s2fieldsofstudy: Biology Medicine

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Qingpeng Ointment Ameliorates Inflammatory Responses and Dysregulation of Itch-Related Molecules for Its Antipruritic Effects in Experimental Allergic Contact Dermatitis

The pathogenesis of itchy skin diseases including allergic contact dermatitis (ACD) is complicated and the treatment of chronic itch is a worldwide problem. One traditional Tibetan medicine, Qingpeng ointment (QP), has been used in treatment of ACD in China for years. In this study we used HPLC and LC/MS analysis, combined with a BATMAN-TCM platform, for detailed HPLC fingerprint analysis and network pharmacology of QP, and investigated the anti-inflammatory and antipruritic activities of QP on ACD induced by squaric acid dibutylester (SADBE) in mice. The BATMAN-TCM analysis provided information of effector molecules of the main ingredients of QP, and possible chronic dermatitis-associated molecules and cell signaling pathways by QP. In ACD mice, QP treatment suppressed the scratching behavior induced by SADBE in a dose-dependent manner and inhibited the production of Th1/2 cytokines in serum and spleen. Also, QP treatment reversed the upregulation of mRNAs levels of itch-related genes in the skin (TRPV4, TSLP, GRP, and MrgprA3) and DRGs (TRPV1, TRPA1, GRP, and MrgprA3). Furthermore, QP suppressed the phosphorylation of Erk and p38 in the skin. In all, our work indicated that QP can significantly attenuate the pathological alterations of Th1/2 cytokines and itch-related mediators, and inhibit the phosphorylation of MAPKs to treat the chronic itch.

INTRODUCTION
Chronic itch has been long known not only as a concomitant manifestation of various skin diseases, such as contact dermatitis, eczema, atopic dermatitis, psoriasis, etc., but also occurs in systemic, neurological, or psychiatric diseases. So far, medications for chronic itch still can be simply categorized into topical (emollients and moisturizers, anesthetics, coolants, glucocorticoids, others) and systemic (antihistamines, neuroactive medications, antidepressants, phototherapy, etc.) forms (Yosipovitch and Bernhard, 2013;Pereira and Stander, 2017;Hachisuka et al., 2018;Shevchenko et al., 2018). Due to its heterogeneity, the causes and pathophysiology of chronic itch are complicated. The treatment of chronic itch, especially intractable itch, is a worldwide problem and a great challenge for clinicians.
Complementary and alternative medicine (CAM), including herbal or botanical therapies, traditional Chinese medicine (TCM), dietary supplements, etc., is clinically used to treat intractable skin diseases like psoriasis. A large amount of evidence indicated those CAM managements could effectively relieve the symptoms of diseases or their underlying causes, which cannot be solved by western medicine (Farahnik et al., 2017;Parker et al., 2017;Svendsen et al., 2017;Na Takuathung et al., 2018). For example, it is estimated that there are about 10-62% of patients with psoriasis in the western world seeking the use of CAM (Farahnik et al., 2017;Svendsen et al., 2017). Consistently animal experimental studies have also accumulated a wealth of data demonstrating the effectiveness of the use of CAM in animal models (Na Takuathung et al., 2018).
Allergic contact dermatitis (ACD) is a skin T cell-mediated immune response caused by one or more non-toxic allergens in contact with the skin, and often occurs together with irritant contact dermatitis significantly affecting the quality of life (Kostner et al., 2017). Existing treatments for ACD are still limited to a few traditional methods such as topical steroids and immunosuppressants, etc., etiologically based and efficient treatments are still lacking. Over centuries Chinese medicines have been used in the treatment of ACD, and their main ingredients and possible molecular mechanisms have been gradually recognized Li et al., , 2018Fu et al., 2015). One traditional Tibetan medicine, Qingpeng ointment (QP), has been used in treatment of ACD in China for many years. QP contains nine main ingredients including Oxytropis falcata Bunge, Rheum lhasaense, Aconitum pendulum Busch, Terminalia chebula Retz (stoned), Terminalia billerica (Gaertn.) Roxb, Phyllanthus emblica Linn., Styrax tonkinensis (Pierre) Craib ex Hartw, Tinospora sinensis (Lour.) Merr., and Synthetic musk (Product Manual, Qizheng Tibetan Medicine Co., Ltd., Lanzhou, China). Two previous works reported that QP exerts anti-inflammation and ameliorates ACD in BALB/c mice Li et al., , 2018Fu et al., 2015). However, similar to other TCM experimental studies on ACD they have some common problems. For example, their ACD model was established by topical 4-dinitrofluorobenzene (DNFB), a model that has been used for a long time but still has actual unknown molecule mechanisms Li et al., , 2018Fu et al., 2015). Secondly without exception, they all focus on anti-inflammatory effect and none of them have comprehensively studied the chronic itch associated with ACD.
Squaric acid dibutylester (SADBE), a small molecule hapten, is commonly used in the treatment of alopecia areata and has been used as a popular ACD inducer in mice (Fu et al., 2014;Feng et al., 2017;Luo et al., 2018). Remarkably in recent years neuroimmune mechanisms of SADBE-induced chronic itch were greatly investigated. It was showed that the increased CXCL10/CXCR3 signaling in sensory neurons may contribute to the pathogenesis of the chronic itch in this model (Fu et al., 2014). Most recently we and our collaborators found transient receptor potential (TRP) channels, TRPV1 and TRPA1-expressing sensory neurons or TRPV4 in the TRPV4-expressing epithelial and immune cells in the skin are molecular and cellular basis for SADBE-induced chronic itch Luo et al., 2018).
In order to fully understand the pharmacological functions of herbal ingredients or components, we also employed a newly developed bioinformatics analysis tool, BATMAN-TCM (a Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine) established by our collaborator. Unlike the traditional concept of "one drug, one goal, " BATMAN-TCM tries to seek the key molecules targeted by the main components of TCM, and discover the biological networks derived from these molecules (Liu Z. et al., 2016;Zhang et al., 2017). Meanwhile, the data will be compared with the molecular targets and signal pathway networks related to diseases established in the database, and will describe the complicated mechanisms and potential therapeutic values of the drug (Liu Z. et al., 2016;Zhang et al., 2017Zhang et al., , 2018. Therefore, this study attempted to introduce BATMANTCM into the ACD study and investigate the anti-inflammatory activities of QP on ACD induced by SADBE in mice, especially whether QP treats the chronic itch, and if it does what the possible mechanism could be.

BATMAN-TCM Target Prediction and Analysis
The target prediction method adopted the BATMAN-TCM platform, which in principle, is a similarity-based method to predict potential drug-target interactions based on the similarity of those known TCMs drug-target interactions. Simply, the combination of QP composition and their corresponding PubChem ID were an input into BATMAN-TCM software for network pharmacological analysis to obtain the results of the "drug-disease" potential target prediction (Liu Z. et al., 2016;Zhang et al., 2017). The analysis was based on multiple source database included TCMID database (providing a giant formula-herb-ingredient association data), DrugBank  (Liu Z. et al., 2016;Zhang et al., 2017).

