In this paper, membrane deflection against fluid flow and opening membrane (threshold) pressure were studied using fluid-structure interaction (FSI) analysis, and compared with experimental data obtained by Jaemin et al. In the current analysis, two different models (I-shaped and V-shaped) were used to perform the FSI simulation. In microvalve modeling, in order to reduce external actuator usage, interconnections are made between two similar microvalves. This typical interconnection creates a pressure distribution in a local environment. Furthermore, to differentiate the volume factor in a microvalve, a length/width (L/W) ratio term was used. Compared with higher- and lower-L/W-ratio models, the higher-L/W model eventually initiates more deflection in a low-pressure regime than the lower-L/W-ratio model. FSI simulations were performed for 4  Microfluidics deals with the volumes of fluids in sizes of nanoliters to picoliters . It is aGenerally, there are two kinds of microvalves: (1) normally open (NO) and (2) normally closed (NC) ,11. ManySeveral researchers performed FSI analyses on various types of microvalves. Most of them selected a two-way FSI analysis to describe the deformation of the microvalve membrane. Yang and Kao analytically and numerically investigated the deformation of a microelectromechanical system (MEMS) diaphragm valve against the effect of fluid flow . Later, In this work, a two-way FSI analysis is performed on an NC microvalve model. This FSI analysis was performed in order to understand the fluid flow inside the microvalve, the membrane deflection against fluid flow, and changes in performance of the microvalve according to changes in parameters. Finally, results from the FSI analysis are compared with experimental data . In part1). Typically, an NC microvalve design contains source  ratio is the change in position of the valve seat and shape in a microvalve design by varying the vertex length (L). In FSI analysis, various L/W-ratio microvalve models are used to understand the behavior of membrane deflection against the fluid flow in the microvalve. The length in the L/W ratio is indicated as the vertex length (L). Width (W) is a constant value, and it is calculated by  is 50 n et al. . As showIn an experimental analysis  to achieThe inlet is connected to the source regions (PS) of microvalves In order to study a coupled field analysis of the fluid flow and structural deformation in microfluidic devices, a fluid-structure interaction was performed using commercial ANSYS software  [In the microvalve FSI analysis, elastic material is used for the structural membrane. This is shown in Coupled two-way fluid-structure interaction problems need to consider every field, i.e., the fluid flow, structural deformations, and moving mesh. The governing equation for all these fields is referred to in ,5,19 andThe conservation laws of momentum and the continuity equation of a viscous, incompressible flow comprising the Navier-Stokes equation with arbitrary Lagrangian-Eulerian (ALE) framework are used to solve the flow problem, which can be written asv is the velocity and p is pressure. Navier-Stokes equationFor the present problem, an incompressible, viscous, constant-input mass flow rate of the fluid is applied between 4 to 12 Thus, Reynolds number of the fluid flow inside a microfluidic valve is predicted before the analysis, and the flow is considered to be laminar throughout the analysis.x asu, The conservation law of linear momentum for a solid continuum is given with respect to spatial coordinate n and The interface conditions between solid and fluid arei, a Dirichlet boundary condition may be given:In the ALE approach to the fluid-structure interaction (FSI) problems, the pseudo-medium’s boundary conditions are the solid deformation in the FSI interface. The governing equation of the displacement of the fluid mesh nodes is written asFSI analysis of the two-way coupling method is illustrated in Initially, the fluid field is solved in ANSYS-CFX based on the given boundary conditions and are applied in the fluid domain. Later, the pressure from the fluid domain is transferred to the structural domain (ANSYS-Transient structure) through the coupling process. The transferred pressure is applied over the FSI interface surface in the structural model. Since external pressure is applied to the elastic structural membrane, it obtains a large deformation and changes its shape in the model. Furthermore, the deformed shape obtained from the solid domain sets the fluid mesh displacement equal to the solid domain deformation on the FSI interface . In lower-L/W-ratio models, the volume of the source region is limited. Hence, the model cannot produce higher pressure and creates a large deflection in the membrane. Even when compared with that at final deformation, the membrane deflection magnitude is lower in lower-L/W-ratio models than in higher-L/W-ratio models. The initial and total deformation of the membrane increase in higher-L/W-ratio models. These results indicate that increasing the L/W ratio of the model increases the initial and total deformation of the membrane.PDMS is one of the most-used materials in nano-electromechanical systems (NEMS) and micro-electomechanical systems (MEMS) because of its elastic properties. Varying the amount of crosslinker in a PDMS network changes the stiffness of the material . In ordeIn order to check the parameter of the change in inlet height, three different inlet region heights are selected and analyzed. The results are shown in The complex fluid-structure interaction between deformable microvalve structural membrane behavior against the fluid flow inside the valves was studied using an FSI model. Using the model, it was observed that an increase in L/W ratio increases the volume in the source region and decreases the volume in the drain region of the microvalve. In addition, an increase in L/W ratio separates the membrane surface area within those regions. Interestingly, the separation completely alters the nature of fluid flow and the behavior of membrane deflection inside the microvalve.Compared with lower-L/W-ratio models, higher-L/W-ratio models exhibit better performance in terms of membrane deflection and low opening threshold pressure. A higher-L/W-ratio model favors a higher volume in the source region, which enables more rapid structural membrane deflection than the lower L/W ratio models. L/W = 0.65 or higher ratio models can deflect the membrane in lower pressure regime favoring the fluid flow than other lower ratio models.Finally, using an FSI simulation, selected parameters such as the material properties of the structural membrane and the inlet region height were analyzed for better rapid deflection and low opening threshold pressure. This creates a new step in understanding better fluid flow inside a microvalve.


Radiomics is a process of extraction and analysis of quantitative features from diagnostic images. Liquid biopsy is a test done on a sample of blood to look for cancer cells or for pieces of tumourigenic DNA circulating in the blood. Radiomics and liquid biopsy have great potential in oncology, since both are minimally invasive, easy to perform, and can be repeated in patient follow-up visits, enabling the extraction of valuable information regarding tumour type, aggressiveness, progression, and response to treatment. Both methods are in their infancy, with major evidence of application in lung and gastrointestinal cancer, while still undergoing evaluation in other cancer types. In this paper, the main oncologic applications of radiomics and liquid biopsy are reviewed, and a synergistic approach incorporating both tests for cancer diagnosis and follow-up is discussed within the context of systems medicine.Radiomics is a process of extraction and analysis of quantitative features from diagnostic images.• Most clinical applications of radiomics are in the field of oncologic imaging.• Radiomics applies to all imaging modalities.• A cluster of radiomic features is a “radiomic signature”.• Machine learning may improve the efficacy of radiomics analysis.•  In the last few years, the term “radiomics” has emerged in the imaging community as a novel field of research, defined by Lambin et al. as a “high-throughput extraction of image features from radiographic images” .Radiomics is the discipline that deals with the extraction and analysis of quantitative features from diagnostic images . The basExamples of features that can be extracted by radiomics analysis include shape/size-based, histogram-based, filtering-based, and texture analysis .Haralick method  to infer the macro-systems dynamics and produce elements of synthesis such as signatures and profiles originated by a variety of information sources and consequently characterised” –35  or circulating tumour-derived nucleic acids thus provided a potential solution for cancer characterization and management [The need for “liquid biopsy” arose from the fact that molecular profiling of tumours relies on invasive surgical procedures, often associated with procedural risks/complications, and tissue collection is unfeasible for many cancer patients. The identification of circulating biomarkers such as nagement .CTCs derive from primary or metastatic tumour cells clusters, with the ability to invade blood vessels through the tissue matrix. CTC analysis offers the advantage of visualizing intact cells for morphological identification of a malignant phenotype; their enumeration is relevant for the assessment of metastatic and primary disease progression, and enables molecular characterization and immunolabelling. Unfortunately, CTCs have a number of disadvantages: they are present in low abundance and are extremely fragile, they may provide false-negative results, their analysis requires extremely sensitive and specific methods, and tumour heterogeneity may not be totally captured by a small number of CTCs .7 leukocytes per ml of whole blood, and their half-life is estimated at about 1–2.5 h [The number of CTCs is around 1 CTC for 10 1–2.5 h . SeveralIsolation methods based on physical properties include CTC size, density, and electric charge, and consist of centrifugation, membrane- or filtration-based systems, and dielectrophoresis. These methods offer the advantage of quick and simple isolation of CTCs, but they are characterised by poor sensitivity and low sample throughput . CTCs caTo date, CTCs have been demonstrated to be an independent prognostic factor in solid tumours, including breast, pancreatic, and prostate cancer –51. In aCirculating tumour nucleic acids are tumour-derived fragments of DNA/RNA in the bloodstream, released by apoptosis and necrosis processes from dying cells or actively released from viable tumour cells . These nTo date, circulating tumour nucleic acid analysis represents the most promising method for the identification and monitoring of molecular tumour-related alterations in cancer patients . In factCirculating free nucleic acid concentrations can be influenced by tumour size, localization, and vascularity, and it is also possible that they are derived in part from CTC lysis . HoweverThe use of liquid biopsy became more important with the discovery of tumour heterogeneity: it is very well known that tumour lesions may be characterised by a non-uniform distribution of genetically distinct tumour-cell subpopulations across and within disease sites  or temporal variations in the molecular makeup of cancer cells  . Tumour The analysis of circulating biomarkers allows clinicians to discover potential predictive and