Technically assisted assessment of volume status before surgery may be useful to direct intraoperative fluid administration. We therefore tested a recently developed whole-body bioimpedance spectroscopy device to determine pre- to postoperative fluid distribution.Using a three-compartment physiologic tissue model, the body composition monitor  measures total body fluid volume, extracellular volume, intracellular volume and fluid overload as surplus or deficit of ‘normal’ extracellular volume. BCM-measurements were performed before and after standardized general anaesthesia for gynaecological procedures . BCM results were blinded to the attending anaesthesiologist and data analysed using the 2-sided, paired Student’s t-test and multiple linear regression.2 = 0.65), but was not associated with change in intracellular volume (r2 = 0.01).In 71 females aged 45±15 years with body weight 67±13 kg and duration of anaesthesia 154±68 min, pre- to postoperative fluid overload increased from −0.7±1.1 L to 0.1±1.0 L, corresponding to −5.1±7.5% and 0.8±6.7% of normal extracellular volume, respectively (both p<0.001), after patients had received 1.9±0.9 L intravenous crystalloid fluid. Perioperative urinary excretion was 0.4±0.3 L. The increase in extracellular volume was paralleled by an increase in total body fluid volume, while intracellular volume increased only slightly and without reaching statistical significance (p = 0.15). Net perioperative fluid balance (administered fluid volume minus urinary excretion) was significantly associated with change in extracellular volume (rRoutine intraoperative fluid administration results in a significant, and clinically meaningful increase in the extracellular compartment. BCM-measurements yielded plausible results and may become useful to guide intraoperative fluid therapy in future studies. All patients undergoing anaesthesia and surgery routinely receive intravenous (iv) fluid therapy intraoperatively for correction of anaesthesia- and surgery-related hemodynamic disturbances. Timing, amount and the specific type of its administration, however, remain controversial Surgery represents a traumatic insult and initiates an acute stress response leading to secretion of proinflammatory mediators and stress hormones, which control the sympathoadrenal system and the hypothalamic-pituitary-adrenal axis Total body fluid volume (TBV) comprises approximately 50% of the female body mass, i.e. 30 liters in a 60 kg woman. Of this amount, about two-thirds (20 liters) are intracellular volume (ICV) and one-third (10 liters) extracellular volume (ECV) in a healthy, young subject To the best of our knowledge, the BCM device has not previously been studied in patients undergoing general anaesthesia, although objective assessment of fluid status before surgery may be useful to optimize intraoperative fluid administration and to prevent postoperative fluid retention. The aim of the present study was to determine the pre- to postoperative fluid distribution using bioimpedance spectroscopy technology, and, by relating changes in volume status to the net administration of iv fluid, to evaluate if BCM-derived measurements yield plausible results.The use of routine BCM-measurements was instituted at the Department of Anaesthesiology of the Medical University of Vienna, as of May 2012, based on the availability of a BCM device provided by the Department of Nephrology. From 12 November 2012 until 31 January 2013, BCM-measurements were consecutively performed directly before general anaesthesia in unselected, conscious females, presenting for various elective gynaecological procedures. Patients with a pacemaker, with a limb amputation or pregnant women were not measured. Patients who had significant blood loss and therefore had received erythrocyte concentrates or colloid solution, and/or all those patients who had not received a urinary catheter were measured, but excluded from the present analysis. All patients fasted overnight for more than 6 hours. All patients received isotonic crystalloid solution  continuously, throughout the duration of anaesthesia. The amount and timing of intraoperative fluid administered was at the discretion of the attending anaesthesiologist, who was not informed about the BCM result, thereby reflecting clinical practice, as influenced by cardiovascular signs and fluid losses, without following a fixed fluid regime.For induction of general anaesthesia propofol, fentanyl and rocuronium were administered intravenously. To secure the airway, an endotracheal tube was inserted as appropriate. Anaesthesia was maintained with sevoflurane in an air/oxygen mixture or with a continuous propofol infusion guided by electroencephalogram . At the discretion of the attending anaesthesiologist, and depending on the anamnesis of the patient, a serotonin antagonist , a glucocorticoid  in low dose (4 mg) and/or a total intravenous anaesthesia (TIVA) were administered intraoperatively to prevent postoperative nausea and vomiting (PONV). Urinary catheterization was performed after induction. Body temperature was maintained with temperature controlling devices like Bair Hugger (3 M) and Hotline 2 Fluid Warmer . The second BCM-measurement was performed as soon as possible after the end of anaesthesia in the postanaesthesia care unit.To evaluate the obtained data in the form of the present open, non-interventional, observational cohort study, we obtained approval from the Ethics Committee of the Medical University of Vienna (EK Nr.: 2059/2012) and anonymised all participant information. The study adhered to the Declaration of Helsinki.BCM-measurements were performed as proposed by the manufacturer. Briefly, four non-recyclable electrode strips were taped to wrist and ankle, respectively hand and foot, on one side of the patient body in supine position. Electrodes were connected to the BCM device using the cable provided by the manufacturer. Body weight and height, patient age and patient gender were entered into the BCM device.About BCM technology: The measurement itself usually requires <2 minutes; the BCM device measures resistance and reactance at 50 discrete frequencies covering the frequency spectrum from 5 to 1000 kHz. Extracellular and intracellular resistance values are obtained on the basis of a Cole model 2), fat mass and fat tissue index (fat tissue mass/height2).Additional information provided by a typical BCM-measurement comprise basic measures of the body composition, such as lean tissue mass, lean tissue index , we primarily use the term fluid overload and fluid status. Similarly, the term extracellular water used in previous publications www.kimcl.at). Preoperative measurements of hemoglobin, serum creatinine and gamma glutamyl transferase were available in all patients. Increased vascular permeability might have influenced the BCM results, and is associated with hypoalbuminaemia and inflammation (manifested by high C-reactive protein [CRP] levels). To assess vascular permeability we therefore calculated the capillary leak index as CRP [in milligrams per deciliter] over albumin [in grams per liter], multiplied by 100 The patient characteristics recorded for this study included age, height, body weight, body mass index, classification per the American Society of Anaesthesiologists’ (ASA) physical status classification system, and New York Heart Association (NYHA) functional classification. All biochemical and hematological analyses (labs) were performed by the Clinical Institute of Laboratory Medicine of the Medical University of Vienna  were used to portray patient characteristics, laboratory values, iv fluid administration and urinary output, as well as all BCM-derived measurements. Changes in BCM-derived measurements from pre- to postoperatively  were obtained by subtracting the preoperative value from the postoperative value.The primary parameter of interest was fluid overload (FO in liters and in % ECV). The change in pre- to postoperative FO was evaluated using the paired, two-sided Student’s t-test, as proposed in the pre-specified statistical analysis plan of the study protocol. Similar exploratory analyses were conducted for all other parameters obtained before and after anaesthesia. When the distribution of the analyzed variables was tested by Kolmogorov-Smirnov test, fluid therapy was found to be non-normally distributed.Sample size considerations were based on association analyses using the Pearson correlation test. To detect a moderate, and thereby clinically meaningful correlation (r = 0.4) between net perioperative fluid balance and change in FO, a sample of 62 analysable subjects was calculated to provide 90% power to discover that the correlation would be significantly different from there being no correlation (i.e. that the correlation would be near zero) at the 0.05 level.We used multiple regression analysis to examine the effect of different predictor variables  on the outcome variable (change in ECV).For calculations we used MS Excel 2011 and IBM SPSS Statistics 22.0.The study population comprised 71 females, aged 45±15 years, with body weight 67±13 kg  after exThe average preoperative iv fluid volume administered amounted to 0.3 L, with 16 patients having received no fluid at all. The average interval between two BCM-measurements was 189 minutes and the average duration of anaesthesia was 154 minutes. During this period, patients received on average 1.9 L iv crystalloid fluid, 6 patients ≥3 L and 5 patients ≤1 L. During the interval between the two BCM-measurements, the average urinary excretion was 0.3 L, with 3 patients excreting ≥1 L. The net perioperative fluid balance amounted to 1.6 L . The overall model (Model 9) explained 71.3% of variance in change in ECV  using BCM. The BCM device measures at 50 frequencies and employs a unique body composition model for determination of fluid overload, respectively for direct prediction of TBV, ECV and ICV. Recently a study by Raimann at al. compared the precision and accuracy of SF-BIA and BIS to direct estimation methods  for body composition analysis in hemodialysis patients. In comparison to DEMs, SF-BIA and BIS were equally precise in determining TBV and ICV. However BIS-determined ECV was closer to DEM-determined ECV, in comparison to SF-BIA. According to Raimann et al., there is no real “gold standard” method with absolute accuracy for determination of the fluid status due the wide limits of agreement Another impedance-based method to estimate fluid distribution is bioelectrical impedance vector analysis (BIVA), where a vector of impedance at one frequency is evaluated against an ordinal scale with tolerance interval percentiles from a reference population 2 measured with DEMs. Specifically, TBV determined by deuterium dilution was equivalent to 46.51% body weight, and ECV determined by bromide dilution was equivalent to 20.71% body weight In the present study the values obtained for relative TBV (47.3%), relative ECV (20.8%) and relative ICV (26.5%) are in contrast to the classical textbook knowledge, stating that TBV comprise approximately 50–60% of the body weight in healthy adults Mechanistically, because fluid content of lean tissue is around 73%, while fluid content of adipose tissue is around 10% While the bioimpedance spectroscopy-based BCM is currently used in various nephrological settings, and specifically for the management of fluid withdrawal in patients with end-stage renal disease, it has not previously been studied perioperatively. Roughly a fourth of our patients were dehydrated (ECV <−10%) and 41% of the patients had a calculated fluid deficit >1 L before surgery, although they were allowed to drink clear fluid up to two hours prior to the operation. Using BCM-derived information as a target for initial fluid management could therefore become useful to guide fluid therapy before anaesthesia in order to avoid hypovolaemia and thereby hypotension after induction The systemic stress response to anaesthesia and surgery is characterized by secretion of corticotrophin, antidiuretic hormone and cortisol, activation of the renin- angiotensin-aldosterone-system and stimulation of the sympathoadrenal axis to maintain hemodynamic stability and to increase salt and fluid retention Appropriate perioperative fluid therapy might limit the incidence and severity of systemic inflammation following surgery Our data showed a significant increase of postoperative FO, TBV and ECV, while ICV increased only slightly. It appears surprising to detect this fluid shift at all, because the gynaecological patients had normal renal function, which may leave one wondering why some of the infused volume accumulates in the periphery. Because this fluid status cannot persist indefinitely, the timing of BCM-measurements is important. In future studies, it would be interesting to measure at additional time points postoperatively to observe the return of fluid overload to normal, at least in those patients who are volume overloaded postoperatively.2 = 0.65), predicting individual changes in fluid status after iv infusions may remain challenging in patients undergoing surgery, unless it can be clarified that an increase of only 60% is physiologically reasonable. Further methodological issues should be ruled out first.A net perioperative fluid balance of 1.6±0.7 L led to a measured increase of only 1.0±1.4 L in TBV and to an increase of 0.8±0.7 L in ECV. Thus, roughly 40% of the administered fluid was “missing”. One reason for not detecting all of the administered volume in the fluid compartments of the patient may be that respiratory-, cutaneous- and wound fluid losses were not considered. Although the correlation between net perioperative fluid balance and change in extracellular fluid volume was found to be quite strong , the limbs contribute 90%, while the trunk with a large cross sectional area adds only 10% to whole body impedance Pneumoperitoneum during laparoscopy increases the intra-abdominal pressure, which affects the cardiovascular system, the respiratory system and renal function Additional study limitations: A net perioperative fluid therapy of 1.6 L would increase the body weight, but the postoperative BCM-measurement was performed with the same patient weight, because it was practically impossible to weigh the supine patient in the recovery room after surgery. Entering a patient weight of plus or minus 1.1 kg into the BCM device before performing the measurement has been shown to result in a minimal change of ECV, ICV and FO  The present study was an open, non-interventional, observational cohort study, and as such, we were unable to compare our routinely generated BCM results with an additional reference method. Nevertheless, the BCM has been validated against direct estimation methods in more than 500 healthy subjects In conclusion, the results of the present study, obtained with whole body bioimpedance spectroscopy using BCM, show that intraoperative crystalloid fluid administration leads to a significant, and clinically meaningful increase of the extracellular volume compartment, while the intracellular fluid volume increased only slightly. The BCM might serve as a simple and non-invasive device for objective assessment of body fluid distribution in the perioperative setting. Further research is now required to evaluate prospectively, whether BCM-guided fluid administration may improve the postoperative volume status, as well as clinical outcomes in surgical patients.