Animals and Drugs
Male C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Animals were single-caged and housed in a SPF-grade environment with water and food ad libitum at temperature (24 ± 2) • C, a 12-h/12-h light/dark cycle lighting off at night (7:00 pm-7:00 am), at least adapted for 1 week before behavior work. All behavioral tests were videotaped above the observation boxes, and assessed by observers blinded to the experimental design. In this study, mice used for behavioral tests were divided into the blank control group (acetone topically), the model group (0.5% SADBE/acetone topically), groups of treatment low-dose, medium-dose and high-dose of QP ointment applied to the shaved nape of the neck (0.625, 1.25, and 2.5 g/kg), or the positive drug (Diphenhydramine Hydrochloride, 10 mg/kg, i.p.). In this study, male mice at 7 weeks old were used for experiments. All animal experimental procedures followed international guidelines for care and use of laboratory animals which were approved by the Animal Ethics Committee of South-central University for Nationalities. SADBE was purchased from J&K Scientific Ltd. (Beijing, China). QP was purchased from Tibet Qizheng Tibetan Medicine Co., Ltd. (Lanzhou, China). Diphenhydramine hydrochloride was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).

Mouse Model of ACD and Treatments
The model was mainly established following previous work Luo et al., 2018). The mice were sensitized once per day for three consecutive days (from day 0 to day 2) by applying 20 µL of 0.5% SADBE/acetone solution topically to the shaved abdominal skin. 5 days later, 20 µL of 0.5% SADBE/acetone solution was applied topically to shaved mouse neck skin about 3 × 3 cm region in the midline backs once a day for three consecutive days. 3 days later, the paw scratching behavior was recorded by the camera under unmanned conditions for 60 min, and the number of scratches to the shaved part of the neck was counted by people blinded to the experimental design. The treatment group was topically applied to the shaved nape of the neck with low, medium and high dose QP (0.625, 1.25, and 2.5 g/kg) twice a day starting from day 0 to day 12, and once a day on day 13. Diphenhydramine (10 mg/kg i.p.) as the positive drug was injected into the mice on day 13. The following scratching behavior was recorded after 30 min at the last QP and diphenhydramine treatment by observers. The control group was used same volume of acetone other than 0.5% SADBE/acetone. The model group mice were applied to the blank substrate (from Tibet Qizheng Tibetan Medicine Co., Ltd.) at the same time Luo et al., 2018).

Skin Morphology Studies
The skin in midline of the mouse neck was collected and fixed in 4% paraformaldehyde, routinely dehydrated, embedded in paraffin, and sectioned. Hematoxylin & eosin (HE) and toluidine blue staining were performed as previously described (Zhao et al., 2013). After the stained sections were dehydrated, cleared and sealed, they were visualized under a microscope, and images were taken. Image J software was used to measure the relative thickness of the epidermis from HE staining images and count the number of mast cells from toluidine blue staining images.

Quantitative RT-PCR
Total RNA was extracted from homogenized DRG neurons or skin samples using the Trizol reagent (TaKaRa, Japan) according to the manufacturer's instructions. RNA was treated with DNase I and cDNA was synthesized in vitro using a ThermoScript RT-PCR reverse transcription kit. Each reaction was performed in a system containing 10 µL SYBR Mix, 2 µL cDNA, 1 µL primer mix and 7 µL RNase-free water, using a real-time PCR system. Relative mRNA expression levels of different target genes compared to GAPDH or β-Actin were calculated using the 2 − Ct method. The primer sequences (5 to 3 ) involved were showed in Table 1.

Measurement of Cytokines
Detection of IgE, Histamine, TNF-α, IFN-γ, IL-1β, IL-4, IL-5, IL-10, etc., in the spleen and serum of mice using an ELISA kit according to the manufacturer's instructions. All mouse ELISA kits were purchased from Shanghai Jianglin Biotechnology Co., Ltd.

Statistical Analysis
All data was expressed as the means ± SEM. GraphPad Prism 6 software (San Diego, CA, United States) was used for the data statistical analysis and graphics. Unpaired t-test was used to analyze statistical comparisons between two groups. Multiple comparisons were compared by one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc tests. p-value < 0.05 was assumed as statistically significant.

The Visualization of QP Ingredient-Target-Pathway/Chronic Dermatitis Association Network
To establish the related target prediction and network/pathway analysis for QP to treat chronic dermatitis, the current work used the platform of BATMAN-TCM and obtained the interactive network diagram of QP-chronic dermatitis (Figure 2). In the view of the association network, based on QP's drug targets and biological pathways, targets of chronic dermatitis diseases and predicted pathways, the prediction results are divided into three types of nodes including composition-targets association, disease-targets association and their overlapping association. A complete gene names of targets and biological pathways for QP's drug and/or chronic dermatitis, see MS Excel worksheets 1 and 2 in Supplementary Table S1.

QP Attenuated Scratching Behavior Induced by SADBE in Mice
Skin allergies and chronic dermatitis have been caused by topical application of SADBE, a popular ACD inducer in mice (Fu et al., 2014;Feng et al., 2017;Luo et al., 2018). In this study, we followed the schematic experimental protocol (Fu et al., 2014;Feng et al., 2017;Luo et al., 2018) and pretreated mice with SADBE in the abdomen for 3 days (sensitization) and re-stimulated after 3 days topically on the neck (challenge), which induced robust spontaneous scratching behavior in mice ( Figure 3A). Previous analyses indicated that no corticosteroid existed in QP  and antihistamines have been used traditionally in the attenuation of experimental and clinical pruritus (Yosipovitch and Bernhard, 2013;Mahapatra et al., 2014;Pereira and Stander, 2017;Hachisuka et al., 2018;Shevchenko et al., 2018), therefore in this study we used a classic antihistamine, diphenhydramine, as the positive control medicine. Similar to previous reports (Fu et al., 2014;Feng et al., 2017;Luo et al., 2018), compared with the blank control mice,  SADBE indeed induced robust scratching behaviors (p < 0.0001) ( Figure 3B). All three QP treatments (0.625, 1.25, and 2.5 g/kg) and the positive drug (diphenhydramine, 10 mg/kg) significantly decreased the scratching behavior than that in the model group (p < 0.05). It is worth noting that QP can work in a dose-dependent manner to suppress the pruritus response in the ACD model ( Figure 3B).

Effect of QP on Skin Histological Changes Induced by SADBE
We next examined the SADBE induced skin lesions and whether the QP treatments were beneficial to them. From the skin general appearance observed, compared with the model group, mice after treated with different doses of QP showed some signs of reduced skin lesions ( Figure 4A). However, from the results of HE (Figures 4B,C) and toluidine blue (Figures 4D,E) staining, there was no significant differences after any QP or diphenhydramine treatment as compared with the model group, and by the measurement of skin thickness and the number of mast cells, which have been frequently used as indicators for the skin inflammation and immune cell infiltration.
The data indicated, in mice SADBE initiated thickening of skin and increased infiltration of mast cells, which could not be significantly inhibited by QP.