prognostic biomarkers and to obtain real-time imaging of tumour dynamics.The molecular profile of human cancer has enriched the understanding of tumourigenic processes, improving tumour diagnosis, prognosis, and prediction of response to treatment. Liquid biopsy enables the identification of tumour mutations responsible for tumour response to treatment , primary or acquired resistance to treatment , and monitoring of tumour dynamics during treatment (i.e. KRAS gene mutation in pancreatic cancer).In terms of knowledge and technical performance, mutation analysis of cftDNA is the easiest way to use liquid biopsy, and many examples are already present in literature. Expression analysis of circulating RNA is still debated although feasible in clinical practice, and requires the most specific techniques, such as the use of exosomes to isolate tumour RNA.The use of liquid biopsy came into clinical practice in 2016, with its introduction in the European Society for Medical Oncology (ESMO) guidelines for EGFR analysis of cftDNA in metastatic NSCLC. In particular, liquid biopsy is considered a valid alternative to tissue biopsy, representing a surrogate source of tumour DNA to monitor disease progression in first-line treatment of EGFR-mutant patients. The positivity of the analysis for the EGFR mutation p.T790 M on cftDNA enables treatment with third-generation EGFR tyrosine kinase inhibitors . AnalysiIn colorectal cancer (CRC), RAS gene mutations may serve as a mechanism of secondary resistance to EGFR inhibitors (EGFR-ab), and cftDNA testing has been shown to be a sensitive method for detecting CRC clonal evolution. Several studies have shown that CRC patients with a detectable cftDNA level after surgery experienced relapse within 1 year, while patients with undetectable cftDNA had no recurrence . CftDNA Prostate cancer has recently entered the arena of the biomarker-addicted tumour, following the discovery that a splice variant of the androgen receptor (AR-V7), tested on CTCs, drove resistance to hormonal treatment . In thisThe use of liquid biopsy enables improved surveillance and patient outcome. However, more complex and different strategies are needed in order to fully understand tumour heterogeneity and tumour dynamics. In this context, a radiomics approach, in addition to pharmacogenetics, may be of help in integrating knowledge for a better personalised medicine approach.Recent progress in cancer genomics has expanded our knowledge of tumour heterogeneity. The genotype-guided approach is a successful strategy in tumours dependent on genetic alterations. However, despite an initial response, most tumours progress, developing resistance to targeted therapies. Thus there is an urgent need for an understanding not only of the static molecular profile, but of cancer dynamics, considering that tumour heterogeneity drives cancer evolution .Advances in radiomics interpretation may make it possible to correlate quantitative features with tissue pathophysiology, linking the imaging phenotypes to the genotype.On the other hand, the analysis of circulating tumour nucleic acids permits the detection of molecular changes at low frequency and in low abundance months before clinical evidence of tumour progression. In this context, the multiparametric pattern analysis of radiomics, combined with molecular information obtained from liquid biopsy, may aid decision-making in clinical practice.As discussed above, radiomics and liquid biopsy have similar advantages: they are both minimally invasive “tests” that can be easily obtained and repeated, enabling the extraction of valuable and early information about tumour type, and can aid in determining tumour aggressiveness to predict progression and recurrence. Both can be used as synergistic tests in the screening, diagnosis, and follow-up of cancer, shifting the therapeutic path from the traditional “one-size-fits-all” concept to the modern personalised treatment of cancer, which will be increasingly dependent on a multidisciplinary approach that combines the different “holons” of systems medicine.The best means of integrating radiomic signatures and liquid biopsy is the collection of data in large repositories of biomarkers, referred to as biobanks . In factLinking imaging biobanks to already existing biobanks is certainly one possible strategy in a synergistic approach to radiomics and liquid biopsy, but prospective data collection must be defined in order to obtain synchronous-linked biomarkers of the same patient. The linking of biomarkers should be followed by correlation analysis that may result in patient stratification based on multi-omic signatures/profiles, among which radiomics and liquid biopsy are essential components.From the radiologist's point of view, the use of radiomics could aid in the interpretation of clinical cases in oncologic imaging, and contemporary evidence of CTCs derived from liquid biopsy could enhance the predictive value of radiomic signatures. For example, a patient with CTCs of colorectal cancer and a radiomic signature of an aggressive, non-responsive tumour could then benefit from high-frequency follow-up after chemoradiotherapy in order to monitor for evidence of local recurrence or metastases.In summary, the combined use of radiomics and liquid biopsy could serve as an alarm signal in patients with a high likelihood of recurrence and metastasis. Future reporting should consider the integration of imaging biomarkers and radiomic profiles in the radiological report; this will be possible only through the adoption of structured reporting tools that allow the integration of quantitative data in the template.Radiomics and liquid biopsy have similar attractive characteristics: they can be collected in a non-invasive manner, are quantifiable, and can be repeated to evaluate tumour progression. Finally, they address two faces of the same problem—cancer diagnosis and treatment decisions.Both provide quantitative biomarkers of most cancer types, and evidence in the literature and in clinical practice suggests their growing role in cancer management.Aggregating information from multiple holons of systems medicine is one strategy in the fight against cancer, and radiomics and liquid biopsy constitute an essential component in addressing this challenge. Further research in the form of large clinical trials and biobanks that include multiple imaging biomarkers is needed to confirm the evidence gather thus far.


Vesicles containing 225Ac were injected intravenously in healthy mice, and intratumourally in tumour-bearing mice, and the relocation of free 213Bi was assessed in different organs upon the injection [225Ac]Ac-polymersomes. The therapeutic effect of 225Ac-containing vesicles was studied upon intratumoural injection, where treatment groups experienced no tumour-related deaths over a 115 day period. While polymersomes containing 225Ac could be suitable agents for long-term irradiation of tumours without causing significant renal toxicity, there is still a significant re-distribution of daughter nuclides throughout the body, signifying the importance of careful evaluation of the effect of daughter nuclides in targeted alpha therapy.Increasing attention is given to personalized tumour therapy, where α-emitters can potentially play an important role. Alpha particles are ideal for localized cell killing because of their high linear energy transfer and short ranges. However, upon the emission of an α particle the daughter nuclide experiences a recoil energy large enough to ensure decoupling from any chemical bond. These ‘free’ daughter nuclides are no longer targeted to the tumour and can accumulate in normal tissue. In this paper, we used polymersomes as model carrier to evaluate the retention of recoiling daughters of  An increasingly popular alternative can be found in the use of α-emitting radionuclides. Only a few α particles passing through the nucleus are sufficient to cause multiple double-strand breaks in the DNA and subsequent cell death. Due to their much higher linear energy transfer (LET), α-emitters are more cytotoxic than β-emitters3. Other advantages of the use of α-emitters in tumour therapy include their independence on tumour oxygenation, and their short tissue range which prevents damage to neighbouring healthy cells.Personalized medicine is a rapidly growing field in cancer therapy research. The use of tumour-specific therapeutics has proven to significantly increase patient survival and decrease side effects. A number of β-emitting radiotherapeutics are routinely being used for the treatment of e.g. metastasized prostate cancer and neuroendocrine tumours213Bi (t1/2 = 45.6 min) which has successfully been used to treat a different tumours5. A lot of attention is currently being paid to 225Ac (t1/2 = 10 days), which provides clear advantages over 213Bi including a long half-life allowing more time for accumulation at the tumour site. Furthermore, 4 α particles are emitted in the decay chain of 225Ac, resulting in a larger dose to the tumour site per mother nuclide. A number of successes have been realized in clinical trials using 225Ac, including treatment of neuroendocrine tumours, prostate cancer, and gliomas5. The use of 225Ac-PSMA-617 for the treatment of metastatic castration-resistant prostate cancer shows some particularly promising results6. Even upon progressive disease during after 2 cycles of 177Lu-PSMA-617, impressive treatment response to treatment with 225Ac-PSMA-617 were observed7. However, one of the potential issues arising when using 225Ac for therapy is unwanted toxicity from recoiled daughter radionuclides. Upon the emission of an α particle the radioactive daughter nuclides experience a recoil energy of about 100–200 keV8 which is much larger than the energy of any chemical bond and will thus always result in the daughter nuclide breaking free from the targeting agent. Furthermore, the different chemical properties of the daughter nuclide can make re-association with the chelator very unlikely9. These ‘free’ daughter nuclides can be a source of dose limiting toxicity10. For instance, long-term renal toxicity has been observed in a study using 225Ac labelled anti-rat HER-2/neu monoclonal antibody due to the relocation of recoiling daughter atoms to the kidney11. In another study by Kennel et al., the potential of killing lung tumours in vivo was clearly demonstrated but at the cost of radiotoxic effects associated with released daughter radionuclides which were likely partially responsible for the death of the animals12.Thus far, promising preclinical and clinical results have been reported for a number of α-emitters, including 225Ac mother nuclide in nanocarriers can help retain the daughter atoms at the tumour site and thus limit damage to healthy organs. This approach has been investigated with a number of nanoparticles, including metal-based particles13, zeolites14 and liposomes15. Woodward et al. demonstrated that LaPO4 based nanoparticles are capable of containing 225Ac daughter nuclides. They observed a near complete retention of 225Ac in the nanoparticles, but found that about 50% of the daughter nuclides were released in vitro. Subsequently, they followed the in vivo release of 213Bi in the lungs, liver, spleen and kidney. Elevated levels of 213Bi were found in the kidneys, indicating some release of this daughter nuclide following nuclear recoil, despite its encapsulation in the nanoparticle16. 