytosis) and the HRP activity, rather than the HRP amount was determined in cell lysates. Association of lipo Tf was two- to three-fold higher than association of lipo C. Surprisingly, the binding of lipo Tf at 4 degrees C was four-fold higher than the association of at 37 degrees C. Most likely this high binding and low endocytosis is explained by intracellular degradation of endocytosed HRP. In conclusion, we have shown targeting of liposomes loaded with protein or peptide drugs to the BCEC and more specifically to the lysosomes. This is an advantage for the treatment of lysosomal storage disease. However, drug targeting to other intracellular targets also results in intracellular degradation of the drug. Our experiments suggest that liposomes release some of their content within the BBB, making targeting of liposomes to the TfR on BCEC an attractive approach for brain drug delivery.     

The quality of communication between parents and adolescent children in the case of parental cancer.

BACKGROUND: This study was designed to investigate: (i) parent-adolescent communication in families of cancer patients; (ii) relationships between parent-adolescent communication and posttraumatic stress symptoms (PTSS) in adolescent children; and (iii) associations between parents' illness characteristics and parent-adolescent communication. PATIENTS AND METHODS: A total of 212 adolescents completed the Impact of Event Scale and Parent-Adolescent Communication Scale. RESULTS: Adolescents communicated less openly with mothers with cancer than controls with mothers; this was the only significant difference with the reference group. Daughters communicated more openly with ill parents than with healthy parents. More open communication with healthy parents was related to fewer PTSS in daughters. More problem communication with both parents was related to more PTSS in both sons and daughters. Sons reported more problems in communication with ill parents in case of more intensive treatment or recurrent disease. Daughters experienced less open communication with both parents when ill parents received more intensive treatment. Time since diagnosis was not related to parent-adolescent communication. Multivariate analyses showed that communication patterns specifically affected PTSS of daughters. Problem communication with the healthy parent was the strongest predictor of intrusion while problem communication with the ill parents was the strongest predictor of avoidance. CONCLUSIONS: Parent-adolescent communication in families of cancer patients differs little from that in families not confronted with parental cancer. Problem communication outweighed lack of openness with respect to development of PTSS. Recurrent disease and intensive treatment regimens affected parent-adolescent communication negatively.     

Solid dispersions based on inulin for the stabilisation and formulation of delta 9-tetrahydrocannabinol.

The aim of this study was to develop a dry powder formulation that stabilises the chemically labile lipophilic Delta(9)-tetrahydrocannabinol (THC), that rapidly dissolves in water in order to increase the bioavailability and that opens new routes of administration. It was investigated whether these aims can be achieved with solid dispersions consisting of a matrix of inulin, an oligo-fructose, in which THC is incorporated. These solid dispersions were prepared by lyophilisation of a solution of THC and inulin in a mixture of water and tertiary butyl alcohol (TBA). Both 4 and 8 wt.% of THC could be incorporated in a glassy matrix of inulin. In the solid dispersions only 0.4-0.5 wt.% of residual TBA was present after storage at 20 degrees C/45% relative humidity (RH) for 7 days. Unprotected THC was completely degraded after 40 days of exposure to 20 degrees C and 45% RH. However, solid dispersions exposed to the same conditions still contained about 80% non-degraded THC after 300 days. Dissolution experiments with tablets compressed from inulin glass dispersion material showed that THC and inulin dissolved at the same rate. Tablets weighing 125 mg and containing 2mg THC were prepared from a mixture of THC containing solid dispersion, polyvinylpolypyrrolidone (PVPP) and mannitol. Dissolution tests revealed that from these tablets 80% of the THC was dissolved within 3 min, which makes them promising for sublingual administration. It was concluded that THC can be strongly stabilized by incorporating it in a matrix of inulin. The aqueous dissolution rate was high which may improve bioavailability.     

Coupling of Metal Containing Homing Devices to Liposomes via a Maleimide Linker: Use of TCEP to Stabilize Thiol-groups without Scavenging Metals

Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe³⁺), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe³⁺). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2–3 h coupling to the liposomes, Fe³⁺ is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes.

Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe³⁺ from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself.

In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe³⁺ from Tf. This method can be applied to other metal-containing homing devices as well.     

Severe congenital neutropenia in a multigenerational family with a novel neutrophil elastase (<Emphasis Type="Italic">ELANE</Emphasis>) mutation

We have analysed a family with nine congenital neutropenia patients in four generations, several of which we have studied in a long-term follow-up of over 25 years. The patients were mild to severe neutropenic and suffered from various recurrent bacterial infections. Mutations in the genes ELANE, CSF3R and GFI1 have been reported in patients with autosomal dominant congenital neutropenias. Using a small-scale linkage analysis with markers around the ELANE, CSF3R, CSF3 and GFI1 genes, we were able to determine that the disease segregated with markers around the ELANE gene. We identified a novel mutation in the ELANE gene in all of the affected family members that was not present in any of the healthy family members. The mutation leads to an A28S missense mutation in the mature protein. None of these patients developed leukaemia. This is the first truly multigenerational family with mutations in ELANE as unambiguous cause of severe congenital neutropenia SCN.     

Implementing Non-violent Resistance,a Method to Cope with Aggression in Child and Adolescent Residential Care: Exploration of Staff Members Experiences

Staff members in residential care for youth are frequently confronted with aggressive behaviour, which has adverse effects on their stress levels and work satisfaction. This paper describes a qualitative evaluation to find out how staff members benefit from Non-violent Resistance (NVR), a method to create an aggression mitigating residential climate. Staff members were positive about NVR and reported feeling more relaxed. Most valued aspects of this method were the focus on being a team, delayed response and giving up the illusion of control. However, training and the intention to use NVR isn't enough, high quality implementation and maintenance are crucial.     

Managing invasive aspergillosis in haematological patients in the era of resistance polymerase chain reaction and increasing triazole resistance: A modelling study of different strategies

Objectives

Triazole resistance in Aspergillus spp. is emerging and complicates prophylaxis and treatment of invasive aspergillosis (IA) worldwide. New polymerase chain reaction (PCR) tests on broncho-alveolar lavage (BAL) fluid allow for detection of triazole resistance at a genetic level, which has opened up new possibilities for targeted therapy. In the absence of clinical trials, a modelling study delivers estimates of the added value of resistance detection with PCR, and which empiric therapy would be optimal when local resistance rates are known.