QP Inhibited Upregulation of Cytokines and Itch Molecules in the Skin of SADBE Mice
In this study we tried to take advantage of the information from our network pharmacological study (Figure 2 and Supplementary Table S1) for molecular analysis. Upon the results of potential effector molecules they provided, we analyzed the mRNA expression levels of IL-5, IL-10, TRPV4, TSLP, GRP, and MrgprA3 in the skin from different groups of mice. The experimental results showed that the mRNA levels of IL-5, IL-10, TRPV4, TSLP, GRP, and MrgprA3 in the skin of the model group were significantly higher than those in the blank control group. Compared with the model group, QP treatments (especially at the high dose group) can significantly reduce the enhanced IL-5, IL-10, TRPV4, TSLP, GRP, and MrgprA3 mRNA levels in the skin of SADBE mice (Figure 5). The positive control drug FIGURE 2 | QP ingredient-target-pathway/chronic dermatitis association network. Red nodes correspond to the predicted key targets of chronic dermatitis, such as TLR4, STAT1, STAT3, PRKACA, PSMB8, IL2RG, CD3G, etc. Blue nodes correspond to the key targets of QP ointment, such as IL-8, CXCR4, JUN, IL-1β, IL-17A, etc., Green nodes represent the key targets associate to both QP and chronic dermatitis, such as IL-4, IL-6, IL-10, TNF, NFκB1, PIK3R3, PIK3CA, EGFR, KIT, ESR1, NR3C1, etc.
diphenhydramine, was able to significantly decrease that of IL-10, GRP, and MrgprA3.

QP Inhibited p-P38 and p-Erk Signaling Augments in the Skin of SADBE Mice
It is well known a number of intracellular kinase cascades would be activated by inflammatory mediators and participate in the generation and maintenance of immune responses in dermatitis (Ayush et al., 2013;Bechara et al., 2017;Oetjen et al., 2017;Li et al., 2018;Xiao and Xiao, 2018). Considering the information from our network pharmacological study (Figure 2 and Supplementary Table S1), expression of several important protein kinases was detected in the skin of mice (Figures 6A-F). Compared with the blank control group, the level of phosphorylated P38 (Figure 6C) in the skin of the model group increased significantly. Compared with the model group, the high-dose QP significantly decreased the expression of p-P38 and p-Erk (Figures 6B,C) in the skin. Detection of the p-Stat3, p-Akt and p-IκBα expression in the skin of mice revealed that there was no significant difference in their expression levels among the groups (Figures 6A,D-F).

QP Inhibited Upregulation of Cytokines and Itch Molecules in the DRGs of SADBE Mice
The DRG is the site where the primary neuronal cells are clustered and also a critical site for the development itch sensory information in the periphery (Han et al., 2013;Liu Z. et al., 2016;Kittaka and Tominaga, 2017;Zhang et al., 2017). Real-time PCR was used to measure the mRNA levels of TRPV1 (Figure 7A),

FIGURE 3 | QP reduced the itching caused by allergic contact dermatitis induced by SADBE. (A)
A Schematic diagram shows the experimental protocol. 0.5% SADBE/acetone was topically treated daily 3 times to the mouse abdomen, and re-stimulated topically daily 3 times to the neck back skin after a 5 days' break. Spontaneous scratching was videotaped and analyzed afterward. (B) The behavior results of SADBE mice in different controls or after three QP (0.625, 1.25, and 2.5 g/kg), and the positive drug (diphenhydramine, 10 mg/kg). Data was expressed as mean ± SEM. * p < 0.05, * * p < 0.01, and * * * * p < 0.0001, compared with the model group. ++++ p < 0.0001, compared with the blank control group. One-way ANOVA followed by post hoc test (n = 5 each group). The method and criteria to present and statistically analyze the data in (B) were followed the same in all other work in this paper unless mentioned specifically. Figure 7B), GRP (Figure 7C), and MrgprA3 ( Figure 7D) in the DRG of mice. As shown in Figure 7, the levels of TRPV1, GRP, and MrgprA3 mRNA in the DRG of model group were significantly higher than those in the blank control group. The positive drug diphenhydramine was effective in reducing TRPA1 and TRPV1 mRNA levels. Even though the low-dose QP had no significant effect on the four itch-related genes, however, compared with that of the model group, TRPV1 and GRP mRNA levels were significantly reduced by the medium-dose of QP, and TRPV1, TRPA1, GRP, and MrgprA3 mRNA levels were markedly reduced by the high-dose of QP.

QP Altered the Levels of Cytokines and IgE in the Serum or Spleen in SADBE Mice
By using the IgE, IL-4, IL-5, IL-10, and TNF-α Elisa kits, the levels of IgE (Figure 8A), IL-4 ( Figure 8B), IL-5 (Figure 8C), IL-10 ( Figure 8D), TNF-α ( Figure 8E) in the serum and TNF-α ( Figure 8F) in the spleen were detected. Compared with that in the blank control mice, the production of IgE, IL-5, IL-10 and TNF-α in the model group mice were significantly increased. Positive control drug could significantly reduce IL-4, IL-5, and IL-10 and TNF-α content in serum, but QP showed good inhibitory effect in reducing the content of all these cytokines in the serum or spleen. Among them, the high-dose of QP showed the most striking effect which significantly to all markers detected.