225Ac-containing liposomes have been shown to be very efficient at selectively killing tumour cells in vitro17. Their efficacy has been tested in 3D tumour models as well as in vivo, introducing a diffusion-assisted approach for full tumour coverage. Here, the liposomes degrade upon entering the lower-pH tumour environment, releasing 225Ac and its daughter nuclides and hence allowing for a better distribution in vitro. This study has shown promising in vivo results, with a significant decrease in tumour volume upon the intravenous administration of the liposomes18. The distribution of the daughter atoms in vivo was not assessed, nor was potential renal toxicity considered, however, very poor 213Bi retention in 100 nm liposomes has been reported in an earlier in vitro study19, suggesting similar problems to occur in vivo.Encapsulation of the 213Bi in a carrier system which has demonstrated good retention of the 225Ac mother nuclide, and determine the in vivo fate of free 213Bi20. We have selected polymersomes as delivery vesicles for 225Ac-based targeted α therapy21, which have shown to retain the daughter nuclides 221Fr and 213Bi to a certain extent20. This retention can be improved by forming small InPO4 nanoparticles around the 225Ac encapsulated within the polymersomes21. Nanocarriers are known to accumulate in the liver and spleen, whereas free 213Bi accumulates in the kidneys, allowing for clearly distinguishable uptake characteristics between the polymersomes themselves and any free daughter nuclides. In this study, we evaluate the retention of 213Bi, one of the daughters in the decay chain of 225Ac, in vivo upon intravenous and intratumoural injection, and looked at the distribution of free 213Bi in select tissues, This is especially important to consider when using long-circulating carriers like nanoparticles or antibodies for tumour targeting. We assessed the effect of the polymersome diameter as well as the presence of InPO4 nanoparticles encapsulated within the vesicles on the retention and distribution of 213Bi. Finally, the effect of [225Ac]Ac-polymersomes and [225Ac]AcDOTA control on tumour growth, proliferation, apoptosis, and double strand DNA breaks upon intratumoural injection are evaluated in BALB/c nude mice.The aim of our study is to assess the retention of the radioactive daughter atom 225Ac in the aqueous cavity of the vesicles containing either DTPA20 or InPO4 nanoparticles21. In this study, we compared the recoil retention of the 213Bi daughter nuclide in vivo of both these polymersome formulations. Throughout this manuscript, ‘free 213Bi’ indicates 213Bi which is no longer encapsulated in the polymersomes following nuclear recoil.It is well known that the recoil energy experienced by the daughter atom is sufficient to break any chemical bond between the daughter atom and a targeting vector. This issue could partially be circumvented through the use of nanocarriers, such as polymersomes, which present a system which is capable of retaining at least part of the daughter nuclides. We have used polymersomes to encapsulate in vivo studies, they were fully characterized by both DLS and Cryo-TEM  could also be nicely visualized within the polymeric nanocarriers.Prior to using the polymersomes in the TEM Fig. . In all 225Ac according to the well-documented procedures as described earlier by Wang et al.20 and de Kruijff et al.21. A loading efficiency of 54 ± 21% has been achieved in the DTPA-containing polymersomes, and 59 ± 6% for co-precipitation in InPO4 containing polymersomes. The labelling efficiency of the [225Ac]AcDOTA compound used for intratumoural injection was 92.3%.Polymersomes were loaded with 225Ac chelated with DTPA or 225Ac coprecipitated with an InPO4 nanoparticle, on the in vivo recoil retention of 213Bi upon intravenous injection. While the 225Ac mother nuclide is very well retained in both systems (retention of approximately 93%)21, this is not the case for the daughter radionuclides. The theoretical recoil distance of the daughter radionuclides is about 100 nm in water8, which means that the probability of the daughter nuclides to be being retained in polymersomes with an optimal diameter of around 100 nm is limited24. The use of larger polymersomes will increase the retention of daughter radionuclides, but this is known to reduce the circulation time and subsequent accumulation in tumour tissue21. Another factor influencing the retention of recoiled daughter is the location of the decaying radionuclide in the vesicle. For instance, if the mother nuclide 217At (second daughter of 225Ac) is located in the polymersome bilayer, the chance of the 213Bi daughter recoiling out of the vesicle is much higher than when 217At would be located in the centre. The loading of 225Ac into polymersomes through co-precipitation with InPO4 nanoparticles has been shown more successful in retaining daughter nuclides compared with 225Ac loaded through chelation with DTPA21. When 225Ac is co-precipitated with InPO4, the recoil distance decreases to about 30 nm, greatly increasing the probability of daughter retention.We assessed the two different polymersome formulations, in vivo system, the distribution of 213Bi after administration of [225Ac]Ac-polymersomes was determined in mice. With a half-life of a little under an hour, the ingrowth of 213Bi was determined through continuous measurement of the ex-vivo organ activity in time. These measurements allowed for extrapolation to the time of sacrifice and a direct comparison of the presence of 213Bi and 225Ac at organ level  and the time it took to perform the biodistributions together with the relatively large distance between the animal facility and gamma counter, we were not able to accurately assess the distribution of 221Fr. Like most nanocarriers, polymersomes accumulate mainly in the spleen and liver25, while free 213Bi is known to accumulate mainly in the kidneys (40%), or is excreted via the urine (30%)26. To be able to measure the 213Bi ingrowth with a certain degree of accuracy, it was important to keep the total number of samples small so that they could be measured frequently. To this end, it was decided to determine the recoil retention of 213Bi in three organs of interest; the blood (injection site), spleen (polymersome accumulation) and kidneys (accumulation of free 213Bi). Immediately upon sacrifice, the organs of interest were collected and continuously measured until the daughter nuclides were in equilibrium with the mother nuclide 225Ac. Back-extrapolation of the obtained data enabled the determination of the amount of 213Bi at the moment of sacrificing the animal, as this is the time point at which blood flow stops, stopping subsequent inter-organ redistribution of free daughters. The activity at this time  as well as in equilibrium with 225Ac is given for selected organs. Furthermore, the ratio of the 213Bi at t = 0 to the equilibrium activity of 213Bi after in-growth to the mother nuclide activity is given. When this ratio is <1, it means that the organ of interest is releasing 213Bi (in this case the blood and spleen), while for a ratio >1, the organ is accumulating free 213Bi. The daughter nuclide distribution presented in Table 225Ac encapsulated in either DTPA-containing polymersomes, or co-precipitated in InPO4 nanoparticles within the polymersomes. In both cases, a significant difference (p < 0.02) between initial 213Bi presence and 213Bi levels at equilibrium with 225Ac was found in the blood. Significantly more (p < 0.05) 213Bi was retained in the blood for the InPO4 containing vesicles vs the DTPA ones (with ratios of 0.14 ± 0.07 and 0.06 ± 0.03 respectively). Based on the blood values, co-precipitation of 225Ac with InPO4 nanoparticles in polymersomes constitutes a definite improvement over DTPA containing polymersomes. However, the ratio of the redistributed 213Bi at time of sacrifice to the equilibrium 213Bi activity found in the spleen and to the kidneys is not significantly different (at a significance level of α = 0.05) when considering the two different polymersome formulations. At first glance this is surprising; a lower amount of free 213Bi would be expected to be present in the mice injected with InPO4-containing polymersomes based on the blood values, which should have resulted in ratios closer to 1 for both the spleen and the kidneys. However, in the kidneys the accumulation of InPO4-containing polymersomes is nearly a factor two lower than for the DTPA-containing polymersomes (2.6 ± 1.1% ID/g vs 4.7 ± 0.8% ID/g respectively), which subsequently increases the ratio of free 213Bi to the equilibrium activity of 213Bi in polymersomes to similar levels. Free 213Bi present in the kidneys at time t = 0 nearly exclusively originates from the release of daughter nuclides from the polymersomes in other organs. The amount of free 213Bi in the kidneys at time of sacrifice therefore more accurately represents the amount of recoiled 213Bi than the ratio does. There is significantly less free 213Bi at t = 0 in the kidneys for the InPO4 containing polymersomes (at a significance level of α = 0.05) which agrees with the differences observed in the blood. For the spleen, similar ratios between the DTPA and InPO4 containing polymersomes were observed. This is most likely explained by the fact that macrophages within the spleen take up the polymersomes, and the macrophages themselves can than act as a secondary barrier against the escape of the 213Bi daughter nuclides from the macrophages. This effect has been described by McDevitt et al., who used 225Ac-labeled antibodies, and found that upon internalization in the cell, the cell aids in the retention of the daughter nuclides27.In Table 213Bi in the polymersomes upon intravenous injection, and looked at the distribution of free 213Bi in organs of interest. However, whereas the daughter radionuclides are retained to at least some degree within the polymersomes themselves, they will always be released when chelated to a tumour-targeting antibody due to the recoil effect28, with amongst others the different chemical properties of the daughter nuclide making re-association very unlikely. Therefore, it is of great interest to compare the results obtained in our study to antibody-targeting studies assessing the recoil retention in vivo. Unfortunately, to date there are only very few studies looking at the free daughter distribution upon decay of 225Ac attached to an antibody. McDevitt et al. recognized that the retention of the daughter alpha-emitters at the target site is critical to the success of the therapy, and therefore specifically focussed on the use of target cell-internalized 225Ac constructs, where internalization by the tumour cell itself resulted in enhanced retention of the daughter nuclides27. The daughter nuclides 221Fr and 213Bi were very well retained at the tumour site at 2 days p.i. (88 ± 9% and 89 ± 2% respectively), although it has to be mentioned that these tumours were counted between 6 and 12 minutes after death and not extrapolated back to time of death, increasing the calculated tumour retention. Kidney values of 213Bi show a significant accumulation of free 213Bi, at least 3 times higher than at equilibrium. However, the equilibrium values of the 225Ac-antibody constructs in the kidneys are much higher compared to the low uptake of [225Ac]Ac-polymersomes found in our study, which significantly decreases the ratio of free 213Bi to that in equilibrium with 225Ac. Furthermore, their values were reported in cpm instead of % ID/g preventing the direct comparison of free 213Bi uptake. Another study by Jaggi et al. looked in detail at the distribution of 225Ac daughter nuclides upon the intravenous injection of 225Ac-labeled antibodies9. While