The detection of heart response to fluid administration is still a challenge in clinical practice. Changes in metabolic parameters may be useful to detect changes in cardiac output (CO) after fluid expansion.2), oxygen consumption (VO2) and carbon dioxide production (VCO2) were calculated according to well-known formulas. Patients were divided into three groups  according to their change in CO .This is a prospective observational study in adult critically ill patients. CO was measured either by echocardiography or by a thermodilution method . Hemodynamic measurements and blood gas analysis were obtained before and after a fluid challenge with either 1,000 ml crystalloid or 500 ml colloid. Arterial and central venous blood gas samples were taken simultaneously. Oxygen delivery  as compared with moderate responders or nonresponders. Furthermore, nonresponders had a decrease in their DO2 , while moderate responders showed no change in their DO2  after fluid challenge. We found no differences in changes in lactate levels and central venous oxygen saturation (ScvO2) between high responders, moderate responders and nonresponders. No differences in the changes of VCO2 or VO2/VCO2 ratio were found between high responders, mild responders and nonresponders too. Changes in DO2/VCO2 ratio were found to be significantly increased only in high responders  and not in mild responders  as compared with nonresponders.We evaluated 27 patients, age 68 (95% CI: 61 to 74) and APACHE II score 22 (95% CI: 18 to 26). Seven patients were high responders, eight patients were moderate responders and 12 were nonresponders. DO2 may be decreased in nonresponders. The changes of ScvO2 and lactate levels do not track the changes of CO after fluid challenge. The DO2/VCO2 ratio may be a useful index to identify significant increases of CO after fluid challenge in cases where CO measurement is not feasible.Only significant increases of CO (>20%), after fluid administration, lead to improved oxygen delivery; DO


So far, the hydrated part of the HPMC matrix has commonly been denoted as a “gel” or “pseudogel” layer. No MRI-based results have been published regarding observation of internal phenomena related to drug dissolution inside swelling polymeric matrices during hydration. The purpose of the study was to detect such phenomena.2 MR relaxometry, in situ, was applied to study formation of the hydration progress in HPMC matrix tablets loaded with L-dopa and ketoprofen using a 11.7 T MRI system. Two spin-echo based pulse sequences were used, one of them specifically designed to study short T2 signals.Multiparametric, spatially and temporally resolved T2 decay envelope were estimated and spatial distributions of their parameters, i.e. amplitudes and T2 values, were obtained. Based on the data, different region formation patterns (i.e. multilayer structure) were registered depending on drug presence and solubility. Inside the matrix with incorporated sparingly soluble drug a specific layer formation due to drug dissolution was detected, whereas a matrix with very slightly soluble drug does not form distinct external “gel-like” layer.Two components in the T1H MRI methods. It reflects molecular mobility and concentration of water inside the hydrated matrix. For the first time, drug dissolution related phenomena, i.e. particular front and region formation, were observed by MRI methods.We have introduced a new paradigm in the characterization of hydrating matrices using  Studies on the behavior of controlled release (CR) hydroxypropylmethylcellulose (HPMC) matrix systems emerged in the late 1960’s . In thisThe development of noninvasive imaging techniques e.g. magnetic resonance imaging (MRI), attenuated total reflection Fourier transformed infrared microscopy (ATR-FTIR) and others, led to a deeper insight into the structure of swelling HPMC matrix systems –5. HowevIt must be pointed out that in the structural analyses of swelling matrices, the presence of an active substance was not discussed systematically. This is connected to fact that photo imaging techniques are not able to distinguish the influence of colorless substances on the structure of the matrix. Other highly sensitive chemical imaging techniques, e.g. FTIR microscopy, were able to detect changes in the concentration of particular components of the matrix  rather than its structure. MRI techniques are noninvasive and allow to study model or commercial dosage forms of various shape and size. For this reason most of the studies used MRI as a method to evaluate matrix tablet hydration –8. But tet al. 2004 studied HPMC tablets with various polymer content  loaded with tetracycline hydrochloride, a drug highly soluble in water. It was stated that drug loading reduced the integrity and the resistance of network structure against the penetration of water molecules  to HPMC matrix loaded with drugs of different solubility.In our previous paper we have discussed a novel multiparametric approach to the investigation of hydrated HPMC matrices. It was based on relaxometric data obtained with magnetic resonance microscopy . In thisThe goal of the study was to apply the previously developed methodology to detect phenomena related to drug presence in the hydrating HPMC matrices, and in consequence, to compare the multilayer structure of HPMC based systems with drug substances of different solubility.Two drug substances: L-dopa (LD) (Sigma-Aldrich) and ketoprofen (KT) (Sigma-Aldrich) were used as model drugs of different solubility. Solubility difference between drugs was chosen to be within two orders of magnitude in 0.1 M hydrochloric acid solutions. According to the European Pharmacopoeia 6.0, ketoprofen is very slightly soluble in water whilst L-Dopa is slightly soluble in water. In 0.1 M hydrochloric acid solutions L-dopa is sparingly soluble (1 g in 30–100 mL) , ketoprovide infra). After measurements the samples were disposed. The procedure was repeated for each of the time points. One sample was measured for one hydration time. A total number of 24 independent samples was measured using the presented method–samples at four different hydration times, of two different polymer viscosities and drug loading were measured.The tablets were kept in 50 mL of hydrochloric acid (0.1 M) at room temperature (~22°C). The tablets were taken for measurements after 30, 60, 120 and 180 min of immersion in hydrochloric acid solution. At these time points the solution was removed and the tablets were protected to minimize the evaporation. The MR measurements were carried out using a home-built dedicated MR probe  was probed using the Multi Slice Multi Echo (MSME) sequence. The short time scale of the T2 decay envelope (up to 12 ms with a resolution of 1 ms) was probed using the Broad Line Imaging Package Modes (BLIP_MODES) MRI pulse sequence . The following MSME sequence parameters were applied: TE/TR = 6.5/4,000 ms, number of accumulations = 2, number of images = 32, matrix size = 256 × 256 × 1, slice thickness = 1 mm, field of view = 15 × 15 mm. A BLIP pulse sequence was used with following parameters: TE/TR = 3/500 ms. Number of accumulations = 2, Number of echoes = 10, Matrix size =256 × 256 × 1, Slice thickness = 1 mm, field of view = 15 × 15 mm. The MSME and BLIP data sets were analyzed by custom developed scripts in MATLAB .Spatially resolved Tng paper  was applet al.  was developed. Details can be found in the article by Kulinowski et al. .The tablets were kept in similar conditions as used for the MRI experiments and removed from the solution after 2 h of soaking. The samples of the external layer were carefully peeled off and precisely weighted in hermetic aluminum pans . Differential scanning calorimetry (DSC) was carried out using the EXSTAR DSC 7020 apparatus (SII NanoTechnology Inc.) equipped with a DSC7020 electric cooling unit and assessed by the Muse Standard Analysis (SII NanoTechnology Inc.) software. The temperature calibration was done with indium and tin . The calibration of enthalpy change was done with Tin . The procedure was carried out at 5.0°C/min.The following thermal protocol was used for cooling/heating experiments: start at 30.0°C; cooling from 30.0 to −50.0°C at 5.0°C/min; equilibrating at −50.0°C for 10 min; heating from −50.0 to 30°C at 5.0°C/min. Flow rate of dynamic nitrogen atmosphere was 50 mL/min.et al. 2012 . Due to2 maps of hydrated matrix fragments. Each matrix is shown at four hydration times, i.e. 0.5, 1, 2 and 3 h. The initial, qualitative assessment shows clearly that all three formulations differ at particular time points despite the similar size of the hydrated area of the matrix. At 3 h, a layer containing water of high mobility (T2~60 ms) is present in pure HPMC matrices and HPMC with L-Dopa but it is absent in the HPMC matrix loaded with ketoprofen.Figure 2 components. Each component is characterized by two parameters: a T2 time constant and an amplitude referred to as proton density (PD). The approach yields the following four estimated parameters: T2 of the shorter component (T2S), amplitude of the shorter component (AS), T2 of the longer component (T2L) and amplitude of the longer component (AL). This approach also appeared to be valid for matrices loaded with drug substances of different solubility. The T2/PD patterns for the investigated matrices differ as shown in Fig. A more comprehensive description of the systems can be given using radial parameter profiles according to the methodology described in our previous paper . It was 2/PD profiles can be assigned to two types of freezable bound water, namely: bound water of higher mobility (T2L/AL) and bound water of lower mobility (T2S/AS).To identify the characteristics of water occurring within the hydrated matrices, DSC studies were carried out. The results of the DSC measurements will be described in details in the subsequent sections of the paper. However, for clarity it is important to note at this point, that thermal analysis confirmed the absence of free (bulk) water in the samples taken from the external part of the matrices. Consequently, the two components in Tversus systems loaded with a very slightly soluble drug. The matrix loaded with the sparingly soluble drug (L-dopa) forms the highest number of distinguishable matrix regions (layers) during hydration. For this reason it will be described first.One of the most important results obtained from the