DISCUSSION
Recent advances in experimental and computational studies have shown that, unlike some single drugs of western medicine, natural medicines or TCM mostly contain multiple components and exert their therapeutic effects by acting on multiple targets (Liu Z. et al., 2016;Zhang et al., 2017Zhang et al., , 2018. Therefore, it is necessary to introduce modern technologies and systematic methods, such as HPLC fingerprint analysis and network pharmacology, to reveal the pharmacological functions of these drugs and predict their action mechanisms (Liu Z. et al., 2016;Zhang et al., 2017Zhang et al., , 2018. In this study the HPLC analysis was used to characterize the phytochemical features of QP ointment and 3 common peaks were proposed as the fingerprints of its multiple chemical constituents. According to the fragment ion at m/z 153 in the mass spectrum indicated that QP contained major phenolic constituents with the central structural unit of gallic acid. Previously this type compounds had been showed treatment effects on ACD or contact dermatitis (Mo et al., 2014;Fu et al., 2015). However, even though gallic acid, corilagin, and ellagic acid were identified as major components of QP, there may be other effective or novel structural compounds in QP since there are several peaks of HPLC fingerprint analysis. Further investigation is needed to investigate what they are and whether they are effective ingredients for the treatment of ACD. On the other hand, BATMAN-TCM is a bioinformatics analysis tool developed by our collaborators (Liu Z. et al., 2016;Zhang et al., 2017). In this study by using BATMAN-TCM, FIGURE 4 | Effect of QP on skin histological changes in SADBE mice. (A) The midline dorsal neck skin images of each group of mice were showed on day 13. The neck skin lesions in QP treatment groups were fewer than those in the model group. (B,C) showed representative HE staining images and measured skin thickness of each group as labeled, respectively. (D,E) showed representative toluidine blue images and cell counts of each group as labeled, respectively. Data was expressed as mean ± SEM. ++++p < 0.0001, compared to the blank group. One-way ANOVA (n = 5 each group). Scale bar: 50 µm.
we analyzed the potential targets of each component of QP, and generated a current ACD's disease-related gene database. Our results suggested that molecules such as IL-8, CXCR4, JUN, IL-1β, IL-17A, etc., are potential effector molecules or downstream molecules of QP, and molecules such as TLR4, STAT1, STAT3, PRKACA, etc., are chronic dermatitis-associated targets, while IL-4, IL-6, IL-10, TNF, NFκB1, etc., may cover by both. As far as we know, this study is the first use of modern methods of network pharmacology to explore the therapeutic effect of Tibetan medicine on allergic contact dermatitis. Certainly, this method still has some limitations. For example, it mainly relies on the continuous updating and improvement of the relevant online database (Liu Z. et al., 2016;Zhang et al., 2017). The results given by BATMAN-TCM were suggested to be combined with the existing literature and research progress, and the results of animal and cytological experiments were to make the most appropriate judgment. The mRNA-seq technology can provide a method for rapidly distinguishing altered mRNA after drug treatment (Duan et al., 2019), if possible, the mRNA-seq work for studying transcriptome expression in a QP-treated ACD mouse model will help to understand and explain the mechanism of action of QP.
Our experimental results highlighted the key role of several Th1/2 cytokines for QP to treat the chronic itch. T helper (Th) cells have been defined into different subsets by different cytokine profiles secreted. For example, Th1 cells are characterized by the secretion of IL-2, IFN-γ and TNF-α, etc., while Th2 cells secrete IL-4, IL-5, and IL-13, etc (Raphael et al., 2015). Th1 cytokines, like TNF-α/TNFR1 signaling, were showed in animal behavior as required for both acute and chronic itch (Miao et al., 2018). More strikingly Th2 cytokines have been reported as highly involving in the inflammation response directly mediating chronic itch. For example, recent data suggest IL-4, IL-31, and IL-33 all have their own receptors expressing in both mouse and human sensory neurons, and these cytokines enhance neuronal responsiveness to multiple pruritogens (Cevikbas et al., 2014;Kaesler et al., 2014;Liu B. et al., 2016;Oetjen et al., 2017). Our data indicate that high level of interleukin IL-5 was detected in ACD mouse skin, and significantly increased IgE, IL-4, IL-5, IL-10 levels in serum, and as wells as elevated TNF-α in both serum and spleen. On the other hand, QP can significantly decrease the upregulation of these molecules in the serum, spleen and skin. In all, these data would not only support some recent chronic itch progress related to our studies (Zhao et al., 2013;FIGURE 5 | The mRNA of IL-5 (A), IL-10 (B), TRPV4 (C), TSLP (D), GRP (E), and MrgprA3 (F) were detected in mouse skin. After mice were treated with different doses of QP ointment and diphenhydramine, the targeted molecular mRNA levels were measured as well as that in the model group or the blank control group. Data was expressed as mean ± SEM. * p < 0.05, * * p < 0.01, and * * * * p < 0.0001, compared with the model group. + p < 0.05, ++ p < 0.01, and ++++ p < 0.0001, compared with the blank control group. One-way ANOVA (n = 5 each group).
Over the past 10 years, effects of various itch mediators expressed by primary sensory neurons and skin cells (like keratinocytes) have been extensively studied. For example, keratinocytes released thymic stromal lymphopoietin (TSLP) could activate sensory neurons and immune cells for itch (Wilson et al., 2013). Several itch specific neuropeptides, like GRP (Sun and Chen, 2007), somatostatin , and specific neurons like MrgprA3 + (Han et al., 2013) and FIGURE 7 | The mRNA of TRPV1 (A), TRPA1 (B), GRP (C) and MrgprA3 (D) were detected in mice DRG. After mice were treated with different doses of QP ointment and diphenhydramine, the targeted molecular mRNA levels were measured as well as that in the model group or the blank control group. Data was expressed as mean ± SEM. * p < 0.05, * * p < 0.01, and * * * p < 0.001, compared to the model group. + p < 0.05 and ++ p < 0.01, compared to the blank control group. ANOVA (n = 5 each group).
FIGURE 8 | Effect of QP on the levels of different cytokines or IgE in the serum or spleen of SADBE mice. Among them, (A-E) showed the levels of IgE, IL-4, IL-5, IL-10, and TNF-α in the serum of each group, and (F) showed of TNF-α in the spleen of each group. * p < 0.05, * * p < 0.01, and * * * p < 0.001, compared to the model group. ++ p < 0.01, +++ p < 0.001, and ++++ p < 0.0001, compared to the blank control group. ANOVA (n = 5 each group). GRPR + (Sun et al., 2009) cells have been identified for the itch signal transmission. TRP channels, especially thermosensitive TRP channels including TRPV1, A1,V4 etc., receive multimodal stimuli and required for itch sensations (Liu Z. et al., 2016;Kittaka and Tominaga, 2017;Zhang et al., 2017). Research studies including our work proved both TRPA1 and TRPV1 channels are required for generating spontaneous scratching induced by SADBE and TRPV4-expressing epithelial and immune cells in the skin dynamically mediate chronic itch (Liu Z. et al., 2016;Zhang et al., 2017). In this study, our work demonstrated the mRNA of TRPV1, TRPA1, GRP, and MrgprA3 were elevated in the SADBE mice DRG and /or skin, which were significantly decreased by QP to a different extent.
Signal transduction originating from membrane-bound Th1/2 cytokine receptors is a complex network that leads to intracellular gene expression and ultimately regulates cellular activities. Among them, the mitogen activated protein kinase (MAPK) (Ayush et al., 2013;Li et al., 2018;Xiao and Xiao, 2018), the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (Xiao and Xiao, 2018), the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) (Bechara et al., 2017;Oetjen et al., 2017) have been intensively studied and strongly implicated with the pathogenesis of skin diseases and inflammation-controlling interventions. Our work supported, probably QP works through p-P38 and p-Erk cell signaling pathways in skin (not DRG, see Supplementary Figures S2, S5) to treat the chronic itch in ACD mice.
The pathogenesis of chronic skin diseases including ACD is complicated, including at least skin barrier dysfunctions, allergy/immunity, and pruritus (Liu Z. et al., 2016;Kittaka and Tominaga, 2017;Kostner et al., 2017;Zhang et al., 2017;Hachisuka et al., 2018). Cytokines can be produced by a wide array of immune cells, including neutrophils, eosinophils, mast cells, macrophages, and T cells (Liu Z. et al., 2016;Kittaka and Tominaga, 2017;Kostner et al., 2017;Zhang et al., 2017;Hachisuka et al., 2018). Therefore, even though we have some understanding about the possible cytokines, itch-related molecules, and related intracellular signaling pathways for QP to work with, the specific subsets of the cell types, what exact ingredients, and how they function through these cells and molecular network systems, etc., need to be further clarified. Future research on experiments in vitro, genetic engineering animals, etc., may help address these issues.
CONCLUSION Via a HPLC system and a BATMAN-TCM platform, for the first time we performed detailed HPLC fingerprint analysis and network pharmacology studies of QP ointment. By in-depth animal and molecular analysis, we identified a number of chronic dermatitis-associated Th1/2 cytokines, itch mediators, and cellular signaling pathways in the skin, DRG and serum for QP to treat the inflammation and chronic itch associated with ACD. Our data clearly suggested, upon stimulation, cytokines and itch-related mediators are released from the skin and neuronal tissues, and QP could effectively work on both the skin and sensory neurons to treat the chronic itch.

ETHICS STATEMENT
All animal experimental procedures followed international guidelines for care and use of laboratory animals which were approved by the Animal Ethics Committee of South-central University for Nationalities.