in most cases they do not specifically assess the ratio of the free daughter nuclide to the equilibrium situation, they do show a kidney-to-femur ratio of 225Ac which is much lower than that of 213Bi (an estimated ratio of approximately 1 for 225Ac vs 20 for 213Bi) indicating significant redistribution of 213Bi to the kidneys. Song et al. used 225Ac-labeled anti-rat HER-2/neu monoclonal antibody and found a difference in kidney activity of 213Bi at time of sacrifice of 2449.8 Bq/g to 887.0 Bq/g at equilibrium11, also concluding that there is significant uptake of free 213Bi in the kidneys. Therefore, while all these studies show that there is indeed redistribution of free 213Bi, confirming the results obtained in our study, the presented ratios cannot be compared directly.In this study, we have assessed the retention of 225Ac-containing polymersomes were intratumourally injected and the distribution of 213Bi was quantified immediately upon sacrifice. In these experiments, 225Ac was encapsulated into polymersomes through chelation with DTPA. Although the polymersomes where 225Ac was co-precipitated with InPO4 yielded better 213Bi retention upon intravenous injection, polymersomes containing DTPA have been studied more extensively22, including their therapeutic potential in vitro29, and were thus selected for the intratumoural experiments presented in this paper. Again, free 213Bi was expected to be transported by the blood to the kidneys, and hence the organs of interest in this study were the blood, kidneys and tumour. Polymersomes with a diameter of either 100 nm or 200 nm were intratumorally injected to study the effect of polymersome diameter on daughter nuclide retention. These larger vesicles were not used in the intravenous study, as polymersomes should ideally have a diameter below 100 nm23 for optimal circulation times.To determine the distribution of free daughter nuclides after accumulation in the tumour, 213Bi in the tumour tissue, giving both the amount of 213Bi present in tissue at time of sacrifice (t = 0) as well as at equilibrium. The ratios of free 213Bi to 213Bi in equilibrium are also displayed in this table, where again a ratio is <1 signifies a release of 213Bi in the tissue of interest, and a ratio >1 an accumulation of free 213Bi. Clearly, the tumour is in all cases the only tissue releasing free 213Bi, where the 213Bi is subsequently transported by the blood to the kidneys amongst others. However, it has to be kept in mind that the ratios for both the blood and the kidneys appear exceedingly high due to near-zero amount of polymersomes present in these organs at equilibrium. Rather, for these organs the activity of free 213Bi at time of sacrifice should be regarded as the main indicator of their free 213Bi accumulation. The very low radionuclide activity in the blood shows that 213Bi is transported by the blood but not retained there. There was no large difference in free 213Bi uptake between the 100 nm and 200 nm vesicles in the blood, although in all cases kidney uptake of free 213Bi was slightly lower for the 200 nm polymersomes. This corresponds to the retention of free 213Bi in the tumour tissue, where although already quite well retained for 100 nm polymersomes, 213Bi is nearly completely retained in the tumour when encapsulated in 200 nm vesicles. However, for both polymersome diameters, no significant increase in tumour retention of 213Bi is seen over time, which indicates that the size of the polymersomes still influences the retention of the daughter nuclides suggesting that part of the vesicles has not yet been taken up by the tumour cells. This corresponds to earlier obtained results in in vitro tumour spheroids, where the polymersomes were still distributing themselves throughout the spheroid more than 4 days after the addition of fluorescently labelled polymersomes to the cell medium29. Looking specifically at potential kidney toxicity, the activity of 213Bi at time of sacrifice is in all cases much higher in the tumour than in the kidneys, with tumour: kidney ratios of 9.9 for 100 nm polymersomes, and 29.5 for 200 nm polymersomes at 7 days p.i. Renal toxicity due to free 225Ac daughter atoms which have recoiled out of the polymersomes was thus not be expected to be a problem for the therapeutic studies20.Table et al., examined the recoil retention of 213Bi in LaPO4 nanoparticles containing 225Ac and showed that the release of 213Bi from the target organ decreased over the course of 5 days16. They attributed this to the potential uptake of their nanoparticle conjugates by the endothelial cells of the lungs, which subsequently trapped any free 213Bi within the cells, retarding its diffusion through the tissue. We did not observe a similar pattern in our study; the retention of 213Bi in the tumour tissue is stable in time for both polymersome sizes. This difference in behaviour could be due to the different injection methodologies. Woodward et al. injected their nanoparticles intravenously allowing them to be taken up in the target tissue in time. Although they did not present any direct measurements of circulation time of the nanoparticles, the activity in the target organs is still increasing up to 48 h p.i., pointing to a long circulation time during which the nanoparticles are not completely taken up in the endothelial cells yet. On the other hand, our polymersomes were injected directly within the tumour tissue and hence did not circulate for an extended period of time, resulting in no significant increase in retention between 1 and 7 days p.i.A study by Woodward 221Fr emits gamma particles with energies suitable for SPECT imaging16, the activity used in this experiment is too low for proper imaging which is why we performed a biodistribution study. The distribution of 225Ac complexed to DTPA in polymersomes was compared to that of 225Ac co-precipitated with InPO4 nanoparticles in polymersomes upon intravenous injection. Biodistribution data of the [225Ac]Ac-polymersomes at 4 h p.i. can be seen in Fig. 225Ac coupled to DTPA or encapsulated in InPO4 nanoparticles. Because the radionuclides are encapsulated within the aqueous core of the polymersomes, the outer surface fo the polymersomes remains the same, logically resulting in the similar organ uptake. The only organ displaying a significant difference in uptake between the two types of vesicles is the liver. At the moment, we have no good explanation for this difference in liver uptake. The two types of polymersomes (containing either InPO4 or DTPA in the aqueous cavity) were composed of the same batch of block copolymers, prepared according to the same method, and injected in mice which had been randomly distributed between the two groups. Since the outer surface of the polymersomes was not changed, a difference in uptake was not expected, and further research would be required to fully understand this difference in uptake. The circulation time of these polymersomes in tumour bearing mice is known to be considerably shorter than in healthy mice, with circulation half-lives of 5 min and 117 min respectively30. The exceedingly short circulation half-life in tumour bearing mice prevents sufficient polymersome accumulation at the tumour site upon the intravenous injection of 111In-containing polymersomes (0.44 ± 0.39% ID/g). We therefore decided to assess therapeutic efficacy of [225Ac]Ac-polymersomes through intratumoural injection.While 225Ac]Ac-polymersomes, vesicles with a diameter of 100 nm were used. Here, the biodistribution of both intratumourally injected 225Ac complexed to DTPA in polymersomes as well as [225Ac]AcDOTA was assessed at 1 and 7 days p.i. The reason for the use of [225Ac]AcDOTA instead of 225Ac-DTPA as a control for the polymersomes is its increased kinetic stability31. [225Ac]AcDTPA has been shown to display substantial toxicity due to 225Ac leakage, whereas [225Ac]AcDOTA constitutes an substantial improvement31. While this lower stability of the [225Ac]AcDTPA complex has been shown not to result in enhanced 225Ac leakage from the polymersomes20, the more stable [225Ac]AcDOTA complex was chosen for the in vivo studies to minimize toxicity due to unbound 225Ac. In Fig. 225Ac]Ac-polymersomes and [225Ac]AcDOTA in selected organs is displayed. When viewing these biodistribution results, it is important to keep in mind that the injected activity as well as volume were very small, due to which the activity measured in various organs was just slightly above background. As expected based on earlier results by Wang et al.25, the polymersomes are very well retained in the tumour tissue, while the [225Ac]AcDOTA compound is rapidly cleared. The tumour retention of the polymersomes was found to be 244 ± 74% ID/g and 289 ± 130% ID/g at 1 and 7 days p.i. respectively, whereas less than 10% ID/g and 5% ID/g of the [225Ac]AcDOTA was retained at the tumour site at both time-points respectively  improved survival as compared with the control groups.The tumour model chosen, MDA-MB-231, is a well-vascularized tumour model. Upon injection of the compounds (the polymersomes as well as the DOTA and PBS), some blood was observed to leak out of the tumours. In Fig. wth Fig. . Five ouwth Fig. . Seven owth Fig. , showingrve Fig. , the the225Ac]Ac-polymersomes are retained very well at the tumour site, whereas [225Ac]AcDOTA is cleared rapidly. The tumour dose induced by the 50 kBq [225Ac]Ac-polymersomes is roughly equivalent to the dose given by 5 kBq 225Ac in polymersomes to a 400 µm tumour spheroid as studied earlier29 taking into account the polymersome uptake in the spheroids of only 0.10–0.15%. There, a significant reduction in tumour size was observed at 3–6 days after addition of the vesicles. In the current in vivo study, no significant increase in tumour size was observed after the injection of the polymersomes. The excellent response in the group injected with 50 kBq [225Ac]AcDOTA, which, similarly to the [225Ac]Ac-polymersome group, experienced no tumour related death over the treatment period, was somewhat unexpected. The biodistribution studies at 1 day p.i. show that at this point nearly all [225Ac]AcDOTA has been cleared from the tumour site . The 225Ac was obtained from the Directorate for Nuclear Safety and Security . The PD10 size exclusion columns were obtained from GE Healthcare . Instant Thin-Layer Chromatography (iTLC) strips were purchased from Varian (USA). For the immunohistochemical analysis, rabbit-anti-H2AX , goat-anti-rabbit , avidin-biotin  and Bright DAB  were used. All other chemicals were purchased at Sigma Aldrich.The block copolymer PBd-PEO with M2PO4 as precipitating agent were prepared for the in vivo studies, according to earlier published procedures21. The vesicles used in the intravenous injection studies were prepared using the direct dissolution method22, where 10 mg/mL block copolymer was added to a 1 mM DTPA PBS solution at pH 7.4, or a 0.5 M KH2PO4 PBS solution at pH 3. The solution was stirred at 300 rpm for a week, upon which the polymersomes were extruded to a diameter of 100 nm by passing them several times through polycarbonate filters with cut-off membrane of 800, 400, 200 and 100 nm. For the intratumoural experiments, the DTPA containing vesicles were prepared according to the solvent displacement method. In short, 1 mM DTPA in 1 mL PBS buffer solution (pH 7.4) was slowly added to 1 mL acetone containing 20 mg/mL block copolymer under continuous stirring. After evaporation of the acetone, 1 mL PBS was added to bring the final concentration to 10 mg/mL block copolymer. In all cases, before loading the polymersomes with the radionuclide 225Ac, the remaining unencapsulated DTPA or KH2PO4 was removed by passing it through a Sephadex G 25 size extrusion column (L × D = 30 × 1 cm). 