Direct quantification of cytosolic delivery of drug nanocarriers using FlAsH-EDT2.

The delivery of therapeutics across the cell membrane and into the cytoplasm is a major challenge that limits the development of new therapies. This challenge is compounded by the lack of a general assay for cytosolic delivery. Here we develop this assay based on the pro-fluorophore CrAsH-EDT2, and provide cytosolic penetration results for a variety of drug delivery agents (polyethyleneimine, poly-arginine, Ferritin, poly [maleic anhydride-alt-isobutene] grafted with dodecylamine, and cationic liposomes) into HeLa and T98G cells. Our results show that this method can be widely applicable to different cells and drug delivery agents, and yield statistically robust results. We later use this method to optimize and improve a model drug delivery agent's (Ferritin) cytosolic penetration.

Does conjugation strategy matter? Cetuximab-conjugated gold nanocages for targeting triple-negative breast cancer cells.

The efficient targeting of cancer cells depends on the success of obtaining the active targeting of overexpressed receptors. A very accurate design of nanoconjugates should be done via the selection of the conjugation strategy to achieve effective targeted nanoconjugates. Here, we present a detailed study of cetuximab-conjugated nonspherical gold nanocages for the active targeting of triple-negative breast cancer cells, including MDA-MB-231 and MDA-MB-468. A few different general strategies were selected for monoclonal antibody conjugation to the nanoparticle surface. By varying the bioconjugation conditions, including antibody orientation or the presence of a polymeric spacer or recombinant protein biolinker, we demonstrate the importance of a rational design of nanoconjugates. A quantitative study of gold content via ICP-AES allowed us to compare the effectiveness of cellular uptake as a function of the conjugation strategy and confirmed the active nature of nanoparticle internalization in cancer cells via epidermal growth factor receptor recognition, corroborating the importance of the rational design of nanomaterials for nanomedicine.

H-Ferritin-nanocaged olaparib: a promising choice for both BRCA-mutated and sporadic triple negative breast cancer.

Poly(ADP-ribose) polymerase (PARP) inhibitors represent a promising strategy toward the treatment of triple-negative breast cancer (TNBC), which is often associated to genomic instability and/or BRCA mutations. However, clinical outcome is controversial and no benefits have been demonstrated in wild type BRCA cancers, possibly due to poor drug bioavailability and low nuclear delivery. In the attempt to overcome these limitations, we have developed H-Ferritin nanoformulated olaparib (HOla) and assessed its anticancer efficacy on both BRCA-mutated and non-mutated TNBC cells. We exploited the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1), and its physiological tropism toward cell nucleus. TNBC cell lines over-expressing TfR-1 were successfully recognized by H-Ferritin, displaying a fast internalization into the cells. HOla induced remarkable cytotoxic effect in cancer cells, exhibiting 1000-fold higher anticancer activity compared to free olaparib (Ola). Accordingly, HOla treatment enhanced PARP-1 cleavage, DNA double strand breaks and Ola delivery into the nuclear compartment. Our findings suggest that H-Ferritin nanoformulation strongly enhances cytotoxic efficacy of Ola as a stand-alone therapy in both BRCA-mutated and wild type TNBC cells, by promoting targeted nuclear delivery.

Diagnosis and laparoscopic management of 12 consecutive cases of ovarian pregnancy and review of literature.

We sought to determine the outcome of the laparoscopic management of ovarian ectopic pregnancy (OEP) in a United Kingdom district general hospital and reviewed the literature. We conducted a 5-year prospective cohort study of the management of OEP cases between January 2003 and January 2008. Twelve patients had OEP confirmed with histology among a cohort of 421 ectopic pregnancies. The mean gestational age was 45 days. All 12 patients had abdominal pain and 4 (33%) had vaginal bleeding. One (8%) patient became hypovolemic before laparoscopy. Four (33%) women had risk factors for ectopic pregnancy, 2 of whom were current intra-uterine contraceptive users. Preoperative diagnosis of ectopic pregnancy was made in 11 (92%) of 12 patients by transvaginal ultrasonography and OEP in 9 (75%) patients. All cases were managed by laparoscopic surgery with no conversion to laparotomy. The ovarian pregnancy was resected and the ovary conserved in 11 (92%) patients with only 1 requiring an oophorectomy. The mean operating time was 49 minutes. None of the patients needed further treatment. No complications occurred after laparoscopic surgery and the mean hospital stay was 2 days. Considering the rarity of ovarian pregnancy, this is one of the largest series of patients with OEP treated exclusively by laparoscopic surgery and highlights our recent experience of performing conservative laparoscopic surgery for most of our patients.