AUTHOR CONTRIBUTIONS
ZM designed and supervised the study. XG, HX, ZZ, and ZM analyzed the data and wrote the manuscript. XG and HX mainly performed the experiments. LY, SL, CT, HW, YL, and WC helped the experiments.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2019.00354/full#supplementary-material DATA SHEET S1 | Three main compounds of QP ( Figure S1). QP suppressed the phosphorylation of MAPKs in the DRG of SADBE mice (Supplementary Figure S2). The data of the original western blot images ( Supplementary  Figures S3-S5). TABLE S1 | The key targets of chronic dermatitis or QP (Sheet 1), and targets corresponding to key signaling pathways (Sheet 2) based on the platform of BATMAN-TCM.


extfieldsofstudy: Medicine Biology

text:
Evaluation of novel formulations for transarterial chemoembolization: combining elements of Lipiodol emulsions with Drug-eluting Beads

There are currently two methods widely used in clinical practice to perform transarterial chemoembolization (TACE). One is based on mixing an aqueous drug with an iodized oil (Lipiodol) and creating an emulsion that is delivered intraarterially, followed by embolization with a particulate agent. The other is based on a one-step TACE using Drug-eluting Beads (DEBs) loaded with drug. It is not recommended to mix Lipiodol with DEBs due to incompatibility. For the first time, novel DEB: Lipiodol: doxorubicin (Dox) emulsions are identified using lyophilized polyvinyl alcohol (PVA) hydrogels (non-iodinated or iodinated) DEBs. Methods: 15 DEB emulsions (50mg Dox) were assessed for stability and deliverability in vitro and in vivo in a swine model. Dox release from selected formulations was measured in vitro using a vascular flow model and in vivo in a VX2 rabbit tumor model. Results: Both DEB formats were shown to be able to form emulsions, however only Iodinated DEBs consistently met defined handling criteria. Those based on the non-iodinated DEB achieved >99%+ Dox loading in <5 minutes but were generally less stable. Those prepared using iodinated DEBs, which are more hydrophobic, were able to form stable Pickering-like emulsions (separation time ≥ 20 minutes) and demonstrated handling, administration and imaging observations more akin to Lipiodol™ TACE emulsions in both embolization models. Controlled Dox release and hence beneficial in vivo pharmacokinetics associated with DEB-TACE were maintained. Conclusions: This study demonstrates that it is possible to formulate novel DEB emulsions suitable for TACE that combine positive elements of both Lipiodol™ based and DEB-TACE procedures.

Introduction
The theory behind conventional Lipiodol Ultra Fluid™ (Guerbet, France) based transarterial chemoembolization (TACE, or cTACE) is founded on the premise that a water-in-oil (w/o) emulsion can be formed when Lipiodol™ is mixed with an aqueous drug-containing solution at an appropriate oil:water ratio [1]. When the emulsion is delivered through a microcatheter into the flowing blood stream, viscous oil droplets form that follow the flow into the tumor microvasculature where the viscosity causes cessation or slowing of flow into the small vessels. The chemotherapeutic is trapped in the aqueous compartment of the emulsion and is released as the oil becomes entrapped in the capillary bed. An additional Ivyspring International Publisher embolic agent is desirable to induce stasis and prevent the oil being washed through prematurely and increase its dwell time. A proportion of the droplets are of a size that can pass through the sinusoids into the portal venous system [2,3] and greater volumes of the liver can therefore be exposed to the chemotherapeutic than intended. The Lipiodol™ is eliminated from the healthy part of the liver by the action of Kupffer cells [4] but as liver tumors do not possess these, the area within the tumor remains stained by the Lipiodol™ appearing as a dense radiopaque region on follow-up X-ray based imaging [3].
Drug-eluting Beads (DEBs) were introduced over a decade ago with the intention of standardizing TACE by providing calibrated microspheres that contain the chemotherapeutic agent that may be used to embolize the vessels of the tumor where they then elute the drug in a locoregional manner, over a number of days to weeks [5]. It has been demonstrated in clinical studies that plasma levels of drug are significantly reduced when comparing DEB with Lipiodol™ TACE, resulting in fewer side effects, less liver toxicity and shorter hospital stays [6][7][8].
Whilst quality of life may therefore be improved, there is still considerable debate as to whether there is any benefit in overall survival for DEB versus cTACE. Indeed, over the years one practice has not superseded the other, physicians choosing one or the other based upon the patients overall health, size and number of tumors, whether discrete or diffuse and their location in the liver [9][10][11][12].
Whilst there are anecdotal reports that some physicians use mixtures of DEBs and Lipiodol™, this is not currently recommended [10] and there are no accounts in the literature to verify this practice. DEBs are suspended in aqueous iodinated contrast agents in order that the suspension can be visualized under fluoroscopic administration. DEBs are highly hydrophilic and if mixed with oil, remain dispersed in the aqueous phase, the admixture disrupting the emulsion stability creating a concoction unsuitable for delivery. A recent study reported on the use of oxaliplatin-loaded polylactide-glycolide copolymer nanoparticles to form stable emulsions with Lipiodol™ suitable for TACE [13]. These relatively hydrophobic nanoparticles adsorb at the water-oil interface and sterically stabilize the droplets preventing coalescence, forming a system known as a Pickering emulsion [14]. Whilst the nanoparticles also acted as a controlled release system for the oxaliplatin, their size was too small to provide for concomitant embolization of the vessels. DEB:Lipiodol™ emulsions therefore present a new paradigm for TACE that currently remains un-investigated. In order to overcome the incompatibility issues experienced with current DEB: Lipiodol™ emulsions and achieve Pickering-like stability, the particles need to have the appropriate balance of hydrophilic and hydrophobic character. This study presents two strategies to enhance the hydrophobic nature of the DEBs and evaluates the stability, deliverability, coherence under flow and drug delivery properties both in vitro and in vivo of the subsequent novel DEB:Lipiodol™ stabilized TACE emulsion formulations.

Preparation of non-iodinated and iodinated PVA hydrogel DEBs
The two DEB types prepared for this study were a non-iodinated DEB based on the PVA-hydrogel used in the fabrication of DC Bead™ [15] (Figure 2A) and an iodinated DEB (containing 155 mg iodine/mL) based on the PVA-hydrogel used in the fabrication of DC Bead LUMI™ [16] (Biocompatibles UK Ltd, Farnham, UK ( Figure 2B). A second iodinated DEB (containing 33 mg iodine/mL) was also prepared for a Contact Atomic Force Spectroscopy (CAFS) study only. Non-iodinated PVA beads were immersed in 10% mannitol solution for 30 minutes and excess solution removed prior to lyophilization in order to prevent collapse of the internal structure (96% water content). The iodinated PVA beads did not require this step as they possess higher solid content (~65% water content). Comparable size ranges of each bead type (70-150 µm) were frozen (-20°C) for 30 minutes and then lyophilized (Wizard 2.0, VirTis, SP Scientific, USA) for 18 hours until visibly dry and free flowing particles were observed (as described previously for the non-iodinated PVA DEBs [17]). This lyophilized format allows for initial mixing with Lipiodol™ oil which can penetrate the DEBs structure and aid in the emulsion compatibility (Figure 2A step 3).

DEB surface property evaluation
Initial qualitative assessments were performed on a Dimension 3000 atomic force microscope (AFM) (Digital Instruments) and data collected using the NanoScope IIIA software (Digital Instruments) [18]. The force distance curve was obtained by measuring the deflection signal (voltage) from the cantilever as the probe approached the surface from approximately 750-1800nm above at a constant scan rate of 1Hz. The cantilever deflection was observed as the difference in signal voltage at the points when the tip is retracting from the particle surface and when the tip is free from the surface. Using a silicon AFM probe, it was expected that the adhesion and cantilever deflection would be highest in particles having a more hydrophilic surface property. Testing was performed in n= 3 replicate samples. The cantilever deflection was measured as a signal voltage and was calculated to give an adhesion force using the spring constant of the cantilever.