225Ac dissolved in pH 2 HCl together with 200 μL 10 mM HEPES buffer was added to a vial containing 0.1 mg A23187 dissolved in 100 μL CHCl3. Upon the evaporation of the CHCl3, 800 μL polymersome solution was added and incubated for 1 hour. A PD10 column was used for purification, and the 7th 0.5 ml fraction was collected for further in vivo experiments at an 225Ac concentration of either 250 kBq/mL for the intravenous experiments, or 2 MBq/mL for the intratumoural experiments. Loading efficiencies were calculated as the amount of activity eluted from the PD10 column in the polymersome-containing fractions divided by the total activity in the sample. The daughter nuclide 221Fr (218 keV) was counted with the Wizard gamma counter (PerkinElmer) when equilibrium was reached and decay-corrected as a representative measurement of 225Ac in the sample. Samples were counted for 1 minute each, with as counting window 170–270 keV for 221Fr and 380–520 keV for 213Bi.Polymersomes containing either DTPA as hydrophilic chelator, or KHin vivo studies, the vesicles were characterized by both dynamic light scattering (DLS) and cryogenic transmission electron microscopy (Cryo-TEM). The DLS consisted of a JDS Uniphase 633 nm 35 mW laser, a fibre detector, an ALV sp 125 s/w 93 goniometer and a Perkin Elmer photon counter, with an ALV-5000/epp correlator and software. A 0.01 mg/mL polymersome solution was placed in a toluene filled, temperature regulated bath (20 °C), where the intensity autocorrelation function was determined at 90°, and the data was fitted using the Contin method. The hydrodynamic radius was obtained using Einstein-Stokes equation.Before being used for the 40.Cryo-TEM images were obtained by depositing 4 μL of the 10 mg/mL polymersome solution on a holey carbon film  supported on a TEM grid. The drop was blotted for four seconds with filter paper in order to obtain a thin layer on the grid, and subsequently vitrified by rapidly immersing in liquid ethane (Leica EM GP version 16222032). The sample was inserted into a cryo-holder (Gatan model 626) and then transferred to a Jeol JEM 1400 TEM. Images were obtained at an acceleration voltage of 120 keV. For the statistical analysis of the polymersome and nanoparticle diameters, about 30–50 images were made of each of the polymersome samples. The diameter of polymersomes and nanoparticles within those images were measured with ImageJ225Ac]AcDOTA, 1.4 MBq 225Ac was added to 8 · 10−9 mol DOTA in 40 μL 0.1 M TRIS buffer at pH 9.0. After an incubation time of 1 h at 37 °C the labelling efficiency was determined with iTLC. A 2 µL droplet of the radiolabelled sample was placed on a 7 cm ITLC strip, with 0.1 M NaOH at pH 12 as mobile phase. The ITLC strip was subsequently imaged with the Phosphor-imager to determine the labelling efficiency, which was calculated as the fraction of the total activity which travelled with the mobile phase divided by the total activity on the sample. The sample was used without further purification, and diluted in PBS to an 225Ac concentration of 250 kBq/mL for intravenous, and 2 MBq/mL for intratumoural studies.For the radiolabelling of Ac-polymersomes, or 50 kBq [225Ac]AcDOTA, and one of the control groups with 25 μL PBS, while the other group was left untreated.All mice used in the intratumoural experiments were subcutaneously inoculated with 5 · 10ex vivo biodistribution and 213Bi recoil retention were analysed at 1 and 7 days p.i. for the mice having been injected with [225Ac]Ac-polymersomes, or [225Ac]AcDOTA. The mice of the PBS and untreated groups were sacrificed for immunohistochemical analysis of the tumour and kidneys. Tumours and kidneys of all animals (3 per treatment group) were fixated in 4% paraformaldehyde in PBS overnight and embedded in paraffin to analyse double strand DNA breaks immunohistochemically. Retention of the daughter nuclide 213Bi in the tumour, and accumulation in the kidneys, was assessed according to the procedure in the intravenous experiment, where 213Bi activity at the time of sacrifice was determined by extrapolating the in-growth back to time t = 0.The 3, ulceration or invasive tumour growth, other signs of clinical discomfort e.g. 15% weight loss in two days, dehydration). Technicians assessing the humane endpoints were blinded for the treatment groups.All groups consisted of 8 mice each, of which the tumour growth and overall survival was monitored up to 115 days after the start of treatment. Tumour volume was measured twice a week. Mice were taken out of the experiment when they reached one of the humane endpoints  staining, paraffin was removed by incubation with xylene, and subsequently sections were incubated with haematoxylin for 20 minutes and with eosin for 5 minutes, followed by dehydration and mounting in Permount.2O2 in methanol at RT. After another two PBS washes, the sections were incubated for 30 minutes at RT with 200 times diluted biotinylated goat-anti-rabbit. Finally, an avidin-biotin complex was applied for 30 minutes at RT. To develop the tumour sections, they were incubated with 50–150 µL Bright DAB for 8 minutes at RT. Nuclei were counterstained with 3x diluted hematoxylin in PBS for 5 seconds. Finally, the sections were dehydrated with consecutively 50%, 70%, and twice 100% ethanol, twice with Xylene, after which they were mounted with Permount, dried and imaged.For the staining for γ-H2AX, first antigen unmasking was performed on the deparaffinated tumour sections by treating the slides with 10 mM citrate buffer (pH 6.0) for 10 minutes at 98 °C. After washing the slides with distilled water and PBS, aspecific binding was blocked by incubating the sections with 50–150 µL 5% normal goat serum (NGS) in PBS for 30 minutes at RT. Subsequently, the tumour sections were incubated overnight with 50–150 µL rabbit-anti-H2AX diluted 1000 times in a PBS solution containing 1% BSA  and 5% NGS at 4 °C. Subsequently, slides were washed 3x with PBS and endogenous peroxidase activity was blocked by a 10-minute incubation in 0.3% HTime-to event data collected during the intratumoural efficacy study were analysed using Graphpad Prism . Statistical significance between groups was analysed by multiple comparisons of the survival curves. Statistical significance was set at p < 0.05 calculated by the Log-rank Mantel-Cox test and corrected for multiple comparisons using a manually calculated Bonferroni-corrected threshold with 6 comparisons and p = 0.05.


Barite sag is a serious problem encountered while drilling high-pressure/high-temperature (HPHT) wells. It occurs when barite particles separate from the base fluid leading to variations in drilling fluid density that may cause a serious well control issue. However, it occurs in vertical and inclined wells under both static and dynamic conditions. This study introduces a combined barite–ilmenite weighting material to prevent the barite sag problem in water-based drilling fluid. Different drilling fluid samples were prepared by adding different percentages of ilmenite  to the base drilling fluid (barite-weighted). Sag tendency of the drilling fluid samples was evaluated under static and dynamic conditions to determine the optimum concentration of ilmenite which was required to prevent the sag issue. A static sag test was conducted under both vertical and inclined conditions. The effect of adding ilmenite to the drilling fluid was evaluated by measuring fluid density and pH at room temperature, and rheological properties at 120 °F and 250 °F. Moreover, a filtration test was performed at 250 °F to study the impact of adding ilmenite on the drilling fluid filtration performance and sealing properties of the formed filter cake. The results of this study showed that adding ilmenite to barite-weighted drilling fluid increased fluid density and slightly reduced the pH within the acceptable pH range (9–11). Ilmenite maintained the rheology of the drilling fluid with a minimal drop in rheological properties due to the HPHT conditions, while a significant drop was observed for the base fluid (without ilmenite). Adding ilmenite to the base drilling fluid significantly reduced sag factor and 50 wt.% ilmenite was adequate to prevent solids sag in both dynamic and static conditions with sag factors of 0.33 and 0.51, respectively. Moreover, HPHT filtration results showed that adding ilmenite had no impact on filtration performance of the drilling fluid. The findings of this study show that the combined barite–ilmenite weighting material can be a good solution to prevent solids sag issues in water-based fluids; thus, drilling HPHT wells with such fluids would be safe and effective. Drilling fluid contains a mixture of solids and polymers. Solids such as weighting materials are used mainly to increase the mud density, while clays and polymers are used to control the rheological and filtration properties. For bentonite-based drilling fluid, the rheological and filtration properties can be improved by using chitin nanocrystals from speckled swimming crab shell waste at different ranges of pH  or by adPolymers are used heavily in the oil industry for controlling the drilling fluid properties or for enhanced oil recovery applications to extract the heavy oil. Polymer flooding is a very important technique to extract the heavy oil from thin and heterogeneous reservoirs . ScreeniDrilling high-pressure/high-temperature (HPHT) oil and gas wells is challenging because it requires a special fluid formulation that can control the high pressure and withstand the elevated downhole temperatures . Weighti3), cheap cost, and low environmental impact. Suspension of weighting agents is critical because settled particulates will not contribute to the hydrostatic column of the drilling fluid, leading to serious well control issues , good performance, availability, and acidity [Because of the high demand and limited resources of barite, many research studies were conducted to find another dense weighting material that can be used to drill HPHT oil and gas wells . Ilmenit acidity ,30. Ilme acidity . To avoi acidity .Elkatatny et al.  evaluateThis study introduces a combined (barite–ilmenite) weighting material as a new solution to the barite sag issue in water-based drilling fluids to be used for drilling HPHT oil and gas wells. The effect of using the combined weighting material on all drilling fluid properties was evaluated in this study. Using the combined weighting agent would reduce sag tendency of barite-weighted fluids and minimize the cost of ilmenite-weighted fluids. Firstly, the methodology followed to conduct this study is presented. Then, the results of this study are discussed. Finally, the findings of this research work are concluded. 50) of around 17 μm, while ilmenite had an average particle size of around 5 μm. Water-based drilling fluid was formulated by mixing the main drilling fluid additives, obtained from local suppliers, at room temperature using a three-speed mixer. These additives were viscosity and pH control additives, a clay stabilizer, a bridging agent, and the weighting material. Barite was used as a weighting agent to attain the high density (15 ppg) of the base drilling fluid. Then, different fluid samples were prepared using a combination of barite and ilmenite with different percentages of ilmenite  from the total weight of the weighting material. A static sag test was performed to study the effect of ilmenite on sag tendency of barite-weighted drilling fluid in static conditions. The test was conducted in vertical and inclined conditions (45°) because the solids sag issue becomes more serious at an inclination angle above 30° . Figure After 24 h, two fluid samples (10 mL) were collected form the top and the bottom of the fluid and weighted; then, the sag factor was calculated using Equation (1). The acceptable sag factor ranges from 0.50 to 0.53 .