presented study is the detection of the micro/meso–scale morphology difference between systems loaded with a sparingly soluble drug 2/PD profiles obtained for HPMC/LD at hydration times of 0.5–3 h  increases slightly in linear manner away from the center of the tablet. Moderate slope ranged from 1.64 A.U./mm at 0.5 h to 0.67 A.U./mm at 3 h of hydration. At the same time the amplitude of the shorter component (AS) decreases linearly. The slope for AS was similar as for AL but of opposite sign. This phenomenon occurs while T2 values of both components (T2L and T2S) increase. The end of the sublayer is associated with a distinct discontinuity in the T2L profile and is preceded with a slope change in AL and AS profiles.Detailed description: The longer TThe second interface layer, the first sublayer (marked dark green)Criteria of selection: two components exist; their parameters change over a wide range but in a systematic manner across this layer.2L profile. Across this layer the amplitude of the longer component (AL) increases linearly away from the center of the tablet. At the same time the amplitude of the shorter component (AS) decreases. The slopes of AL and AS change in the second sublayer and are steeper than in the first sublayer. For example, the AL slope ranges from 4.95 A.U./mm at 1 h to 3.57 A.U./mm at 3 h of hydration and is 3–6 times steeper than in the first sublayer. This phenomenon occurs while T2 values of both components (T2L and T2S) increase. “Amplitude crossing” occurs approximately when the T2 value of the shorter component (T2S) reaches a plateau. The amplitude of both components reach a plateau when the T2 value of longer component (T2L) reaches its plateau.Detailed description: It starts at the position of a discontinuity in the TThe second interface layer, the second sublayer (marked light green)The layers in the radial profiles were identified due to the criteria described in details in our previous article . Identif2 profile of the longer T2 component (T2L)  . T2 profile shows a discontinuity even at 0.5 h of hydration, however, the “gel-like” (diffusion) layer formation is of negligible size (time factor for “gel-like” layer formation is needed).The threshold initiating a slope change in the Ain the system loaded with LD, the 2nd interface region is thicker than in a pure HPMC matrix, at the expense of the external “gel-like” (diffusion) layer.sublayers in the 2nd interface layer appear and existence of these layers can be associated with the presence of undissolved and dissolved drug substance in the matrix.Key differences between matrix hydration of HPMC/LD and pure HPMC can be summarized briefly as:2/PD pattern in the hydrated HPMC/KT system is a subset/part of that registered for HPMC/LD formulations. Starting from the position of full hydration (at maximum of the AS) the longer component, characterized by AL and T2L, appears (exactly as in the HPMC/LD case). Then, the amplitude of the longer component (AL) increases slightly (moderate slope) away from the center of the tablet. At the same time the amplitude of the shorter component (AS) decreases in a similar manner. The drug dissolution related effects in the matrix are absent. Amplitude (AL) of the longer T2 component (water of higher mobility content) for HPMC/KT reaches higher values than threshold for HPMC/LD (2.36 A.U. vs. 1.59 A.U. at 2 h of hydration), but neither a discontinuity in T2 profile nor a slope change in PD were observed. It can be hypothesized that there are no sufficient conditions to trigger/enable a transition to the “gel like” state. The corollary is that the HPMC matrix with the very slightly soluble drug substance (ketoprofen) is not able to form the most external two regions observed for HPMC with sparingly soluble drug. i.e.2nd sublayer of 2nd interface layer associated with dissolved drug;homogeneous external “gel like” layer.As it can be seen from Fig. Properties of the external layer in the swelling matrix are a crucial factor for drug release. As a result of different layer formation, the external layers in matrices loaded with sparingly and very slightly soluble drug have different properties see Fig. . reflect2 relaxation times of the most external parts of the pure HPMC, HPMC/LD and HPMC/KT matrices are presented in Table 2L profile discontinuity for HPMC/LD matrix are also presented in Table Molecular mobility of water expressed by T2L was 46.3 ± 3.8 ms while for HPMC/KT matrix was 33.1 ± 1.8 ms. The T2L for HPMC/KT at the external border of the matrix has very similar values as for the HPMC/LD matrix at the T2L discontinuity. It shows that the water molecular mobility at these points is similar. In case of the matrix with very slightly soluble drug (HPMC/KT) the external layer is similar to the 1st sublayer of the 2nd interface layer that was found in the HPMC/LD matrix.In case of the HPMC/LD matrix, the external layer is a “gel-like” layer, while for the HPMC/KT system the external layer is the 1st sublayer of the 2nd interface layer with the undissolved drug as can be found in the HPMC/LD matrix. For the HPMC/LD external layer an average TWith the DSC method three states of water can successfully be identified: free water, freezable “bound” water and non-freezable “bound” water. The first two states can be distinguished comparing the phase transitions during a heating and cooling runs. The crystallization and melting processes of free water undergo a transition near 0°C while the main exo- and endothermic peaks are shifted toward lower temperatures, representing freezable “bound” water. It is clear that if the interactions between water and the polymer matrix are getting stronger, the crystallization temperatures will be decreasing. The non-freezable bound water absorbed in polymer does not crystallize as a typical exothermic peak, even when the swollen sample is cooled down to −70°C .2 component) and a less mobile water fractions. Chemical exchange between the polymer water of lower mobility (shorter T2 component) and the more mobile fraction is expected in such a system  , where DCP is a practically insoluble in phosphate buffer, the drug release follows the erosion of the matrix (the release of the polymer). If, as we suggest, for the matrix with a poorly/insoluble substance “gel-like” (diffusion) layer was not formed, there is no diffusion component in the mechanism of drug release across/from outer layer–in this case the release of active substance should be purely erosion driven. But surprisingly, in contrast to the their release tests or our results, Tajarobi et al.  , in whici et al.  did not  , “gel layer thickness” or overall swelling of the matrix was sometimes presented ,11,19. I2‘s as well as PD’s and consequently in terms of particular layer formation are unambiguous. It means that overall swelling (or hydration) cannot be used for comparison of different matrix systems. Only similar (“specific” as denoted by Dahlberg) matrices can be compared this way. Moreover, drug release is merely a result of synergy of many factors/mechanisms and cannot be used for deducing mechanisms. While physico-chemical properties between matrices may vary, deep insight into the physical parameters of the hydrating system is necessary for proper matrix dosage form evaluation.Our results confirm that the approach presented by Dahlberg was valid. Physicochemical properties of the swollen region vary with varying solubility of additives (drug). There are no noticeable differences in solvent penetration at consecutive hydration times between all three formulations. But differences in properties of the hydrated matrices in terms of Tet al.  based o    imaging of the hydration of pure HPMC matrices have been presented by Li . (2010) . The ide. (2010)  were as   is required, and this level is dependent on the solubility of the drug substance. For the HPMC/KT system, this level was not reached, even after 3 h of hydration. Considering the time factor, for the HPMC/LD matrix, the gel-like layer does not form immediately after reaching the threshold level of water of higher mobility. It is negligible at 0.5 h of hydration, but appears at 1 h  suggests that the mechanism of region formation is the same in both cases. The key difference is that in the case of HPMC/KT the system is lacking conditions to form a gel-like layer. Gel-like (diffusion layer) layer formation for matrices with incorporated drugs requires dissolution of the drug substance as for the HPMC/LD matrix at 1, 2 and 3 h of hydration  as presented in this study, provides new, consistent insight into the hydration of HMPC based polymeric matrices–in this case: pure polymer, and polymer loaded with drugs of different solubility.We have introduced a new paradigm in the characterization of hydrating matrices by Magnetic Resonance Imaging methods. High resolution MR relaxometry imaging  was obtained comparing data with previous MRI studies while preserving the possibility to noninvasively image real (various size and shape) pharmaceutical products. We have shown that the morphology of the hydrating HPMC matrix is complex and depends on the solubility of incorporated drug substances. In the most general case (HPMC/LD), the matrix was characterized in terms of a dry core, a minimally hydrated zone, two interface layers (one with two separate sublayers) and an external gel-like (diffusion) layer.For the first time, this drug dissolution related phenomena inside the HPMC matrix, i.e. discontinuity in TIn case of HPMC loaded with sparingly soluble L-Dopa all layers/zones were present.Pure HPMC matrix does not form the first sublayer in the second interface layer as presented in both drug loaded matrices–this zone is associated with the presence of undissolved drug.In case of the HPMC matrix loaded with an very slightly soluble drug substance (Ketoprofen) the system does not form a distinct gel-like layer–only the first two internal interface layers (transition zones) are present.In particular cases layer formation was different:From a controlled release point of view it could be concluded that drug release from polymeric matrices is merely a consequence of specific matrix formation and the properties of the external part/layer of the matrix. Corollary, nature/morphology of the hydrated part of the matrix should be taken into account when designing (comparing) dosage forms and modeling dosage form behavior.