Diagnosis and laparoscopic management of 11 consecutive cases of cornual ectopic pregnancy.

OBJECTIVE: To determine the pre-operative diagnosis by two dimensional ultrasound scan and the outcome of the laparoscopic management of cornual ectopic pregnancy. DESIGN: Prospective database cohort study. SETTING: Whipps Cross University Hospital, UK (District General Hospital). PATIENTS: Eleven patients with cornual ectopic pregnancy presenting in our hospital between January 2003 and December 2007. INTERVENTIONS: Laparoscopic cornuostomy or cornual resection. OUTCOME MEASURES: Pre-operative diagnosis by ultrasound scan, conversion rate to laparotomy, successful laparoscopy (not requiring further treatment), complication rate and duration of hospital stay. RESULTS: The mean gestational age was 8 +/- 2 weeks. All 11 patients presented with abdominal pain and vaginal bleeding and two (18%) patients became haemodynamically unstable before laparoscopy. There were five (45%) patients with risk factors for ectopic pregnancy. The mean serum beta-human chorionic gonadotropin (beta-hcg) was 15,263 +/- 12,045 microm/ml. One patient did not have a transvaginal scan as it was decided to proceed to surgery on clinical grounds. The diagnosis of ectopic pregnancy was correct at initial scan in nine (90%) of the ten patients who had transvaginal scans as one patient was misdiagnosed at the first scan. However, an ectopic pregnancy was diagnosed on a second ultrasound scan assessment. Initial laparoscopy was negative in one of the nine patients diagnosed as having an ectopic pregnancy. The diagnosis was later confirmed following serial serum beta-hcg monitoring, a repeat scan and a second laparoscopy. Ten (91%) of the 11 patients had successful operative laparoscopy as one (9%) patient had conversion to laparotomy. Among patients who had laparoscopic surgery, cornuostomy was performed in three (30%) patients while cornual resection was performed in the other seven (70%) patients. One (10%) of the patients who had laparoscopic surgery needed further treatment with systemic methotrexate. This patient had a cornual resection and was the only complication following laparoscopic surgery. The mean hospital stay was 2 days. CONCLUSION: This presentation of one of the larger series of patients with cornual ectopic pregnancy managed by laparoscopic surgery reveals that experience at ultrasonography and laparoscopic technique can lead to earlier diagnosis and few cases requiring laparotomy or further treatment. In addition laparoscopic surgery for cornual ectopic is safe and lends itself to conservative approach (cornuostomy) in selected cases.

Changing trends in the laparoscopic management of ectopic pregnancy in a London district general hospital: 7-years experience.

We set out to evaluate the effect of a programme introduced in January 2003 to make operative laparoscopy the standard surgical treatment for women requiring surgery for ectopic pregnancy. This was a retrospective and prospective clinical data analysis performed at The Whipps Cross University Hospital in London, with a comparison of data taken before and after the introduction of the programme. A total of 116 women who had surgical management for ectopic pregnancy from January 2000 to December 2002 and 313 women who had surgery for ectopic pregnancy between January 2003 and December 2006 took part in the study. A programme was started in January 2003 to make operative laparoscopy the surgical management of choice. The main outcome measure was the proportion of women requiring surgery who had operative laparoscopy in the two study periods. The chi(2)-test was used to determine if there was any statistically significant difference between proportions. A difference was deemed statistically significant if p < 0.05. The results showed that there was a progressive rise in the proportion of ectopic pregnancies managed by operative laparoscopy following the change in January 2003. A total of 34% of women were managed laparoscopically between 2000 and 2002, increasing to 90% between 2003 and 2006. This difference was statistically significant (p < 0.001). In 2006, some 96% of women requiring surgery were managed by laparoscopic surgery. The findings of this study indicate that it is possible to implement changes which increase and sustain a high rate of laparoscopic surgery for women with ectopic pregnancy requiring surgery in a district general hospital setting.