(1)Sag The sag shoe was put in the bottom of the cup and the drilling fluid sample (140 mL) was poured in the cup;The fluid sample was heated until 120 °F and a viscometer was run at 100 RPM;1);Then, a 10-mL sample was collected from the collection well in the bottom of the sag shoe using a syringe with a cannula, and the weight was recorded (W2);After 30 min, the viscometer was stopped, and another sample was taken from the bottom and weighted (WThe dynamic sag factor (VSST) was calculated in ppg using Equation (2).The sag tendency of drilling fluid samples was evaluated under dynamic conditions using a viscometer sag shoe test (VSST). The test was conducted in standard conditions, i.e., atmospheric pressure and 120 °F . Figure A drilling fluid that has a VSST value equal to or less than 1.0 ppg will exhibit good performance, and solids sag is unlikely to occur , while aDrilling fluid rheology was measured in low- and high-temperature conditions (120 °F and 250 °F) to investigate the effect of adding ilmenite on the rheological properties of the drilling fluid formulation. These rheological properties were yield point, plastic viscosity, and gel strength after 10 s, 10 min, and 30 min. Plastic viscosity (PV) and yield point (YP) were calculated from dial reading data (Ø) at 600 and 300 RPM using Equations (3) and (4), respectively. On the other hand, gel strength data were obtained directly from dial readings at 3 RPM after 10 s, 10 min, and 30 min of gel time.Viscoelastic properties of drilling fluid samples were measured by conducting an oscillatory amplitude test and an oscillatory frequency test using an Anton Paar 302 rheometer. Firstly, the oscillatory amplitude test was conducted to identify the linear viscoelastic range. The test was run at 250 ℉ with a constant frequency of 0.2 rad/s. Then, the oscillatory frequency test was run to obtain the storage modulus G′ and loss modulus G″. The storage modulus can be an indicative tool to detect solids sag tendency .Filtration tests were performed to evaluate the effect of adding ilmenite on the filtration performance of the drilling fluid and the sealing properties of the formed filter cake. The test was performed at 250 °F and 300 psi differential pressure, which was applied using nitrogen gas. A 10-μm ceramic filter disc was used as the filtration medium. The test was started, and the filtrate volume was recorded with time for 30 min. Then, the weight and the thickness of the formed filter cake were measured. 3, and ilmenite density, 4.79 g/cm3 [Ilmenite was added to the base barite-weighted drilling fluid with different percentages from the total weighting material . 79 g/cm3 . Therefo79 g/cm3 .3 and a 2.5-mm filter cake was formed in all fluid samples  and it can be maintained by adding pH control additives.Adding ilmenite enhanced the stability of the drilling fluid and significantly reduced the sag factor for both static and dynamic conditions. The sag factor decreased as the concentration of ilmenite increased, and 50 wt.% ilmenite was adequate to prevent barite sag in both conditions. The sag factor dropped from 0.59 to 0.51 in static conditions, while VSST dropped from 1.75 to 0.33 in dynamic conditions.Ilmenite improved the rheology of the drilling fluid and maintained rheological properties under HPHT conditions. In contrast, a huge drop in rheological properties of the base fluid (without ilmenite) was observed because of the high temperature. Similarly, ilmenite enhanced the viscoelastic properties of the drilling fluid by increasing the elasticity of the drilling fluid, which validates the sag test results.3 for both fluids (100 wt.% barite and 50 wt.% ilmenite), while a 2.5-mm filter cake was formed in both cases.From the HPHT filtration results, adding ilmenite to the base drilling fluid had no impact on the filtration performance and filter cake properties. The total filtrate volume was 6.2 cmBased on the rheology and sag test results, 50 wt.% is the optimum concentration of ilmenite, and adding more than this concentration is not recommended as it would add more cost to the drilling operation.The combined barite–ilmenite weighting material could be a good option to safely kill or drill HPHT oil and gas wells with minimal sag tendency; however, more research work is needed to remove the composite filter cake in the case of drilling, because the removal process of the filter cake would be more complex as two different materials need to be dissolved. Therefore, a new approach to remove the composite filter cake is required.Extensive experimental work was conducted to evaluate the effect of combining barite–ilmenite weighting materials to mitigate the barite sag issue in water-based drilling fluid. From the results of this work, the following conclusions can be drawn:


Triticum aestivum L.) chromosomes are still limited. In this study, the whole genome shotgun assembly sequences (IWGSC WGA v0.4) and the first version of the reference sequences (IWGSC RefSeq v1.0) of Chinese Spring (T. aestivum L.) were used to find new tandem repeats. One hundred and twenty oligo probes were designed according to the new tandem repeats and used for ND-FISH analysis of chromosomes of wheat Chinese Spring. Twenty nine of the 120 oligo probes produce clear or strong signals on wheat chromosomes. Two of the 29 oligo probes can be used to conveniently distinguish wheat A-, B-, and D-genome chromosomes. Sixteen of the 29 oligo probes only produce clear or strong signals on the subtelomeric regions of 1AS, 5AS, 7AL, 4BS, 5BS, and 3DS arms, on the telomeric regions of 1AL, 5AL, 2BS, 3BL, 6DS, and 7DL arms, on the intercalary regions of 4AL and 2DL arms, and on the pericentromeric regions of 3DL and 6DS arms. Eleven of the 29 oligo probes generate distinct signal bands on several chromosomes and they are different from those previously reported. In addition, the short and long arms of 6D chromosome have been confirmed. The new oligo probes developed in this study are useful and convenient for distinguishing wheat chromosomes or specific segments of wheat chromosomes.Non-denaturing FISH (ND-FISH) technology has been widely used to study the chromosomes of Triticeae species because of its convenience. The oligo probes for ND-FISH analysis of wheat ( Althougkgrounds , they arcv. Chinese Spring (T. aestivum L.) that was kept in our laboratory was used in this study.Common wheat 1. Tandem Repeat Finder  (The whole genome shotgun assembly sequences (IWGSC WGA v0.4) and the first version of the reference sequences (IWGSC RefSeq v1.0) of wheat Chinese Spring were downloaded from the IWGSCon 4.09)  was usedon 4.09)  and the on 4.09)  with theSupplementary Table S1.Finally, the consensus sequences obtained through the two strategies were used for Nucleotide BLAST searches against the Nucleotide collection (nr/nt) database using blastn tool in National Center for Biotechnology Information. The consensus sequences that have the identity ≥70% with the published tandem repeats such as 45S rDNA, 5S rDNA, pSc119.2, pAs1, pTa-535, pTa-713, pTa-86, pTa-465, pTa-k566, pTa-s120, pTa-s126, pTa-k288, and pTa-k229 etc. were omitted, and the rest ones were used for designing oligo probes. The new oligo probes that can work are listed in Supplementary Table S1 were synthesized by Tsingke Biological Technology Co. Ltd. . The oligonucleotide sequences were 5′- or 3′-end-labeled with 6-carboxyfluorescein (6-FAM) or 6-carboxytetramethylrhodamine (TAMRA) (Supplementary Table S1). In addition, oligo probes Oligo-pSc119.2-1 and Oligo-pTa535-1 developed by Supplementary Table S1. For each probe, three ND-FISH experiments were repeated. Images were made and processed according to the methods described by The oligo probes listed in 2 against IWGSC RefSeq v1.0 of wheat Chinese Spring, and the chromosome plot was drawn using Plot tool in B2DSC.The consensus sequence of the oligo probe that only produced hybridization signal on the telomeric region of one arm of 6D chromosome was aligned using blastn tool in B2DSCSupplementary Table S1.One hundred and fourteen new tandem repeats were detected from whole genome shotgun assembly sequences (IWGSC WGA v0.4) and the first version of the reference sequences (IWGSC RefSeq v1.0) of wheat Chinese Spring. According to these tandem repeats, 120 oligo probes were designed and used for ND-FISH analysis of the root tip metaphase chromosomes of wheat Chinese Spring. Twenty-seven of the 120 oligo probes produced strong or distinct signal bands on wheat chromosomes, and two of the 120 oligo probes produced clear signals mainly on the whole B- or D-genome chromosomes. The ND-FISH signals of the 29 oligo probes are reproducible. The information of the 29 probes is listed in Supplementary Figures S1–S12). Sixteen of the 29 new oligo probes only produced clear or strong signals on specific segments of wheat chromosomes . Probes Oligo-1AS, Oligo-5AS, Oligo-7AL, Oligo-4BS, Oligo-5BS, and Oligo-3DS only generated hybridization signals on the subtelomeric regions of 1AS, 5AS, 7AL, 4BS, 5BS, and 3DS arms, respectively . Probes Oligo-1AL, Oligo-5AL, Oligo-2BS, Oligo-3BL, Oligo-6DS.1, and Oligo-7DL only produced strong or distinct hybridization signals on the telomeric regions of 1AL, 5AL, 2BS, 3BL, 6DS, and 7DL arms, respectively . The signals of probes Oligo-4AL and Oligo-2DL only occurred on the intercalary regions of 4AL and 2DL arms, respectively . The signal of probe Oligo-6DS.2 only appeared on the pericentromeric region of 6DS arm , and probe Oligo-3D only produced signals on the subtelomeric region of 3DS arm and the pericentromeric region of 3DL arm .Combined with oligo probes Oligo-pSc119.2-1 and Oligo-pTa535-1, all the new designed oligo probes were used for ND-FISH analysis of chromosomes of wheat Chinese Spring. The signal patterns of Oligo-pSc119.2-1 and Oligo-pTa535-1 can help for the recognition of individual wheat chromosome . All three of the probes Oligo-440.1, Oligo-440.2, and Oligo-37 produced signals on two wheat chromosomes . Both of the probes Oligo-60 and Oligo-107 generated signals on three chromosomes, and four chromosomes carried the signals of Oligo-119 . Both of the probes Oligo-18 and Oligo-44 produced signals on five chromosomes . All of the three probes Oligo-42, Oligo-45 and Oligo-428 generated signals on six chromosomes .Eleven of the 29 oligo probes generated distinct signal bands on several chromosomes and they are different from those previously reported . The combination of the two probes Oligo-B and Oligo-D can be used to distinguish wheat A-, B-, and D-genome chromosomes . Therefore, it is convenient to use the two oligo probes and ND-FISH analysis to identify wheat A-, B-, and D-genome chromosomes.Probe Oligo-B mainly produced signals on the whole B-genome chromosomes, and probe Oligo-D mainly generated signals on the whole D-genome chromosomes . The consensus sequence of Oligo-6DS.1 hits the sequences between 17 and 18 Mbp regions of 6D chromosome . From Figure 4A, it can be noted that the 17–18 Mbp region should be located on the short arm of 6D chromosome (6DS). Therefore, the signal of probe Oligo-6DS.1 should be located on the telomeric region of 6DS arm . The 6D chromosome can be identified using probe Oligo-pTa535-1 . Probe Oligo-pTa535-1 produces a very strong signal band on one arm of 6D chromosome, and two clear signal bands on the other arm . According to the Figures 4A,B, therefore, the arm carrying one signal band of Oligo-pTa535-1 should be the 6DS arm.To determine that the arm of 6D containing Oligo-6DS.1 signal is short or long arm, the consensus sequences of probe Oligo-6DS.1 was used for Nucleotide BLAST searches against the IWGSC RefSeq v1.0 of wheat Chinese Spring using blastn tool in B2DSC, and chromosome plots were drawn to view the locations of the consensus sequence on chromosomes (Hordeum vulgare L.) , a bioinformatic pipeline was used to develop bulked oligo probes, in which repetitive DNA sequences were omitted (Cucumis species (ND-FISH technology has provided a convenient and efficient way to identify the chromosomes of Triticeae species . The keygare L.) . This prgare L.) . With thdetected . Based o omitted . The bul species . The sam species . The rel species . In this species . The gra species . In this species  was usedn, (AAC)n, Oligo-pSc119.2, Oligo-pTa535, Oligo-pTa71, Oligo-s120, Oligo-275, Oligo-k566, and Oligo-713 have already been used to identify individual wheat chromosome (in situ hybridization in suspension (FISHIS) has been developed to effectively isolate individual chromosome of wheat and its relatives (Simple sequence repeat and non-SSR oligo probes such as (AAG)romosome . Howeverromosome . In the elatives . In thiselatives . TherefoIt has already been reported that chromosomal rearrangements are ubiquitous among polyploidy wheat accessions . MulticoFor the other new oligo probes in this study, they produced the FISH signal patterns that are different from those previously reported . The FISTandem repeats pAs1 mainly produce signal bands on D-genome chromosomes, and the arm of 6D chromosome that contains one signal band of pAs1 was regarded as the long arm . Tandem The whole genome shotgun assembly sequences (IWGSC WGA v0.4) and the first version of the reference sequences (IWGSC RefSeq v1.0) of wheat Chinese Spring were used to develop new oligo probes for ND-FISH analysis of wheat chromosomes. Two oligo probes can be used to identify wheat A-, B-, and D-genome chromosomes, 16 oligo probes can be used to identify specific segments of wheat chromosomes, and 11 oligo probes give new chromosomal landmarks of common wheat. These oligo probes are useful and convenient for distinguishing wheat chromosomes or specific segments of wheat chromosomes. In addition, the short and long arms of 6D chromosome have been confirmed.SF and ZT designed the study, analyzed the data, and wrote the manuscript. SF, ZT, ST, TL, and ZY analyzed the genomic sequences of wheat Chinese Spring and designed the oligo probes. LQ, GL, WZ, and JZ performed the experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The piezoelectric micro-jet, which can achieve the drop-on-demand requirement, is based on ink-jet technology and small droplets can be ejected out by precise control. The droplets are driven out of the nozzle by the acoustic pressure waves which are generated by the piezoelectric vibrator. The propagation processes of the acoustic pressure waves are affected by the acoustic properties of the fluid and the shell material of the micro-jet, as well as the excitations and the structure sizes. The influences of the fluid density and acoustic velocity in the fluid on the nozzle pressure and support reaction force of the vibrator are analyzed in this paper. The effects of the shell material on the ejection performance are studied as well. In order to improve the ejection performance of the micro-jet, for ejecting a given fluid, the recommended methods of selecting the shell material and adjusting excitations are provided based on the results, and the influences of the factors on working frequencies are obtained as well. The working process of the piezoelectric micro-jet is based on the inverse piezoelectric effect and the core component is the piezoelectric vibrator. Acoustic pressure waves are generated in the fluid when pulse voltages are applied on the piezoelectric material, and then the droplets are ejected out of the nozzle by the pressure waves. The piezoelectric micro-jet technology, which can realize drop-on-demand, is widely used in electric and electronic fields ,2, the bThe influences of the pulse voltage parameters on the ejection performance of the micro-jet are widely studied by researchers based on the analysis of the hydrodynamic characteristics ,12,13,14The influences of the fluid acoustic properties and acoustic impedance of the shell on the ejection performance are analyzed in this paper. Based on the analysis results, the methods of shell material selection and excitation adjustment are given, when ejecting fluid with different properties, so as to ensure the ejection intensity of the micro-jet. In order to ensure the restraint stiffness of the vibrator, the reaction forces of the vibrator under different conditions are studied as well.As for different applications, the designed structure of the piezoelectric micro-jet are different. The results shown in this paper are mainly used for the given common structure style shown in the text. For other structures, results can be obtained according to the proposed analysis simulation method.A piezoelectric micro-jet, which is simple and common, as shown in The acoustic pressure distribution in the fluid field of the piezoelectric micro-jet, after the vibration of the piezoelectric vibrator, is shown in The model used in simulations is shown in cE], piezoelectric stress constant matrix [e], and dielectric constant matrix [εT]) in the simulation are set as:The type of the piezoelectric ceramic is selected as PZT-5H, and its physical properties (elastic stiffness constant matrix [10 N/m2, 7.5 × 103 kg/m3, and 0.32, respectively. The outer ring of the copper diagram is set as a fixed constraint. The nozzle is the interface between fluid and air; therefore, the acoustic impedance at the nozzle is set as 400 kg·m−2·s−1 according to the related data [The elasticity modulus, density, and Poisson ratio of the copper film are set as 7.65 × 10ted data . In ordeWhen the cavity of the micro-jet is filled with liquid, the vibration of the vibrator will be subjected to the reaction force of the fluid at the fluid-solid interface due to the fluid-solid coupling effect. The obvious influence of the fluid-solid coupling effect on the vibration of the vibrator is that the resonant frequencies of the vibrator are all reduced.When the cavity is filled with air, the mode shapes and the corresponding resonant frequencies of the first four order modes of the vibrator are shown in When the cavity is filled with fluid, the mode shapes and the corresponding resonant frequencies are shown in As the constraint stiffness of the vibrator should meet the requirements, which are determined by the reaction force of the vibrator, so as to ensure the stability of the vibrator, the frequency response curves of the reaction force under different conditions are obtained. The frequency response characteristics of nozzle pressure are analyzed, as the ejection intensity is reflected by the acoustic pressure at the nozzle. To obtain better ejection performance, the vibration frequency of the vibrator, which determines the working frequency of the micro-jet, should be consistent with its resonance frequency. Thus, the influences of fluid properties and shell material on the resonant frequency of the vibrator are analyzed as well.The frequency response curves of the nozzle pressure of the micro-jet, when the acoustic velocity in the fluid is set as 1400 m/s and the material of the shell is set as aluminum, and when the fluids with different densities are ejected, are shown in As the micro-jet generally operates at high frequency, so as to obtain high working efficiency, only the influences of the density on the nozzle pressure of the micro-jet, when the vibrator vibrates in the fourth-order mode, are analyzed. The effects of the densities on the amplitude of the nozzle pressure, when the vibrator vibrates in the fourth-order mode, are shown in The response curves of the reaction force of the vibrator, when fluids with different densities are ejected, are shown in As we can see from As we can see from 3, the material of the shell is set as aluminum, and the acoustic velocity in the fluid is set differently, the frequency response curves of the nozzle pressure are shown in When the density of the fluid is set as 1000 kg/mAs the acoustic velocity in the fluid has little influence on the nozzle pressure when the micro-jet is working at low frequency, only the relationships between the acoustic velocity and the nozzle pressure when the micro-jet is working at high frequency are obtained. The curve of the nozzle pressure amplitude along with the increase of the acoustic velocity of the fluid is shown in The response curves of the reaction force of the vibrator, when fluids with different acoustic velocity are ejected, are shown in The relationship between the reaction force amplitude of the vibrator working in the fourth-order mode and the acoustic velocity in the fluid is shown in When the vibrator vibrates in the fourth-order mode, the influences of the fluid acoustic velocity on the resonant frequency of the vibrator are shown in 3 and 1400 m/s, respectively, the frequency response curves of the nozzle pressure of the micro-jet are shown in When selecting different shell materials and the density and acoustic velocity of the fluid are set as 1000 kg/m6 kg·m−2·s−1, the nozzle pressure amplitudes of the micro-jet when working in the first-order mode are greater than that of working in the fourth-order mode. With the increase of the acoustic impedance of the shell, the nozzle pressure amplitude increases gradually and the change tends to be stable when the micro-jet is working in the first-order mode. The nozzle pressure amplitude of the micro-jet, working in the fourth-order mode, also increases along with the increase of the acoustic impedance of the shell; however, the change tends to be enhanced.The relationship curves between the material impedance of the shell and the nozzle pressure, when the vibrator works in the first-order mode and fourth-order mode, are shown in When the shell materials with different acoustic impedances are selected, the response curves of the reaction force of the vibrator are shown in The influences of the impedance of the shell material on the reaction force amplitude of the vibrator, when the vibrator works in the fourth-order mode, are shown in When fluid with relatively low density and low acoustic velocity is ejected, the shell material with high acoustic impedance should be selected and the pulse voltage excitation should be enhanced, so as to ensure the strength of the ejection.In order to guarantee the required restraint stiffness and stability of the vibrator, when fluid with relatively low density and high acoustic velocity is ejected, the shell material with small acoustic impedance is recommended to reduce the reaction force amplitude of the vibrator.Ejecting fluid with relatively small density and high acoustic velocity, when there are a variety of fluids that can be selected, can increase the working frequency of the micro-jet, which means the working efficiency of the micro-jet can be improved. In addition, the influences of the acoustic impedance of the shell on the operating frequency of the micro-jet can be neglected.