In previous experiments an enhanced anti-proliterative effect of the EGFR/ErbB tyrosine kinase inhibitor (TKI) BIBW 2992 with single dose irradiation was observed in FaDu tumour xenografts. Aim of the present experiment was to determine if this effect can also be seen in combination with a fractionated radiotherapy. Secondly we investigate the efficacy of BIBW 2992 on local tumour control for UT-SCC-15.Tumour pieces of FaDu, UT-SCC-14, A431, UT-SCC-15 (squamous cell carcinomas) and A7 (glioma) tumour models were transplanted onto the right hind leg of NMRI (nu/nu) nude mice. For evaluation of tumour growth mice were either treated daily orally with BIBW 2992 (30 mg/kg body weight), or carrier up to a final tumour size of 15 mm or with a fractionated radiotherapy  with simultaneous application of BIBW 2992 or carrier. For local tumour control UT-SCC-15 tumours were treated with a fractionated radiotherapy (30f/6weeks) or received 30f/6 weeks in combination with daily orally BIBW 2992 (22.5 mg/kg b.w.) during RT.A significant effect on tumour growth time was observed in all tumour models for BIBW 2992 application alone. However, substantial intertumoural heterogeneity could be seen. In the UT-SCC-14, UT-SCC-15 and A431 tumour models a total regression of the tumours and no recurrence during treatment time (73 days) were determined where as for the A7 tumour only a slight effect was noticeable. For the combined treatment of fractionated radiotherapy (15f/15d) and BIBW 2992 administration a significant effect on tumour growth time was seen compared to irradiation alone for A7, UT-SCC-15 and A431 (ER 1.2 – 3.7), this advantage could not be demonstrated for FaDu and UT-SCC-14. However, the local tumour control was not altered for the UT-SCC-15 tumour model when adding BIBW 2992 to fractionated irradiation (30f/6weeks).A heterogeneous effect on tumour growth time of BIBW 2992 alone as well as in combination with fractionated irradiation could be demonstrated for all tumour models. However, the significant effect on tumour growth time did not translate into an improvement of local tumour control for the UT-SCC-15 tumour model. Overexpression of the epidermal growth factor receptor (EGFR) on tumour cells has been shown to increase chemo- and radioresistance and therefore is associated with a poor outcome -3. InhibBIBW 2992 is an irreversible ErbB-family (EGFR/ErbB2/ErbB4) inhibitor, which in previous experiments demonstrated a significant prolongation of tumour growth time in a combined setting with single dose irradiation in FaDu tumour xenografts. In vitro but not in vivo a radiosensitizing effect could be shown for this tumour model. The antiproliferative effects are in line with a clear G0/G1 arrest of the tumour cells . Based o7 to14-week-old male and female NMRI (nu/nu) mice from the specific pathogen-free animal breeding facility of the Experimental Center of the Medical Faculty Carl Gustav Carus, Technische Universität Dresden were used for the experiments. The facility veterinarians checked the microbiological status of the animals regularly. The experiments and animal facilities were approved according to the German animal welfare regulations. Water ad libitum and a commercial laboratory animal diet were provided for the animals. A constant temperature of 26°C, daylight in addition to a 12 h light–12h dark electric cycle (light-on time 07.00 a.m.) and a relative humidity of 50-60% were supplied within the animal room. A whole-body irradiation 2 – 5 days before tumour transplantation with 4 Gy using 200 kV X-rays (0.5 mm Cu) at a dose rate of about 1 Gy/min was given for further immunosuppression of the animals.50 for single dose irradiation under clamp conditions with whole body irradiation was 58.6 Gy  and without whole body irradiation 67.8Gy  (p = 0.577).Four established human squamous cell carcinoma lines  and a glioma cell line (A7) were used in this study. UT-SCC-14 and UT-SCC-15 were established by Prof. Reidar Grenman . UT-SCC-14 ,18 was dIn the experiments, tumour pieces of 1–2 mm diameter were transplanted subcutaneously into the right hind leg of anesthetised (16 mg/kg body weight (b.w.) xylazine  and 120 mg/kg b.w. ketamine [i.p.]) animals. All procedures for tumour transplantation have been described previously in detail ,26-28.Animal welfare approval number: 24D-9168.11-1/2006-20.in vitro and in vivo has been shown to be greater than that of the first generation TKIs (e.g. erlotinib) [BIBW2992, a second generation tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) family binds irreversibly to the intracellular tyrosine kinase of the EGFR and ErbB2 and ErbB4-receptor . Its efflotinib)  and resiFor evaluation of the drug effect alone, carrier or BIBW 2992 was administered daily orally at a concentration of 30 mg/kg b. w. up to the final size of the tumour (one diameter reaching 15 mm). Within the combined treatment, carrier or BIBW 2992 were only given simultaneously during fractionated irradiation using the same application and concentration schedule mentioned above, with a 4 hour interval before each irradiation fraction. For the local control experiment a lower BIBW 2992 concentration (22.5 mg/kg b.w.) was administered due to observed toxicity within the previous experiments.Local tumour irradiation was carried out under ambient conditions to air-breathing animals without anaesthesia . Specially designed jigs were able to hold 5 animals for simultaneous irradiation. The tumour-bearing leg was held positioned in the irradiation field while mice were immobilized in a plastic tube which was fixed on a lucite plate by a foot-holder distal to the tumour.The first experiment was divided into 2 arms Figure : in arm For the local tumour control experiment (C), the UT-SCC-15 tumour model was selected as the best responding model regarding tumour growth time Figure . UT-SCC-3 .Tumour volumes for both experiments at start of treatments were approximately 100 mmAnimals were observed up to final tumour size (mean diameter exceeded 15 mm), until day 150 after the end of treatment or until death. For UT-SCC-15 tumours that were evaluated for local tumour control, it has been shown before that almost all recurrences occur within that follow-up time after radiotherapy . In the 2, where a is the longest and b is the perpendicular shorter tumour axis, tumour volumes were calculated. For each treatment arm the median tumour volumes and their standard errors were computed as a function of time after start of treatment. Evaluations were stopped when < 50% of the animals of each treatment arm were alive. From individual growth curves the median tumour growth time was calculated as the time that a regrowing tumour needed to reach 2 and 5 fold of the starting volume . Enhancement ratios (ER) were computed as the quotient of a GD value of BIBW 2992 treated tumours and the GD value in the carrier treated group.Tumour diameters were determined twice per week. With the formula for the rotational ellipsoid V = π/6 * a * bx = vector of covariates that define the treatment, β = vector of parameters describing radiosensitivity of the tumours, and f is a (possibly nonlinear) function of these. Parameters were estimated using maximum likelihood as implemented in STATA 7.0 software . Quoted confidence limits are asymptotic estimates from the results of the likelihood fits. Comparison of maximum likelihood fits was performed using the likelihood ratio test. Tumour control dose 50% (TCD50) at day 150 after end of irradiation and associated dose–response curve were determined from:150 days after end of irradiation tumour control rates were calculated for each dose group after correction for censored animals according to the method given by Walker and Suit . When an2, 95% humidity) in 25 cm2 tissue culture flasks. For the A7 tumour model MEM-EARLE with 10% fetal calf serum and 1 mM pyruvate was used as medium. After 24 hours cells were incubated with BIBW 2992 or for control with dimethylsulfoxid (DMSO). Three days later, cells were irradiated with doses of 2, 4, 6 or 8 Gy. Directly after irradiation  cells were trypsinised and counted. Appropriately diluted single cell suspensions were incubated in petri dishes for 14 or 10 days (A431), fixed and stained with crystal violet.Colonies with ≥ 50 cells were scored as survivors. Mean values of the surviving fraction and their standard deviations (SD) were determined for each treatment group and dose level. Cell survival curves were fitted according to the LQ-model. Plating efficiencies (PE) and surviving fractions (SF) were calculated using the following formulas:Tumour cells of the different cell lines were grown in Dulbeccos modified Eagle’s medium with glutamine, 10% fetal calf serum, 1 mM pyruvate, 1% non-essential amino acids, 20 mM HEPES and 1% Penicillin-Streptomycin at 37°C (5% CO2) and 5-fold (GDV5) of the starting volume. The application of BIBW 2992 alone leads to a significant prolongation of tumour growth time in all tumour models with considerable intertumoural heterogeneity. While for the A7 tumour model only a slight prolongation of tumour growth can be seen, a distinct effect was observed for the FaDu-, A431-, UT-SCC-14 and UT-SCC-15 tumour models. A complete regression of all tumours during continuous application of BIBW 2992 alone without recurrence seen during treatment time (mean 73 days) was determined in the UT-SCC-14, A431 and UT-SCC-15 tumours. During fractionated irradiation  a significant prolongation of tumour growth by BIBW 2992 could be observed for the A7, A431 and UT-SCC-15 tumour models where as this effect was not evident in FaDu and UT-SCC-14 tumours .Figure Figure Table V5 and for BIBW 2992 an ER of 7.8 for the same endpoint was determined [The present experiments are the first to test the effect of combined fractionated irradiation and an EGFR/ErbB-TK inhibitor on tumour growth time and local tumour control. 4 different squamous cell carcinoma cell lines with heterogeneous radiosensitivity and a radioresistant glioma cell line were selected. We could show a heterogeneous effect on tumour growth time in these 5 different tumour xenografts tested for the administration of BIBW 2992 alone or in combination with fractionated irradiation. These findings are in line with previous experiments, where a prolongation of tumour growth time was seen for the treatment of BIBW 2992 with or without single dose irradiation in the FaDu tumour model  or bladdin vitro and with the largest effect of the combined treatment on tumour growth in vivo. The A7 cell line showed also a minimal but significant radiosensitizing effect in vitro. However, because of the small amount of this effect and the lower response of this cell line regarding the other endpoints, this was not further followed up. Regarding the effect on local tumour control in UT-SCC-15, there was no benefit seen by the simultaneous ErbB family inhibition with BIBW 2992 during fractionated irradiation . Common side effects were erythema of the mouth and diarrhea as described before by Schütze et al. . After ain-vitro experiments, the induced resistance to EGFR-TKI could be overcome by IGF1R-inhibitors [in vitro does inactivate clonogenic cells in UT-SCC-15 and UT-SCC-14 independent from the irradiation effect (Table It can only be speculated which reasons underly the missing translation of the positive effect on tumour growth into improvement of local tumour control for TK-inhibitors in comparison to antibodies like for example cetuximab. One potential reason could still be an alternative signal transduction. Hu et al.  reportedhibitors ,41. Indehibitors . It is ict Table . If thisThe present experiments show a heterogeneous effect of the ErbB family TK inhibitor BIBW 2992 on tumour growth in 5 different tumour models for drug application alone as well as in combination with a fractionated radiotherapy. The major effect on tumour growth in UT-SCC-15 tumours did not translate into an improvement of local tumour control. Along with previous experiments on combined fractionated irradiation and EGFR-TK inhibition, it appears likely that TKI do not affect survival of tumour cells that can cause recurrences but can lead to a good palliative effect by proloning tumour growth for a reasonable amount of time.