DEB:Lipiodol™ emulsion formulation
Emulsions (containing DEBs) with different oil and water ratios were formulated by the mixing of proportionate volumes of Lipiodol Ultra Fluid™ (Guerbet, France) and aqueous solution, in the presence of the DEBs. The aqueous solution was comprised of doxorubicin hydrochloride (Dox, Hisun, China)) solution and aqueous Omnipaque 350™ contrast agent (GE Healthcare, UK). The overall ratios of oil to aqueous investigated ranged from 53:47%v/v to 83:17%v/v, the volume of the DEBs were not expressed in the Oil:Water ratio as this was fixed at 2mL. Both iodinated and non-iodinated DEBs were analysed over this range.
Depending on the desired oil:water ratio, an amount of Lipiodol™ (Guerbet, France) was added directly to vials of each bead type and the sample allowed to stand for 5 minutes before aspirating into a 20 mL syringe. The desired amount of an aqueous Dox solution (50 mg total Dox) was added to a 10 mL syringe and if required, a further volume of aqueous contrast agent was added to this to increase the density of the aqueous phase and further improve stability of the emulsion. The emulsion was formed by purging the aqueous contents of the 10mL syringe into a 20 mL syringe via a 3 way connector, and then back again into the original 20 mL syringe. This was repeated 10 times, i.e. a total of 20 passes of the mixture through the 3-way connector. The emulsion was left to stand for up to 60 minutes (with resuspension of the emulsion every 10 minutes) to allow drug load into the DEBs and was subsequently resuspended one final time before being analysed for emulsion stability or flow properties.
Visual light microscopy of the emulsions was performed using a microscope at 10x magnification (BX50, Olympus, Japan) and using methods adapted from Pichot et al. [19]. Emulsions were prepared and dispensed into a petri dish, a cover slip added (22x22 mm Manzel-Glaser, Avonchem LtD, UK) and then a drop of aqueous blue dye (Reactive Blue 4, Sigma Aldrich) was applied to the edge of the cover slip to identify the aqueous phase and hence the preferred location for the DEBs as shown in the micrographs in Figure 1. In some experiments the emulsion was sampled periodically to determine the extent of Dox loading into the DEB by spectrophotometric determination of the residual drug remaining [15] and visual confirmation of loading by light microscopy. cTACE emulsion formation cTACE emulsions were prepared by direct mixing of proportionate volumes of Lipiodol and aqueous doxorubicin hydrochloride solution. The emulsion was formed by purging the aqueous doxorubicin solution (10mL syringe) into the Lipiodol (20mL syringe) via a 3 way connector, and then back again 10 times. Although in clinical practice this would be followed up with an embolic particle to finalise the embolization, we attempt to show the burst effect of drug associated with purely the emulsions, prior to any additional embolisation.

DEB Emulsion separation time
Emulsion stability was first assessed by recording the time it took for phase separation to become evident in the syringe immediately after it had been mixed through the 3-way connector. Separation time was taken as the onset of inhomogeneity in emulsion appearance as distinct layers began to form as the phases separated (see Figure 3D).

DEB Emulsion drop test
DEB emulsions were injected in a steady stream via an 18G blunt fill needle (BD, US) into saline solution in an adaptation of the pendant method presented by Pichot et al. [19]. The droplet integrity and formation of strings were evaluated as a measure of emulsion stability and cohesion in response to limited shear and aqueous contact. Good emulsions were classified as having discreet droplets that remained intact following delivery from the needle as visually observed ( Figure 5B& E). Poor indicators of stability were defined as streams of loose DEBs and separated oil droplets containing few or no DEBs ( Figure 5A&D). Assessments were repeated a minimum of 3 times for each formulation.

DEB Emulsion performance assessment under dynamic flow in a Vascular Flow Model
In order to evaluate DEB emulsions under flow, a modified set-up using a previously described Vascular Flow Model (VFM) was employed [20]. Emulsions were injected into flowing saline solution under various clinically representative flow rates, with the catheter (2.4Fr Progreat, Terumo Tokyo) positioned in a location that was agreed to be an approximation of a typical injection site arterial lumen diameter (channel diameter between 2 mm-4 mm). This was confirmed visually with the assistance of an interventional oncologist with >20 years' experience in TACE procedures and who had also previously used similar versions of the flow model to evaluate TACE emulsion behaviour [21]. Each emulsion was repeated a minimum of 3 times. The rate was selected as visually representative of blood flow observed under fluoroscopy ranging from 120 mL/min, periodically reduced to 60-30 mL/min to recreate the effect of embolization and flow restriction. The site of the injection was varied between a distal and proximal location (Distal= 2 mm channel diameter, proximal= 4 mm channel diameter) to visually assess the influence of channel aspect ratio, shear rate and flow rate of the fluid surrounding the catheter tip. Optical video microscopy (Dinolite digital microscope (AnMo Electronics Cop. Taiwan) was also used to record DEB emulsion behaviour under flow conditions in the VFM (Videos in supplemental S1). An in-line pressure transducer (Research grade BP, Harvard instruments, UK) was linked to real-time flow measurements during delivery as a measure of the embolic effect of the emulsions on flow ( Figure  5C).

Grading of DEB emulsions
DEB emulsion performance was graded (1* (poorest) -5* (best)) based upon the results from the separation time, drop test and flow properties ( Table  2). Together these provide a measure of the ability of the microspheres to stabilize the oil:water interface and influence the oil droplet size (streaming or discreet droplet formation) and integrity (its tendency to breakup under flow). Such factors also affect the resistance experienced during delivery through the microcatheter, which is also a predictor of whether the formulation would be suitable for a TACE procedure.

Drug elution properties of DEB emulsions
For Dox elution determination, the microcatheter was positioned into a single channel of the VFM, covered with a 25 µm microporous mesh (Sefar AG, Switzerland) at the outlet to prevent DEB passage and allow accumulation of an occlusion ( Figure 6A). The flow was set to 120 mL/min as per previous flow studies [22], and injection rate was determined to be 0.5 mL/min from previous emulsion bench testing. The eluent was collected from this outlet, starting from the initiation of the DEB delivery and every minute for the first ten minutes then every five minutes until a 30 minute test period was complete. Dox concentration in the eluent was determined using UV-visible spectrophotometry (Varian CaryBio50, UV/Vis Spectrophotometer) set at an absorbance maximum of 483 nm and an elution curve was constructed from the data. The experiment was repeated 3 times for each emulsion tested.