Mammary retention of DOX and PEG‐DOX nanocarriers was quantified using in vivo imaging. Histopathologic effects of DOX and PEG‐DOX nanocarriers on mammary ductal structure were also investigated. Cytotoxicities of small linear PEG‐DOX nanocarriers (5 and 10 kDa) were not different from DOX whereas larger PEG‐DOX nanocarriers showed reduced potency. The order of mammary retention was 40 kDa PEG‐(DOX)4 > 5 kDa PEG‐DOX >> DOX, in normal and tumour‐bearing rats. Intraductally administered PEG‐DOX nanocarriers and DOX were effective in reducing tumour incidence and increasing survival rate, with no significant differences found among the three treatment groups. However, nanocarriers administered intravenously at the same doses were not effective, and intraductally administered free DOX caused severe local toxicity. Intraductal administration of PEG‐DOX nanocarriers is effective and less toxic than that of free DOX, as well as IV DOX/PEG‐DOX. Furthermore, PEG‐DOX nanocarriers demonstrate the added benefit of prolonging DOX ductal retention, which would necessitate less frequent dosing.Ductal carcinoma in situ is the most commonly diagnosed early stage breast cancer. The efficacy of intraductally delivered poly(ethylene glycol)‐doxorubicin (PEG‐DOX) nanocarriers, composed of one or more DOX conjugated to various PEG polymers, was investigated in an orthotopic ductal carcinoma in situ‐like rat model. In vitro cytotoxicity was evaluated against 13762 Mat B III cells using MTT assay. The orthotopic model was developed by inoculating cancer cells into mammary ducts of female Fischer 344 retired breeder rats. The ductal retention and in vivo antitumour efficacy of two of the six nanocarriers (5 kDa PEG‐DOX and 40 kDa PEG‐(DOX) Most breast cancers are initiated in the mammary ducts. Ductal carcinoma in situ (DCIS), the most common type of noninvasive breast cancer, results from the proliferation of malignant epithelial cells within the lumen of the breast duct, generally without penetrating the basement membrane s (PEGs) were promising nanocarriers for transpapillary administration and intraductal delivery  and the M2™ Compact High‐Performance MRI (1T) system from Aspect Imaging . PEG‐DOX nanocarriers were synthesized and characterized according to our previously published method  was obtained from Baxter Healthcare Corporation . The high‐performance liquid chromatography system, equipped with a Symmetry 300™ C18 column  as well as ultraviolet and fluorescence detectors, was purchased from Waters . The In‐Vivo MS FX PRO2 and 95% humidity at 37°C. Cells with a confluence of less than 90% were used.The 13762 Mat B III cell line, a rat mammary adenocarcinoma cell line, was obtained from American Type Culture Collection. Cells were maintained in RPMI 1640 medium  supplemented with 10% fetal bovine serum, penicillin (200 units/mL), and streptomycin (200 mg/mL) under condition of 5% COFemale retired breeder F344 rats were purchased from Hilltop Lab Animals, Inc. . The rats were housed in a room with a 12‐h light/dark cycle, allowed to acclimate for at least a week before the study, and were fed a standard diet and water ad libitum. All animal studies were performed in Association for Assessment and Accreditation of Laboratory Animal Care International accredited animal facilities, under approved protocols from the Rutgers University Animal Use and Care Committee.The PEG‐DOX nanocarriers were synthesized as described in our previous paper  were dispensed into 96‐well plates and incubated with PEG‐DOX nanocarriers and DOX for 48 h. Cell viability was evaluated using a CCK‐8 kit , by measuring the absorbance at 450 nm with a microplate reader.5/duct) suspended in 0.1 mL serum‐free RPMI 1640 medium were inoculated intraductally, using a 33 G needle attached to a Hamilton syringe . Tumour dimensions were monitored, and tumour volumes were calculated as follows: evaluated tumour volume = 0.5 × length × width2. End point criteria were determined as follows: (1) a maximum tumour volume larger than 10% of the net bodyweight (raw bodyweight minus tumour weight), (2) a mean tumour diameter exceeding 40 mm in rats, (3) ulceration of the tumour, (4) tumour interference with normal movement or function of vital organs , and (5) inability of the animal to eat or drink. All animals meeting end point criteria were euthanized with CO2 and counted as death events.Rats were anesthetized with isoflurane and placed under a dissection microscope. The fourth nipple on the left side was cleaned with 70% ethanol. After dilation of the nipple orifice, 13762 Mat B III cells  prepared in saline were intraductally injected into the same duct on day 2. The mammary contents of the test articles were then measured fluorometrically, via optical imaging.Retention of DOX and PEG‐DOX nanocarriers was evaluated in mammary glands of both normal and tumour‐bearing rats. In the tumour model, 2.5 × 105 cells/rat). At day 2 after initial cell inoculation, 5 kDa PEG‐DOX, 40 kDa PEG‐(DOX)4, and free DOX were injected into the tumour‐bearing rats either intravenously, or intraductally into the same mammary gland as the cell inoculum, at a dose of 0.83 mg/kg DOX equivalent (10 rats per group). The control group received no treatment. Tumour growth and rat bodyweights were monitored. The rats were euthanized via CO2 once the tumour size reached the end point criteria. Tumour, liver, lung, spleen, and lymph nodes were excised upon killing and fixed in 10% neutral formalin. The fixed tissue samples were then embedded in paraffin and processed for histological evaluation by routine procedures with hematoxylin and eosin staining.The F344 female rats were intraductally injected with 13762 Mat B III cells  were estimated by non‐compartmental pharmacokinetics, using PKSolver [4].A day before the start of the study, the rats were shaved with electric clippers and depilated with Veet depilatory lotion. During the experiment, the rats were anesthetized via isoflurane and placed into the imaging method. Whole‐body images were captured by an In‐Vivo MS FX PROvivoquant , an analytical software program. Within vivoquant, the images were examined for activity . The images were also compared with the prior scans (self‐control) to compare any noted growth or activity.Magnetic resonance imaging (MRI) images were acquired using two scans per rat, with one scan covering the lower body starting at the duct and the other covering the upper torso. All scans were run at a 256–250 matrix, with the echo time/repetition time at 80/3787 and a flip angle of 180°. Five averages  were taken each time. The data were transferred from the MRI to The rats were euthanized at different days after intraductal inoculation of 13762 Mat B III cells. The mammary glands were excised and fixed in 10% neutral formalin for 24 h. They were then rinsed in distilled water for 15 min. The glands were then dehydrated in serial concentrations of ethanol  for 1 h each and defatted in two changes of xylene for 1 h each. The specimens were thereafter rehydrated and stained with alum carmine. The stained whole‐mounts were dehydrated with ethanol, cleared with xylene, and stored in biopsy pouches filled with methyl salicylate. Whole‐mounts were photographed at a uniform magnification.Whole‐mount specimens were trimmed and subjected to paraffin embedding. The paraffin blocks were sectioned to 5‐micron thickness and then stained with hematoxylin and eosin.graphpad prism v6 . Non‐compartmental pharmacokinetic parameters were calculated using PKSolver [4].Experimental values were expressed as mean ± standard deviation. Nonlinear regression, survival analysis, and other statistical analyses were conducted using 50) were calculated based on the best‐fit model. As shown in Table 50 value of DOX was 0.025 μM. The IC50 values of linear 5, 10, 20, and 40 kDa PEG‐DOX, as well as the 40 kDa PEG‐(DOX)4 and 40 kDa PEG‐(DOX)8 branched nanocarriers, were 0.071, 0.24, 0.76, 2.42, 0.16, and 0.15 μM, respectively. Among the linear structures of nanocarriers, the 20 and 40 kDa PEG‐DOX were significantly less potent than free DOX and 5 or 10 kDa PEG‐DOX. The ratio of IC50 values of linear PEG‐DOX nanocarriers versus DOX displayed a linear correlation to PEG molecular weight . Thus, intraductal inoculation of 13762 Mat B III cells was demonstrated to be a valid breast cancer model mimicking DCIS‐like lesions and progression of DCIS to invasive breast cancer, for further efficacy studies.Various cell loads of 13762 Mat B III were also tested, and tumour growth consistently displayed cell load‐dependent and time‐dependent characteristics throughout all studies (data not shown). A cell load of 2.5 × 104 nanocarriers in the mammary gland exhibited a rapid increase and subsequent reduction with time, which was consistent in both normal and tumour‐bearing rats t1/2), defined as the time required for fluorescence intensity to reach 50% of its peak value, was estimated by non‐compartmental pharmacokinetic model 4 were 5.9 ± 1.2 and 14.0 ± 1.9, respectively. Conversely, in tumour‐bearing rats, the t1/2 of free DOX, 5 kDa PEG‐DOX, and 40 kDa PEG‐(DOX)4 were 3.8 ± 1.2, 5.1 ± 0.9, and 14.6 ± 1.8 h, respectively. The nanocarriers exhibited significantly longer mammary retention than the free DOX in both normal rats and tumour‐bearing groups. Under both normal and tumour conditions, 40 kDa PEG‐(DOX)4 manifested the longest retention time. On the other hand, all test articles showed similar mammary retention between the normal and tumour groups 4 groups, respectively. These results demonstrate that the intraductal approach is more effective than IV in treating intraductal mammary tumours.The effect of PEG‐DOX nanocarriers was investigated using the orthotopic breast cancer model. Free DOX and PEG‐DOX nanocarriers were injected either intraductally or intravenously, at day 2 post‐tumour cell inoculation into the mammary duct. Tumour growth, represented as tumour volume, is shown in Figure Magnetic resonance imaging was performed to monitor mammary tumour development upon intraductal administration of PEG‐DOX nanocarriers and free DOX. In free DOX‐treated rats, we found that soft lumps developed in both normal and tumour‐bearing rats Fig. . The sofFigure Even though DCIS lesions may stay localized for years, 50% of untreated DCIS cases eventually progress to invasive breast cancer. The intraductal drug delivery approach to treating DCIS is an attractive opportunity to enhance local exposure while limiting systemic toxicity, as well as reducing the need for surgery and/or radiation. In addition to several animal studies, a recent human study has showed the effectiveness and potential of intraductal delivery for curing localized breast cancer. While this is promising, further investigation into the characteristics of the mammary duct  is critical to improve the efficacy of ductal therapy.2 every 21 days, with the cumulative dose not exceeding 550 mg/m2 by intravenous route, in the form of hydrochloride salt , a locaAll PEG‐DOX nanocarriers with various molecular weights and architectures exhibited cytotoxicity in vitro against 13762 Mat B III, a rat breast cancer cell line. The potency of small PEG nanocarriers (5 and 10 kDa) was not different from that of free DOX, while larger PEG‐DOX (20 and 40 kDa) showed reduced potency. Surprisingly, branched PEG‐DOX nanocarriers were more effective than their linear counterparts. This trend was consistent with our prior findings using MCF7, a human breast cancer cell line , PEG‐DOX nanocarriers exhibited prolonged ductal retention half‐lives with respect to free DOX, which was consistent with the results we obtained previously in SD rats  rats was investigated. Because F344 retired breeders were used instead of SD rats to establish a breast cancer tumour model, the influence of molecular size of nanocarriers on the ductal retention in normal and tumour‐bearing F344 rats was reassessed. Because of the larger molecular size and inherent viscosity and/or aggregate formation of their aqueous solution  nominal proteolytic activity in normal tissues as compared with cancerous tissue  nanocarriers in treating mammary tumours in the F344 rat tumour model. Unlike free DOX, no local inflammation or alteration of ductal structure was observed in the intraductal groups treated with PEGylated DOX nanocarriers, suggesting a reduced local toxicity. In addition, we have noninvasively assessed the retention of intraductally administered nanocarriers in both normal F344 rats and the F344 tumour model. There was no significant difference of retention between normal F344 animals and the F344 tumour model. The data presented here will aid in the design of further drug delivery systems for intraductal therapy, to treat DCIS in humans.This work was supported by the grant from National Institutes of Health HIT IT (R01AI084137‐01).