Forficula auricularia), and was found to be an effective contact toxicant with residual activity on substrates commonly encountered in urban environments. Within 16 h of being directly sprayed with indoxacarb, ≥90% of earwigs from two populations were either ataxic, moribund, or dead, and 100% displayed these symptoms of severe intoxication at 1 d. Brief exposure (5 min or 1 h) to dried residues on either a porous (pine wood) or non-porous (ceramic tile) substrate also was sufficient to cause severe intoxication of earwigs within 1 d. In all bioassays, indoxacarb-treated earwigs showed no signs of recovery during the 21-d observation period. In outdoor urban habitats, intoxicated earwigs would be more vulnerable to desiccation, predation, or pathogens leading to higher mortality than in a laboratory setting.Indoxacarb (Arilon 20WG) was evaluated against a nuisance pest, the European earwig ( Forficula auricularia L. (Dermaptera: Forficulidae), was initially reported from North America in the early 1900s, and extensive outbreaks were first noted in Newport, RI, in 1911 and in Seattle, WA, in 1915  followed a predictable sequence of disorientation, ataxia, and moribundity followed by death over a period of 8 to 23 d depending on concentration and exposure time [Blattella germanica (L.)], untreated cockroaches succumbed after being exposed to bait-fed cockroaches; mortality was attributed to consumption of the excretions and regurgitate of indoxacarb-intoxicated cockroaches as well as cannibalism. Furthermore, the horizontal transfer of indoxacarb continued beyond secondary mortality, with significant tertiary mortality observed [Other studies with indoxacarb likewise have shown that this insecticide is a slow-acting toxicant against various urban insect pests. Intoxication of termites [ure time . In the Indoxacarb was an effective insecticide against the European earwig, both as a direct spray and as a residual deposit on porous and non-porous substrates commonly encountered in urban environments. All earwigs were ataxic, moribund, or dead within 1 d after being directly sprayed with indoxacarb, and the majority were similarly affected after briefly contacting (5 min or 1 h) dried indoxacarb residues on pine wood and ceramic tile. Recovery of directly sprayed earwigs or those exposed to dried residues was not evident during the 21-d observation period. Hence, indoxacarb, an oxadizine, has good potential for earwig control in urban environments.


To compare facial appearance and dento-alveolar relationship outcomes from the CSAG (1998) and CCUK (2013) studies.Five-year-olds born with non-syndromic unilateral cleft lip and palate. Those in the original CSAG were treated in a dispersed model of care with low-volume operators. Those in CCUK were treated in a more centralized, high-volume operator model.We compared facial appearance using frontal view photographs  and dental relationships using study models . Facial appearance was scored by a panel of six assessors using a standardized and validated outcome tool. Dento-alveolar relationships were scored by two assessors using the 5-Year-Olds’ Index. Ordinal regression was used to compare results between surveys.Excellent or good facial appearance was seen in 36.2% of CCUK compared with 31.9% in CSAG. In CCUK, 21.6% were rated as having poor or very poor facial appearance compared with 27.6% in CSAG. The percentage rated as having excellent or good dento-alveolar relationships was 53.0% in CCUK compared with 29.6% in CSAG. In CCUK, 19.2% were rated as having poor or very poor dento-alveolar relationships compared to 36.3% in CSAG. The odds ratios for improved outcome in CCUK compared to CSAG were 1.43  for facial appearance and 2.29  for dento-alveolar relationships.Facial and dento-alveolar outcomes were better in CCUK children compared to those in CSAG. A child born with a cleft lip and/or palate (CLP) will receive treatments from a range of specialties as the anomaly affects several anatomical areas. Correction of anatomical structures is a key determinant of facial appearance, dento-alveolar relations and function. Appearance and dento-alveolar relationships are thus important outcome measures in these children.A number of methods are available to evaluate appearance. These include photographs, videotapes, projected cine films, black and white drawings, identikit pictures  and computer generated pictures Several systems have been described for measuring dento-alveolar relationships as an outcome measure for CLP. The first widely adopted dento-alveolar outcome measure was the Great Ormond Street, London and Oslo Yardstick, more commonly known as GOSLON Following CSAG, the 57 units which provided cleft care before have now been reduced to 11 centres or managed clinical networks The data came from two cross-sectional studies in the UK, the CSAG All of the photographic prints from CSAG were taken using a standardized protocol. There is no internationally accepted system for aesthetic assessment of individuals with cleft lip and palate http://stattrek.com/statistics/random-number-generator.aspx), ensuring there was no systematic clustering of images into CSAG or CCUK groups. The assessment of the collected images was made by a panel of six assessors comprising an adult with a cleft, a lay person and four cleft professional assessors from a single cleft unit . Each assessor was asked to rate a total of 491 frontal images (239 CSAG and 252 CCUK). After 4 weeks, 10% of randomly selected images from both the CSAG and CCUK were rescored. All of the data were then entered into an Excel spreadsheet.The cropped and coded images were arranged for assessment, with a random number generator (www.ablebits.com) and were scored once by each assessor in 1 day. One week later, all the models were rearranged into a different random order and scored for a second time by both assessors. All of the data were then entered into an Excel spreadsheet.The collection of the CCUK study models is described in the methods paper in this supplement The four scores for each of the CCUK study models (i.e. two assessors on two measurement occasions) were reduced to a single score for the main analysis. The coding rule was that if at least three of four assessment scores were the same, then that score was selected. If there was greater disagreement, then the study models were re-examined by both assessors together and a consensus reached. For CSAG, single scores  were already available.For both photographs (CSAG and CCUK) and models (CCUK only), agreement between and within assessors was determined using Cohen’s weighted kappa (κ) statistic. Linear weights were used to allow comparability with previous studies http://www.stata.com/.Comparisons between CSAG and CCUK were firstly made by combining photograph and model outcome categories into excellent (category 1), good, fair, poor  and very poor (category 5), then calculating the percentages of photographs and models in each category. For facial appearance, data from all assessors were combined to describe the distribution of categories in each survey. A mixed-effects ordinal logistic regression model was then used to compare the outcome in the CSAG vs. CCUK group using individual-level data from each observer. The mixed-effects model accounts for the fact that observations from each assessor were not independent. Ordinal logistic regression was also used to assess whether facial appearance had improved from the CSAG to CCUK group for each assessor individually. For dental relationships, an ordinal logistic regression model was used to compare the outcome in CSAG and CCUK using the single score from both groups. For all models, the outcome variables were reversed such that a higher score represented a better outcome. Hence, if the odds ratio was greater than one, then the odds of a better outcome were higher among children in the CCUK study vs. CSAG study. Data were analysed using Stata (version 12) Tablep = 0.032]. It should be noted that children in the CCUK study were on average 10 months younger than those in the CSAG study, and younger children tended to be rated with better facial appearance.Fig.The odds ratios for a better outcome in CCUK compared to CSAG from each assessor are shown in TableTablep < 0.001]. Data on age of children were available for 218/223 of the CSAG models and 197/198 of the CCUK models. When we adjusted for age, the odds ratio for a better outcome was moderately attenuated from 2.69  to 2.29  among those individuals with information on age. Unfortunately, we were not able to link the photographs to the age of the children and so were unable to examine whether age affected the difference we report between surveys for this finding.As seen in Fig.This study repeated, as closely as possible, the original CSAG study which was conducted some 15 years ago. We have reported two outcomes  in 5-year-old children born with unilateral cleft lip and palate. In repeating the study, we needed to be confident that recruitment to both studies was similar and that the collection of photographs and models was not biased. The recruitment issues have been discussed in the first paper of this series Although all efforts were made to ensure equivalent quality of facial photographs in both groups, such as the use of same resolution and dimension adjustments, some aspects could not be standardized. For example, the CSAG sample comprised photographic prints that were scanned to derive a digital format. Some loss of detail would have occurred during the scanning process. In addition, although a standardized photography protocol was applied to both groups, the equipment used differed. In the CSAG groups, photographs were taken using Pentax 35 mm film-based SLR Camera, which is less sophisticated than the cameras used in the CCUK group . Moreover, there was a difference in the magnification used in the CSAG group (1:6) and the CCUK group (1:8). It has been shown that evaluation errors using digital photographs may result because of the magnification factor The agreement between the members of the assessment panel rating frontal views was fair in both the CSAG group and the CCUK group. There was heterogeneity in the assessment panel which included a lay person, an adult with a cleft and professionals . Elsewhere, similar groups have evaluated facial appearance in adults with repaired UCLP with no correlation between professional and lay assessments of nasolabial appearance, perhaps not surprisingly given that the evaluation of facial appearance on cropped photographs is not a task familiar to the lay person It is worth highlighting that in other intercentre studies The 1998 cleft service reorganization appears to have resulted in improved facial appearance of UK 5-year-olds born with complete unilateral cleft lip and palate. The measurement error indicated by the, at best, moderate intra- and interassessor agreement for photographs would likely bias our comparisons towards the null. The odds ratio for a better facial appearance in CCUK children is thus likely to be underestimated in these analyses. That said, younger children rate better for facial appearance and children in the CCUK study were on average 10 months younger than those in the CSAG study. In addition, in the CSAG study, analogue pictures were used and these were then scanned, which may have resulted in loss of detail in making these assessments. Furthermore, the intra- and interobserver agreement was weaker than for the observations on dento-alveolar relations.In the original CSAG study, dento-alveolar relationships were determined with the 5-Year-Olds’ Index and the interassessor agreement (weighted kappa) was 0.94 The improvements in outcomes for CCUK are clear when compared to CSAG. Over 50% of children included in the CCUK study model sample were recorded as having a good dento-alveolar relationship and <20% had a poor relationship. By contrast, <30% of the CSAG sample was assessed as good and over 36% were poor. As the outcomes were measured in children aged around 5 years old, there are relatively few treatment factors which could have an effect on the observed result. Primary surgery is usually carried out before the child is 1 year old and is often the only major intervention before 5 years of age. Both alveolar bone grafting and orthodontics are undertaken at a later age and would not account for the differences seen. Although facial growth patterns can influence results, there is no reason to believe that these would be different between the CSAG and CCUK groups. Furthermore, at 5 years of age facial, growth patterns are not fully expressed and would not distort the result of the primary surgery. This leaves primary surgery, used to correct the anatomical relationships, as the most likely cause of the differences in dento-alveolar relationships in the CSAG and CCUK samples. There have been no major changes in the surgical techniques used in the UK. If anything, the surgical techniques used currently are more similar across all centres and there is a smaller range of surgical techniques employed in UK centres than in the past Three main changes as a result of CSAG may account for the improvements in these anatomical outcomes. First, centralization of cleft care has increased the volume of cases that each surgeon treats per year. Seventeen of the 19 primary cleft surgeons in the UK met a target of operating on between 40 and 50 cases annually in 2009–2010 The 5-year-old index has face validity and can predict outcomes in older children With regard to improved outcomes of dento-alveolar relationships in children born with UCLP in the UK post-centralization, the national registry, the Craniofacial Anomalies Network, known as CRANE reported on 5-year-old study casts (364) between 2004 and 2007 There is evidence from this study that outcomes of anatomical correction of children born with UCLP have improved since 1998 when they were still being treated in a dispersed model of care. There is strong evidence of improved outcomes from study models of children treated pre- and post-CSAG (CCUK) at the age of 5 years, and although overall facial appearance improved from CSAG to CCUK, the evidence is less convincing but is supportive of this improvement. Service reconfiguration seems to have improved anatomy and appearance, and this might form the basis to predict that this would translate into benefits in function and psychological adaptation. These issues are covered in subsequent papers in this supplement. The evidence reported here indicates that the centralization of cleft services in the UK has improved appearance outcomes for children born with a cleft.Centralization of cleft services in the UK has been ongoing for the last 15 years with the reduction of the number of cleft centres from 57 to 11. This process was predicated on a belief, and some evidence, that outcomes would be optimized in a model of high-volume operators and concentrated care. Two key outcomes in children born with a cleft are facial appearance and dento-alveolar relations. In the previous Clinical Standards Advisory Group study, these outcomes were poor. The implementation of centralized multidisciplinary care appears to have resulted in improved outcomes for dento-alveolar relations and facial appearance.