Drug elution properties in rabbit VX2 model
New Zealand white rabbits (N=7) aged more than 5 months weighing between 3 and 4 kg were implanted with VX2 donor tissues and grown in tibial cranial muscle. Following maturation, 4-5 mm sized tumor sections were harvested and transplanted directly into test liver parenchyma (7 animals per test arm) and allowed to further implant for 12-14 days. Vascular access to the right femoral artery was performed surgically after incision of the hindmuscle, exposure of the femoral artery and insertion of a 4 French vascular sheath. Selective catheterization of the hepatic artery and super-selective catheterization of the left branch of the hepatic artery was performed using a 1.7 Fr microcatheter under fluoroscopy guidance (Discovery IGS 730, GE Healthcare). An angiogram and a 3D C-arm CBCT imaging was performed and recorded to visualize the tumor vasculature. The embolic agent was slowly administered under fluoroscopy monitoring with care being taken to avoid reflux in non-catheterized arteries. The endpoint for injection was fixed at 1mg Dox as calculated from the formulation composition in all the groups. If stasis of the tumor-feeding artery is not reached after injection of the formulation, bland particles were injected to complete the devascularisation. Due to anatomical restrictions a smaller size of radiopaque DEBs in the range 40-90 µm (Biocompatibles UK Ltd, Farnham, UK), were employed in VX2 model, these were also utilised in the swine model for handling comparison for both iodinated and non-iodinated. A control angiography and a 3D C-arm CBCT imaging was performed after embolization and after 7 days.
Qualitative comparisons of proportional drug release and bio-distribution were compared to in vitro observations and quality indicators for a measure of the predictive capabilities of the profiling experiments. Plasma analysis was performed at T= 0 min, 5 min, 15 min, 30 min, 60 min, 2 h, 6 h, 24h, 72 h and 7 days after the end of injection (10 samples per animal). Tissue analysis was performed at 7 days after injection in the tumor and in the peritumoral healthy parenchyma (2 samples per animal).

DEB emulsion handing and in vivo flow properties in healthy swine model
A total of 4 swine (FBM minipigs) were used for the study (2 female and 2 castrated males). Animals were aged 7 months with a mean weight of 38.7 ± 4.5 kg (min-max 34 -44 kg). Each animal was identified by a unique national identification number attached to the ear. The number of the animal was verified before anaesthesia and before transfer to the angiography suite. A right femoral artery puncture was performed percutaneously and a 5 French vascular sheath was placed. Alternatively, a surgical access to the femoral artery was gained after incision of the hind muscle and exposure of the femoral artery for one animal. The angiography suite was equipped with a Discovery IGS 730 Image Guided System (GE Healthcare, Little Chalfont, UK). A 5 French catheter (Glidecath® vertebral, Terumo, Guyancourt France) with a 0.035'' angled hydrophilic wire (Terumo, Japan) was used to reach the celiac artery. A super-selective catheterization of one branch of the hepatic artery was performed using a 2.4 French Progreat® microcatheter (Terumo, Japan) and a 0.016" 45° GT wire (Terumo, Japan). The formulation volume prepared depended upon the ratio of the various components (see Table 4) and was between 7.5 mL and 20 mL. When ready for testing, 0.5 mL of formulation was transferred into a 1 mL syringe for ease of delivery. The formulation was administered as intermittent small "puffs" and the 1mL syringe recharged with the resuspended emulsion once emptied, until complete stasis of the catheterized arterial territory was achieved or injection of the full volume of formulation prepared. The microcatheter was then moved to a second hepatic artery branch and a second formulation was injected with the same endpoint. Selective catheterization of the renal artery or of the upper/lower branch of the renal was performed successively in each kidney and the same formulations as in the liver were injected until complete stasis. All animals were sacrificed at the end of the embolization procedure by intravenous injection of 20 mL pentobarbital (Dolethal® 200 mg/mL, Vetoquinol, Lure France) followed by exsanguination. Liver and kidneys were explanted and fixed in 10% formalin. All sequences of injection were recorded. 3D-CBCT sequences were recorded after every 1.0 mL injection.
Emulsions tested included iodinated and non-iodinated beads of two sizes across a range of oil:water ratios (Table 4), selected to cover the working range identified in Figure 5, Table 3. The following parameters were graded by the interventional radiologist (who had been blinded to the formulation compositions), in order to provide an overall handling rank: Syringe stability, Catheter delivery, Radio-opacity, Stability of emulsions during injection, portal opacification during injection, degree of embolization and distality of embolization.

Surface characterization of non-iodinated and iodinated PVA hydrogel DEBs
Measurements were performed on the two DEB types using CAFS and force-distance-amplitude protocols (see Methods, Figure Table 1), suggesting their surface chemistries were different and that they may behave differently when formulated into emulsions.

DEB emulsion stability under static conditions
Lyophilization of the DEBs allowed for mixing with Lipiodol™ oil without premature separation. In a Lipiodol™ cTACE emulsion, sufficient oil phase is present in the emulsion formulation such that aqueous drug droplets are confined within the oil phase, the latter subsequently forming oil droplets within the continuous aqueous-based blood phase during catheter delivery (hence forming a water-in-oil-in water emulsion (w/o/w) in the body, Figure 3A). The objective of modifying the DEB surface properties was to encourage the DEBs to reside at the oil:water interface and to stabilize the emulsion droplets ( Figure 3B). Optical microscopy of the DEB emulsions showed non-iodinated DEBs to have a greater affinity for the aqueous phase of the emulsion, whereas the iodinated DEBs were found to reside more preferentially in the oil phase and at the oil:water interface ( Figure 3C). Optical microscopy also confirmed that the Dox was taken up into both DEB types preferentially, rather than residing in the aqueous compartment of the emulsion. Interestingly, Dox uptake from the emulsion containing the non-iodinated DEB was extremely rapid (>98% loaded in <2 mins) compared to the equivalent size of DC Bead prepared by the recommended hospital pharmacy loading methods (>98% in 30 mins). There was no change however, in the rate of Dox uptake from the emulsion into the more hydrophobic iodinated DEB compared to the equivalent size of DC Bead LUMI prepared by standard loading methods (>98% loaded in ~90 mins). Stability of the DEB emulsions in a syringe was defined as an important quality parameter ( Figure 3D). The time taken for the emulsions to separate in the syringe was assessed and used to generate an overall ranking combined with static and flow stability observations ( Table 2). This ranking was used to decide which formulations to take forward for further evaluation.   A wide range of emulsion formulations were prepared and evaluated (Table 3). Stable o/w emulsions were observed more prominently in the iodinated DEB samples and with low aqueous content (Table 3 and Figure 4). The iodinated DEB samples indicated partition preference into the oil phase, whereas the non-iodinated samples remained in the aqueous phase, even in low aqueous ratio emulsions. There was increased presence of non-iodinated DEBs at the oil:water interface in low aqueous content, which helped to stabilize these emulsions compared to those with higher aqueous content. This is clearly observed in Figure 3C under the microscope for 1:1 versus 1:3 water:oil ratios.

DEB emulsion stability under flow conditions
The emulsion drop-test analysis reinforced the occurrence of phase partitioning for non-iodinated DEBs observed in the optical microscopy, again showing the beads separate from the poor stability emulsions upon dilution in an aqueous continuous phase ( Figure 5A). Iodinated DEBs remained in the oil phase, stabilizing the oil as descrete, intact droplets as they entered the aqueous continuous phase ( Figure  5B); this is more reminiscent of a cTACE emulsion behavior. The dynamic stability of emulsions was assessed in terms of droplet formation under flow following release from the catheter tip under various injection rates using a Vascular Flow Model (VFM, Figure 5C [20]). Droplet size is influenced by flow and shear rates in the delivery channel upon administration into the continuous aqueous phase [23]. Through this method it was possible to identify development of droplets and their integrity as they flowed through a series of bifurcations. This was used to identify formulations that would be robust and stable for use in pre-clinical testing. VFM droplet stability was consistently higher in iodinated bead containing emulsions compared against non-iodinated bead (Figure 5 D&E). Selection of "best case" conditions was based on a scored 1-5 index for each of the test parameters relating to their influence on clinical performance (Table 2). Iodinated DEBs showed the widest stable formulation range, with the lowest recorded flow stability for iodinated DEBs being observed with those formulations containing a higher aqueous content (Figure 4). The DEB emulsions demonstrate a clear ability to progressively occlude the flow in the VFM channels (with increasing hydrostatic pressure) due to the embolic effect of the particle, compared to the cTACE emulsion where Lipiodol™ passed freely through the 25 µm VFM filter ports without any occlusive effect ( Figure 6B). This is aligned with both the established literature [8,24] and the proposed mechanistic action of the novel emulsions [25]. In clinical practice cTACE would be followed by some form of embolic particle, however it is worth noting that prior to embolisation the systemic drug release profile shown in vitro would be experienced in vivo.