Laparoscopic management of ectopic pregnancy in the presence of a significant haemoperitoneum.

OBJECTIVE: To assess the trend in the use of operative laparoscopy in the management of patients with ruptured ectopic pregnancy and significant haemoperitoneum. METHOD: Four-year prospective observational study of the operative management of women with ruptured ectopic pregnancy and significant haemoperitoneum at the Whipps Cross University Hospital from January 2003 to December 2006. The inclusion criteria were patients with a clinical or laparoscopic assessment of significant haemoperitoneum (>800 ml). The amount of haemoperitoneum was determined at surgery. RESULTS: The blood loss ranged from 800 to 3,500 ml. The laparoscopic approach in this specific clinical scenario increased from 40% in 2003 to 100% in 2006. In 2003 there were five patients, two (40%) were treated by laparoscopy, one (20%) was converted from laparoscopy to laparotomy and two (40%) had laparotomy. In 2004 there were six patients, five (85%) underwent laparoscopic management and one (15%) had laparotomy. In 2005 we had 14 patients, ten (72%) had laparoscopic management, 2 (14%) were converted to laparotomy and 2 (14%) had laparotomies. There were 12 patients in 2006 and all (100%) were treated by laparoscopy. CONCLUSION: Our study demonstrates that with highly skilled anaesthetic and surgical teams, operative laparoscopy with its recognized advantages over laparotomy and is feasible in women with ruptured ectopic pregnancy and significant haemoperitoneum.

A bio-dosemeter that utilises isolated enzymes to detect ionising radiation.

The development of a biosensor for the detection of ionising radiation (biodosemeter) utilising the advantageous properties of the photosystem II (PSII) complex and its response to ionising radiation is reported. The transducer signal for this biosensor can be fluorescence, which is dependent on photosynthetic activity. Exposure of biological material to ionising radiation leads to a loss of function due to the destruction of critical structures. Radiation target theory predicts an exponential decrease in biochemical activity that is dependent on the absorbed radiation energy and directly proportional to the mass of the individual molecules possessing this activity. The activity is lost whenever the protein is hit since very high energy is transferred through the chain. Several approaches were used to optimise the immobilisation of PSII complexes to improve the sensitivity of the bio-dosemeter.

External PIGE-PIXE measurements at the São Paulo 8UD tandem accelerator / Medidas externas do PIGE-PIXE em São Paulo 8UD acelerador tandem

An external PIGE-PIXE setup was installed on a beam line of the 8 MV tandem Pelletron accelerator of the Open Nuclear Physics Laboratory (LAFN). Proton beam energy was chosen in the 8-12 MeV range, sufficient to get an acceptable gamma ray yield but not so high as to prevent us from measuring X-rays. This also allowed the use of a thick aluminum exit window (0.5 mm) instead of the usual thin and sometimes fragile plastic windows. This external PIXE-PIGE system was used to analyze trace element concentrations in the enamel of human and animal teeth. The main interest was to find compatible human teeth substitutes for dentistry laboratory practice and chemical tests. In spite of their morpho-histological similarity, trace element concentrations in human and animal teeth have not yet been compared. Teeth from humans, cattle and swine collected primary at São Paulo region were analyzed. The elements Cu, K, Zn, Fe, Ti, Sr, V, Mn and Zr were detected by high energy external beam PIXE technique. Though preliminary, the results showed that the trace element concentrations observed in the enamel of human and swine are more similar to each other than to cattle teeth