DClolivine/fluid varying between 10−5 and 10−3, whereas DClorthopyroxene/fluid and DClclinopyroxene/fluid are ~10−4 and DClamphibole/fluid is ~5 × 10−3. Furthermore, partitioning results for incompatible trace element show that compatibilities of trace elements are generally ordered as Damph/fluid ≈ Dcpx/fluid > Dopx/fluid > Dol/fluid but that Dmineral/fluid for Li and P is very similar for all observed silicate phases. Infrared spectra of olivine synthesized in a F-free Ti-bearing system show absorption bands at 3,525 and ~3,570 cm−1. In F ± TiO2-bearing systems, additional absorption bands appear at ~3,535, ~3,595, 3,640 and 3,670 cm−1. Absorption bands at ~3,530 and ~3,570 cm−1, previously assigned to humite-like point defects, profit from low synthesis temperatures and the presence of F. The presence of planar defects could not be proved by TEM investigations, but dislocations in the olivine lattice were observed and are suggested to be an important site for halogen incorporation in olivine.We present new partition coefficients for various trace elements including Cl between olivine, pyroxenes, amphibole and coexisting chlorine-bearing aqueous fluid in a series of high-pressure experiments at 2 GPa between 900 and 1,300 °C in natural and synthetic systems. Diamond aggregates were added to the experimental capsule set-up in order to separate the fluid from the solid residue and enable in situ analysis of the quenched solute by LA–ICP–MS. The chlorine and fluorine contents in mantle minerals were measured by electron microprobe, and the nature of OH defects was investigated by infrared spectroscopy. Furthermore, a fluorine-rich olivine from one selected sample was investigated by TEM. Results reveal average Cl concentrations in olivine and pyroxenes around 20 ppm and up to 900 ppm F in olivine, making olivine an important repository of halogens in the mantle. Chlorine is always incompatible with Cl partition coefficients The online version of this article (doi:10.1007/s00410-013-0902-5) contains supplementary material, which is available to authorized users. Mantle minerals such as olivine, ortho- and clinopyroxene can host up to several hundreds ppm water  F is less incompatible than Cl  was used to investigate the relations between the OH-related IR absorption bands and humite-type defects.In this study, we present new experimental results derived from synthesis experiments at 2 GPa and 900–1,300 °C for F and Cl incorporation in olivine, ortho- , clinopyroxene, and amphibole and mineral/fluid partition coefficient for Cl in the system MgO–SiO2 = 44.2 wt%; Al2O3 = 1.3 wt%; Fe2O3 = 9.4 wt%; MnO = 0.1 wt%; MgO = 43.6 wt%; CaO = 1.3 wt%) and of a synthetic MSH  powder both doped with TiO2, NaCl, CaF2 or MgF2 2O was added to the capsule, corresponding to 15 wt% of the starting powder + water mixture. The capsule was then filled with 6–7 mg of synthetic diamond crystals (grain size 20 μm) kindly provided by Servsix GmbH  and with 35–55 mg of the starting material powder. For experiments without the diamond trap, the set-up of the capsule was the same except for the Pt ring and for the diamond crystals that were not used. For experiments with the MSH mixtures, 2 μl H2O was added to the capsule. The capsule was welded while cooled in water, and the weight of the capsule was compared before and after welding to ensure that no water was lost during welding. The sealed capsule was squeezed to a final cylindrical shape with a length of 5.5–6.5 mm and then held at 120 °C for 24 h, in order to check whether the capsule was sealed properly.As sample containers, we used Pt capsules with an outer (inner) diameter of 3.0 (2.6) mm. For experiments with the diamond trap, a Pt ring with an inner diameter of 2.1 mm and a height of 1 mm was inserted into the Pt capsule. Then, ~6 μl HfO2 (Table fO2 for the controlling reaction Fe–FeO is ~3 log units below the QFM buffer (QFM-3) and for the reaction Ni–NiO is ~1.5 log units above the QFM buffer (QFM + 1.5)  and weighed again. Usually, the weight loss upon drying corresponds to 90–100 % of the initial water content. In the two samples buffered by the reactions Fe–FeO and Ni–NiO, remnant metal foil from the oxygen buffer material and brightly colored crystalline material was recovered from the capsule. The metal foil was analyzed by EDS during the EMPA session. All recovered capsules were embedded in epoxy and ground by hand. As soon as the trap became visible, the sample was impregnated with epoxy to prevent destruction during preparation. Later, the capsule was ground until its maximum cross section was exposed.Experiments were carried out in an end-loaded piston-cylinder apparatus at 2 GPa and temperatures in the range of 900–1,300 °C for durations between 1 and 6 days at Innsbruck University  were measured. Since the differences in the isotope ratio were always below 3 %, the influence of this interference was considered negligible. The detection limit for chlorine was below 100 ppm. For minerals analysis, the NIST SRM610 glass was used to calibrate elements. Element concentration calculations were based on the Si concentration as determined by electron microprobe analysis. For the analysis of the crystalline phases, the diameter of the laser was adjusted as function of the crystal size  a usual nitrogen-cooled MCT-D316-025 (mercury cadmium telluride) detector  and (2) a nitrogen-cooled focal plane array (FPA) detector consisting of 64 × 64 MCT-D364 detectors. For measurements with the single-element detector, each spectrum was acquired by 64 scans in the 550–7,500 cm2 F20 X-Twin transmission electron microscope at GeoForschungsZentrum Potsdam, Germany, operated at 200 kV. Selected area electron diffraction (SAED), combined with bright field (BF), high-resolution (HR-TEM) and scanning transmission electron microscopy using a high-angle annular dark field detector in scanning transmission mode (STEM-HAADF), was performed to investigate the local defect structure of forsterite. The sample for TEM was prepared by first determining the orientation of the olivine using electron-backscattered diffraction (EBSD) with an Ultra 55 Plus (Carl Zeiss SMT) field emission scanning electron microscope. The foil for TEM was prepared by the focused ion beam technique (FIB)  Wirth  using a B) Wirth . It was 2O) molar ratio of 0.2 and 0.4 increases the temperature of the solidus in the system Mg2SiO4 + MgSiO3 + H2O + KCl at 5 GPa, respectively, of ~200 and ~350 °C  molar ratio of 0.3, they can be considered being subsolidus. The textures of the experimental charges can serve as additional constraint of the absence of a melt during the runs. Textures of representative run products are shown in Fig. Experimental conditions and results are summarized in Tables 97) is present at temperature above 1,100 °C, at 1,100 °C orthopyroxene appears, at 1,000 °C olivine coexists with ortho- and clinopyroxene and at 900 °C amphibole occurred in addition. Spinel coexists with olivine in the run buffered by the reaction Fe–FeO. The Cl concentrations in olivine are 20 ± 14 ppm, in opx 26 ± 10 ppm and in cpx 20 ± 4 ppm, whereas the Cl contents of amphibole is 585 ± 36 ppm.Run products were mainly composed of olivine, pyroxenes, salt and aqueous fluid. In single cases, additional phases such as clinohumite, amphibole or spinel were observed. Individual phase assemblages are listed in Table 100) coexists with enstatite in all runs. At 1,200 °C, clinohumite coexists with forsterite and enstatite and at 1,000 °C F-tremolite (4.2 wt% F), diopside (Di85En15 containing 690 ppm F) and an unidentified F-bearing phase—supposedly talc—occurred. At 1,200 °C, forsterite has a F content of 900 ± 40 ppm and a Cl content of 170 ± 70 ppm. Enstatite shows a Cl content of 30 ± 20 ppm, similar to the opx in the run with natural starting composition. The aqueous fluids are homogeneous with respect to their major element composition. Most trace elements were below detection limit both in minerals and in the quenched solute. Concentrations measured with LA–ICP–MS and calculated partition coefficients  are illustrated in Fig. In the synthetic system, forsterite (FoThe compositions of the aqueous fluids were determined with LA–ICP–MS (Online Resource 1). The aqueous fluids of each run are homogenous with respect to their major element composition and contain large amount of silicate components . Measured fluid compositions are generally consistent from run to run with the temperature of the run.2 molar ratios of the fluids versus the temperature of the runs  and higher values for Ce (~10−2). Olivine/fluid values for Li, B, Ti, Lu, V and P are ~10−1.Measured olivine/fluid partition coefficients show that olivine strongly depletes Cl, Na, Ce and Sr relative to the fluid Fig. , with lo−4, Sr ~10−3, Na and Ce ~10−2, Li, Y, V and P ~10−1, Ti and Lu ~5.Orthopyroxene/fluid values Fig.  are give2, for Ti, Y, Nd, Pr and Zr in the range 1–10. Clinopyroxene/fluid values for Na, Li, La, Sr, V and P are in the range 10−1–1 and for Cl are ~10−4.Clinopyroxene/fluid partition coefficients show a strong enrichment of Pr, Nd, Zr, Ti, Y and Lu in clinopyroxene relative to the fluid Fig. , with va−1–1 for Li, B, La, Sr, P and ~5 × 10−3 for Cl.Amphibole is enriched in Nb, Ce, Pr, Nd, Zr, Ti, Y, Lu and V with respect to the fluid with amphibole/fluid values larger than 1 Fig. . AmphiboDol/fluid. This trend is extremely pronounced for some cations , with Dol/fluid dropping by 1–2 log units as the solute content increases to a similar extent. Similar effects on Dmineral/fluid were reported by Brenan and Watson  were calculated from the Cl concentration in the mineral measured by EMPA and the Cl concentration of the coexisting fluid measured by LA–ICP–MS. The DClol/fl is between 9 × 10−5 and 2 × 10−4. Chlorine in orthopyroxene is more compatible than in olivine with DClopx/fl values between 1.5 × 10−4 and 3.7 × 10−4. Clinopyroxene has DClcpx/fl values of 1.4 × 10−4 and 2 × 10−4 similar to orthopyroxene, and amphibole shows the highest DClamph/fl value of 4.9 × 10−3. For fluorine and for runs performed without diamond trap, the partition coefficients can be estimated as the ratio between the concentration in mineral versus the concentration in the starting mixture. We obtain DFfo/startmix ~0.1 for forsterite characterized by the presence of humite-type point defects, DFchu/startmix = 6.25 and DFDi/startmix = 0.14 for diopsidic pyroxene . Olivine crystallized under relatively oxidizing conditions (fO2 = QFM + 1.5) shows an IR spectrum similar to that of olivine crystallized with no constrain on fO2, whereas the IR spectra of olivine crystallized under reducing conditions (fO2 = QFM − 3) do not have bands at 3,326 and 3,354 cm−1 and those over 3,500 cm−1 tend to be less intense. This observation supports the idea that the bands around 3,350 cm−1 can be assigned to Fe3+-related OH defects  contains much less water with respect to crystals from relatively more oxidized experiments  are observed in the BSE image , major elements in minerals by EMPA, fluid and trace elements in minerals by LA-ICP-MS (DOC 61 kb)Supplementary material Quantification of water dissolved in olivine (DOC 40 kb)Below is the link to the electronic supplementary material.


However, pre-existing hypoxia in tumours and oxygen consumption during PDT can result in an inadequate oxygen supply, which in turn hampers photodynamic efficacy. Here to overcome this problem, we create oxygen self-enriching photodynamic therapy (Oxy-PDT) by loading a photosensitizer into perfluorocarbon nanodroplets. Because of the higher oxygen capacity and longer 1O2 lifetime of perfluorocarbon, the photodynamic effect of the loaded photosensitizer is significantly enhanced, as demonstrated by the accelerated generation of 1O2 and elevated cytotoxicity. Following direct injection into tumours, in vivo studies reveal tumour growth inhibition in the Oxy-PDT-treated mice. In addition, a single-dose intravenous injection of Oxy-PDT into tumour-bearing mice significantly inhibits tumour growth, whereas traditional PDT has no effect. Oxy-PDT may enable the enhancement of existing clinical PDT and future PDT design.Photodynamic therapy (PDT) kills cancer cells by converting tumour oxygen into reactive singlet oxygen ( Photodynamic therapy is used in cancer treatment and generates reactive oxygen species to kill tumour cells but is limited by the availability of oxygen. Here, the authors modify a photodynamic sensitiser so that it produces excess oxygen species and show enhanced tumour cell killing in vitro and in vivo. Because of its high oxygen capacityefficacy . This tyThe near-infrared photosensitizer IR780  and perf1O2 generation by Oxy-PDT with traditional PDT, as determined by the fluorescence intensity of oxidized SOSG (an indicator of 1O2). In the presence of 1O2, the oxidation of SOSG results in increased fluorescence and therefore provides a means of monitoring 1O2 production. We measured the varying extent of fluorescence gain in water in the presence of LIP(IR780+PFH) or LIP(IR780) irradiated by an 808-nm laser for 5-s intervals . The singlet oxygen quantum yields (Фso) of both LIP(IR780) and LIP(IR780+PFH) were also calculated  is higher than for LIP(IR780) or IR780, indicating that the Oxy-PDT platform can increase the quantum yield of photosensitizer. Moreover, this phenomenon can also be achieved using other photosensitizers, suggesting that the enhancement is not specific to IR780  and LIP(IR780) to a series of solutions with concentrations of PFH ranging from 30 v/v% to 1.88 v/v% . The dil2) and then received 20 s irradiation. The results suggested that LIP(IR780+PFH) significantly enhanced 1O2 production under different oxygen pressures. Meanwhile, LIP(IR780+PFH) in extremely hypoxic conditions (0.1 kPa O2) showed higher 1O2 generation than LIP(IR780) in non-hypoxic conditions (21 kPa O2), indicating that during Oxy-PDT treatment, the PFC can successfully enrich oxygen to accelerate 1O2 generation.We then tested the photodynamic efficacy of both LIP(IR780) and LIP(IR780+PFH) in different hypoxic conditions . The sam1O2 from Oxy-PDT and traditional PDT in live cells using carboxy-H2DCFDA as a fluorogenic marker for ROS. Carboxy-H2DCFDA permeates cell membranes to be oxidized, emitting bright green fluorescence in the presence of 1O2. The resulting oxidatively stressed cells showed green fluorescence, indicating increased 1O2 gain. We treated cells with PBS, LIP(IR780) or LIP(IR780+PFH), with and without 20 s continual 808-nm laser irradiation. Without laser irradiation, MCF-7 cells treated with PBS, LIP(IR780) or LIP(IR780+PFH) showed negligible fluorescence  such as rescence . After irescence . To verirescence . Cells t1O2 producers, we tested Oxy-PDT and traditional PDT for their photodynamic effects on inducing cell death. Without laser irradiation, we found no significant cytotoxicity in MCF-7 cells treated with LIP(IR780) or LIP(IR780+PFH) . Similar results were also observed in CT26 murine colon adenocarcinoma cells (After comparing their performances as 780+PFH) . After 2ma cells . In addima cells .P<0.05, two-sided Student's t-test), indicating that Oxy-PDT can improve the efficacy of PDT in both normal and hypoxic conditions.The effects of Oxy-PDT in hypoxia were also studied . CT26 cein vivo1O2 generation of both LIP(IR780) and LIP(IR780+PFH) was measured. SOSG was also used as an indicator because of its green fluorescence after oxidization by the generated 1O2. Without laser irradiation, tumours treated with LIP(IR780) or LIP(IR780+PFH) showed negligible fluorescence. After laser irradiation, tumours treated with LIP(IR780+PFH) showed significant green fluorescence, whereas tumours treated with LIP(IR780) showed weak fluorescence  and LIP(IR780+PFH) groups with and without 808-nm laser irradiation. To avoid the additional efficacy induced by photothermal effects, the temperature of the tumour during irradiation was controlled by dividing the irradiation time into two consecutive 10 s applications with a 1-min interval in between +NIR, two-sided Student's t-test, P<0.05, two-sided Student's t-test), suggesting the improved efficacy of Oxy-PDT.Mice were divided into five groups, including the saline control group and the LIP (IR780) and LIP (IR780+PFH) groups with and without laser irradiation. To avoid the confounding effects induced by photothermal radiation, the temperature of the tumour during irradiation was controlled within a safe range by dividing the irradiation time into two consecutive 10-s applications with a 1-min interval . MonitorAlthough the tumours were significantly inhibited, they were not completely regressed as a result of intravenous injection plus laser irradiation. Indeed, significant tumour inhibition was obtained by a mere single-dose injection and 20-s irradiation, compared with traditional PDT, in which the dosage was too low to show any therapeutic effect. In light of the current results, further optimization of administration frequency is warranted to exploit the full potential of Oxy-PDT-enhanced photosensitizer as a new PDT clinical approach1O2 to kill cancer cells, and thus the 1O2 generation rate determines PDT efficacy. In the Oxy-PDT agent, both PS and oxygen are enriched in the nanodroplet, and thus the oxygen available for the photodynamic reaction are increased compared with traditional PDT in the tumour matrix. Moreover, the lifetime of 1O2 varies with the polarity of the solvent: the half-life of 1O2 is ∼6 × 10−7 s in the cellular environment, 5 × 10−6 s in water, and 5 × 10−2 s in PFH1O2 is extended. These phenomena may be two major reasons that Oxy-PDT can enhance the reactive oxygen level.In this study, we demonstrate a novel Oxy-PDT platform for achieving enhanced efficacy. PDT depends strongly on 1O2 quantum yield (Фso) than visible dyes because the wavelength of NIR light (>800 nm) is longer than the wavelength of visible light and may have insufficient energy to excite oxygen to its singlet state12soLIP(IR780+PFH) was found to be higher than ФsoLIP(IR780), demonstrating that the Oxy-PDT platform can increase the 1O2 quantum yield of NIR dyes. Furthermore, our results showed that Oxy-PDT could increase the dark stability of IR780 and reduce its self-quenching.NIR dyes (such as IR780) typically have a lower in vitro and in vivo. With direct injection into tumours, the in vivo studies showed complete tumour growth inhibition in Oxy-PDT mice treated with a low photosensitizer dosage and 20-s laser irradiation, whereas traditional PDT showed negligible tumour inhibition. These results suggested that in Oxy-PDT, PFH can help the photosensitizer achieve improved effects. The ultraviolet–visible absorption of the NIR dye is reduced after laser irradiation, possibly due to the degradation of the NIR dye by exposure to 1O2. Although the degradation products might also be toxic to cells, the enhanced tumour growth effect of LIP(IR780+PFH) is still attributed to PFH because the possible degradation product is also present in LIP(IR780). Interestingly, with only a single-dose intravenous injection into tumour-bearing mice, Oxy-PDT still exhibited significant tumour inhibition, indicating that passive targeting plays an important role in the therapeutic efficacy. It should be noted that in all our experiments, the influence of photothermal therapy has been excluded to examine only photodynamic effects. Moreover, previous studies have already confirmed the high oxygen content in perfluorocarbons, which are used as artificial blood in clinical applications and can be used as ultrasonography24in vivo.The results also showed a higher therapeutic efficacy of Oxy-PDT than traditional PDT both in vivo and anticipate its wide clinical application after dose optimization.As previously discussed, Oxy-PDT is considered to be the first PDT design that can realize high efficacy in hypoxic conditions. This Oxy-PDT approach with self-enriching oxygen offers a simple yet effective treatment option for cancer patients. We have provided a proof of concept for the PFC/photosensitizer-loaded emulsion as an agent of improved PDT Lecithin and cholesterol were purchased from Aladdin Industrial Corporation, and DSPE-PEG2000 was obtained from A.V.T. Pharm. Ltd. . The 99% perfluorohexane was purchased from Bailingwei Tech Co., Ltd. . IR780 was purchased from Sigma-Aldrich Chemical Corporation . Singlet Oxygen Sensor Green was obtained from Molecular Probes, Inc. Carboxy-H2DCFDA was purchased from Invitrogen (USA). Cell counting kit-8 (CCK-8) was supplied by Dojindo Laboratories (Japan). MCF-7 and CT-26 cells were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences . All of the BALB/c mice were purchased from Yangzhou University Medical Centre . All of the chemicals were used as supplied without further purification.First, 24.65 mg lecithin, 4.28 mg cholesterol, 3.79 mg DSPE-PEG2000, and the specified IR780 were dissolved in 5 ml dichloromethane. The dichloromethane was removed from a 25-ml round flask by rotary evaporation to form lipid films. Using 10-min sonication, 1.4 ml pure water was added, and the film was peeled off. Next, 0.6 ml PFH was added gradually under high-speed dispersion  at 24,000 r.p.m. in an ice bath for 10 min to form 2 ml LIP(IR780+PFH) (30 v/v% PFH). LIP(IR780) can be formed by the addition of pure water instead of PFH.−2), the oxidized SOSG was quantified by measuring the fluorescence intensity (excited at 504 nm and measured at 525 nm) using a multifunctional microplate reader . As for the production of singlet oxygen in hypoxia, samples were kept in transparent box with different hypoxic air . After laser irradiation , the oxidized SOSG was quantified by measuring the fluorescence intensity. All operations were performed without light exposure. The experiments for each group were run in triplicate.First, 0.1 ml samples and 0.02 ml of 50 μM SOSG were mixed in black 96-well plates (Costar). After irradiation  or LIP(IR780+PFH) was added to the well. The cells were further incubated for 30 min at 37 °C and 5% CO2. The final concentration of IR780 was 4 μg ml−1. After washing once with PBS, the cells were incubated with 600 μl carboxy-H2DCFDA (25 μM) for 10 min. Subsequently, the cells were washed once with PBS and irradiated by an 808-nm laser (2 W cm−2) for 20 s per well. Then, the cells were fixed by 4% formaldehyde polymer for 10 min and labelled with 600 μl Hoechst 33342 (1 μM) for 5 min. Finally, the cells were replaced with 1 ml PBS. The fluorescence emission spectrum of carboxy-DCF (Ex/Em=495/529 nm) and Hoechst 33342 (Ex/Em=350/461 nm) were immediately captured on a confocal fluorescence microscope (OLYMPUS FV1000).The MCF-7 cells were seeded with a density of 2 × 105 per well in 12-well plates. After incubation for 24 h, the medium was replaced with 1.4 ml fresh culture medium. Then, 700 μl PBS, LIP(IR780), or LIP(IR780+PFH) was added to each well, as appropriate. The cells were further incubated for 50 min at 37 °C in an atmosphere containing 5% CO2. The final concentration of IR780 was 4 μg ml−1. After washing once with PBS, the cells were incubated with 550 μl carboxy-H2DCFDA (25 μM) for 10 min. Subsequently, the cells were washed once with PBS and irradiated by an 808-nm laser (2 W cm−2) for 20 s per well. The cells were then centrifuged, resuspended in 500 μl PBS, and analysed by flow cytometry . Green fluorescence was collected on the FL1 channel due to intracellular carboxy-DCF. Data were obtained and analysed using the CELL QUEST and FLOWJO programs.The MCF-7 cells were seeded with a density of 5 × 104 cells per well. After incubation for 24 h, the cells were treated with LIP(IR780) or LIP(IR780+PFH) at different concentrations . For cytotoxicity in hypoxia, the cells were put into a GENbox Jar, where GENbox anaer and anaerobic atmosphere indicator were included. As the oxygen was consumed by GENbox anaer, the indicator changed its colour from blue to colourless. The Jar was approximately oxygen-free 4 h later. Then, the cells were immediately irradiated by an 808-nm laser (2 W cm−2) for 20 s per well. After co-incubation for 2 h, the drugs were removed, and fresh culture medium was added. After further incubation for 24 h, a mixed solution consisting of CCK-8 (10 μl) and fresh culture medium (100 μl) was added to each well and incubated for an additional 2 h at 37 °C and 5% CO2. Finally, the absorbance was measured at 450 nm using the microplate reader. Cells without any drugs or NIR irradiation were used as a negative control.The MCF-7 or CT26 cells were seeded into 96-well plates at a density of 4 × 103), then the loss of 20% of body weight relative to the start of the study, or the appearance of large or open ulceration in the xenograft before scheduled killing, it was killed by CO2 inhalation. In several instances, however, tumour volumes were allowed to exceed the stated limit of 3,000 mm3 because the affected mice appeared otherwise healthy and had lost <5% of their original body weight. This implementation of the protocol has been confirmed by NJU-IACUC. Hairs on the flanks of the mice were removed before further treatments. Tumours were first developed in BALB/C mice by subcutaneously implanting 1 × 107 CT26 cells suspended in 100 μl of serum-free DMEM in the lower flanks of the mice. When the tumour volume reached ∼200 mm3, the tumour mass was removed and cut into small pieces of approximately 2–6 mm3, which were subcutaneously implanted into other mice.BALB/C male mice aged 4–6 weeks were purchased from Yangzhou University Medical Centre  and were used in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) of Nanjing University. During the study, animals were observed for any clinically relevant abnormalities daily or once every other day. If any animal was moribund due to treatment-associated toxicity, tumour over-growth , 25 μl saline, and LIP(IR780) (156 μg ml−1 IR780) or LIP(IR780+PFH)  was directly injected into tumours. Subsequently, laser treatment (with an 808-nm laser (2 W cm−2) for two consecutive exposures of 10 s each, with a 1-min interval between the two irradiations) was performed on the LIP(IR780)+NIR and LIP(IR780+PFH)+NIR groups to generate 1O2. Then, tumours from the five groups were collected and cryosectioned onto slides at a 7-μm thickness. The fluorescence emission of the oxidized SOSG (Ex/Em=504/525 nm) was captured by fluorescence microscope.Tumours were developed in 4- to 6-week-old BALB/C male mice. When the tumour volume reached ∼1,000 mm3 tumour pieces. The groups were as follows: group 1: saline; group 2: LIP(IR780); group 3: LIP(IR780)+NIR; group 4: LIP(IR780+PFH); group 5: LIP(IR780+PFH)+NIR. First, 50 μl of LIP(IR780) (156 μg ml−1 IR780), LIP(IR780+PFH)  or saline alone was directly injected into the tumour mass. Then, laser treatment was immediately performed on groups 3 and 5 by irradiating the tumour regions with an 808-nm laser (2 W cm−2) for two consecutive exposures of 10 s each, with a 1-min interval between exposures for tumour cooling. At day 0, tumours from the five groups were cryosectioned at 5-μm thickness onto slides and stained with TUNEL  according to the manufacturer's instructions. Tumour size was measured daily using a vernier calliper for 14 days after the first PDT treatment. The maximum width (X) and length (Y) of the tumours were measured, and the tumour volumes (V) were calculated using the formula V=(X2Y)/2. Changes in tumour volume as a function of time were determined for each mouse by normalizing the tumour volume at day X to their respective tumour volume at day 0 after treatment. Mice were randomly selected in each group, and the tumours were photographed at days 7 and 14. At 14 days after treatment, the tumour, heart, liver, spleen, lung, kidney and brain tissues of mice from group 5 were collected and detected under in vivo small animal imaging for the biodistribution study. Tumours from the five groups were cryosectioned at 7-μm thickness onto slides and stained with H&E according to the manufacturer's instructions.PDT treatments were then performed on 4- to 6-week-old BALB/C male mice 7 days after inoculation of the 2–6 mm3. The testing groups were as follows: group 1: saline; group 2: LIP(IR780); group 3: LIP(IR780)+NIR; group 4: LIP(IR780+PFH); group 5: LIP(IR780+PFH)+NIR. First, 200 μl of LIP(IR780) (60 μg ml−1 IR780), LIP(PFH+IR780)  or saline alone was intravenously injected. NIR images of IR780 accumulation in the tumours of the mice in the LIP(IR780+PFH) group were taken at 0, 12, 24 and 32 h, and ultrasound images of the tumour accumulation of PFH were taken pre- and 24-h postinjection. Twenty-four hours later, laser treatment was performed on groups 3 and 5 by irradiating the tumour region with an 808-nm laser (2 W cm−2) for two consecutive exposures of 10 s each, with a 1-min interval between the two irradiations for tumour cooling. Tumour size was measured every two days using a vernier calliper for 10 days after the first PDT treatment. The tumours were measured for the maximum width (X) and length (Y) and the tumour volumes (V) were calculated using the formula: V=(X2Y)/2. Changes in tumour volume as a function of time were determined for each mouse by normalizing the tumour volume at day X to the respective tumour volume at day 0 after treatment. Mice were randomly selected in each group, and the tumours were photographed at day 0, 5 and 10. At 10 days after treatment, the mice in each group were killed, and the tumours were removed, photographed and weighed.The CT26 subcutaneous tumours were first developed in the mice as described above. PDT treatment was then performed at 8 days after inoculation of the tumour pieces. The average initial tumour volume was 144.4 mmt-test for two groups, and one-way analysis of variance for multiple groups. A value of P<0.05 was considered statistically significant.Statistical analysis was performed by two-sided Student's How to cite this article: Cheng, Y. et al. Perfluorocarbon nanoparticles enhance reactive oxygen levels and tumour growth inhibition in photodynamic therapy. Nat. Commun. 6:8785 doi: 10.1038/ncomms9785 (2015).Supplementary Figures 1-8, Supplementary Table 1 and Supplementary ReferencesRaw data for Fig5c_Intratumoral injectionRaw data for Fig6c_Intravenous injection