Drug elution in vitro
A modified VFM set-up was employed to profile the stability of drug-containing emulsions in terms of the drug elution properties of a selection of the previously characterised formulations ( Figure 6A). The DEB emulsion formulations were based upon those tested in the stability assessment and were those highlighted in Figure 5, consisting of a low and high aqueous formulation of each bead type to evaluate the two extremes of the working stability range. The cTACE emulsion consisted of a 10mL Lipiodol™: 2mL aqueous Dox (25 mg/mL) mixture. The drug burst effect is ultimately associated with systemic free-drug and it is critical to understand the relationship between the emulsion stability and the in vitro pseudopharmacokinetic profile generated using the VFM. The low aqueous DEB emulsions, with increased level of droplet stability under flow conditions, showed slower Dox elution compared to their high aqueous DEB emulsion counterparts. All DEB emulsions show significantly slower release of drug compared to the cTACE (non-embolic) formulation (Figure 7).
Close examination of the emulsion flow through the VFM channels shows that the more stable low aqueous DEB emulsions remain as intact droplets throughout their journey from catheter tip to distal end-channel, where the bead-oil droplet accumulate without separation at the filter but still as discrete droplets that allow some residual flow ( Figure 5C(d & e) and Supplementary videos S1). The relatively less stable high aqueous DEB emulsions showed a tendency to separate, particularly at bifurcations where higher shear forces are experienced, with the beads alone accumulating at the distal exit, whilst most the Lipiodol™ passed through the filter ( Figure  6C(d & e)). Interestingly these emulsions reduce the residual flow more effectively and result in a slightly higher hydrostatic pressure ( Figure 6B).

Emulsion drug release, flow properties and imaging in vivo
Rabbit VX2 model for PK and radiopacity distribution A rabbit VX2 tumour model was used to carry out a pharmacokinetic (PK) study of Dox release from the same formulations tested in the VFM. Figure 7A-C shows the in vitro pseudopharmacokinetic drug release profiles on a log scale for the emulsions tested in the VFM. These profiles clearly show the high quantity of Dox that is released as a burst in the first 100 seconds during administration of a cTACE emulsion (~150 µg/mL). This is in contrast to the very much reduced peak Dox concentrations (~25 µg/mL) that are observed with the DEB emulsions. These results compare very favourably with the findings of the PK study in the rabbit VX2 tumour embolization ( Figure 7D(log) & E(linear)), where in this case the peak concentrations (Cmax) of Dox are observed at the first measured time point around 5 mins and are much greater for the cTACE treated animals (~275 ng/mL) compared to the much reduced C max seen for the DEB Emulsions (15-30 ng/mL). In this study there was very little difference between the data measured for the iodinated versus the non-iodinated low aqueous DEB emulsions, both behaving similarly in terms of stability and handling during administration.   Animals treated with all formulations were survived for 7 days after which spatial distribution of the radiopaque components of the remnant emulsions in the liver was assessed under cone beam computed tomography (CBCT). Figure 8A-C show single slice images from the CBCT in the sagittal plane thresholded to remove the surrounding tissues that show the outline of the liver and the nodular mass on the right hand side of the organ that is the tumour. The white areas in this image are the radiopaque components that remain post-treatment with the various emulsions. Figure 8D-F shows a volume rendered image to portray the three-dimensional distribution of radiopaque matter. There was highly concentrated tumour filling of radiopaque matter in both cTACE and iodinated DEB emulsions ( Figure 8A & C, D & F) indicating good tumour vessel filling. Non-iodinated DEB emulsions presented a more diffuse filling pattern, with some of the more proximal feeding vessels containing significant amounts of radiopaque material ( Figure 8B & F). These emulsions also presented more difficulties in delivery via microcatheter with increased resistance and intermittent catheter clogging indicative of the stability issues encountered during in vitro testing. Consequently, only an average of 0.63 ± 0.15 mg of the 1mg planned dose could be delivered with the non-iodinated DEB emulsions compared to 0.95 ± 0.19 mg for the iodinated DEB emulsion. As shown in Figure 7D & E, the average plasma dose of Dox is higher for the non-iodinated emulsions (27.2 ± 26.7 ng/mL when compared to the iodinated DEB emulsion (13.6 ± 10.9 ng/mL) at the 5 minute time point, further illustrating the disparity in stability between the two emulsion composed of different DEB chemistries.

Swine handling and flow properties
A series of formulations were reconstituted with both iodinated and non-iodinated DEBs as described in Table 4. These included some emulsions formulated with smaller DEB size (40-90 µm) in order to assess their handling as these were used in the VX2 study due to the anatomic restrictions in vessel size. In general, non-iodinated DEB emulsions were ranked less stable in the syringe and handled more poorly during delivery compared to cTACE or the formulations using iodinated DEBs ( Table 4). The in vivo stability however, appeared good under fluoroscopic imaging and the formulation tended to form droplets in the vessels more rapidly than the Lipiodol™-Dox cTACE mixture. The embolic effect was similar to cTACE but the occlusion was more proximal with the DEB-emulsion formulation compared to cTACE. The aspect of the product under fluoroscopy was also different: the formulation with the non-iodinated DEBs showed a more scattered deposition in the arteries compared to cTACE.
The stability in the syringe for all formulations reconstituted with iodinated DEBs was good. There was a strong effect of dilution on all the parameters assessed: increasing the dilution with contrast agent decreased the in vivo stability and the embolic effect of the formulation and increased the distality of occlusion. The performance of formulations with iodinated DEBs (containing between 4 mL and 6 mL of contrast agent) seemed to be close to the performances of cTACE. For the formulation with the lower dilution, the embolic effect seemed to be higher than for cTACE or formulations using non-iodinated DEBs. However after waiting a few minutes, flow was regained, more product could be injected and the total volume of formulation injected was in the same range as for cTACE and formulations with non-iodinated DEBs. The flow and visual appearance of the introduced emulsions echoed that of the VFM pre-selection testing and it was possible to identify droplets and string formations under fluoroscopy as shown in Figure 8G-I (Supplementary video S1).

Discussion
All of the currently available commercial DEBs are composed of hydrogel materials that are substantially swollen in aqueous media and highly hydrophilic in nature. Mixing these directly with Lipiodol™ creates a incompatible concoction unsuitable for TACE consisting of an unstable multiphase system in which the DEBs reside in the aqueous phase. The rationale for the preparation of the novel DEBs used in this study was to modify the surface properties to make them more hydrophobic in an effort to encourage their accumulation at the oil:water interface and to help in the steric stabilization of the resulting emulsions. The non-radiopaque DEB based on DC Bead™ was lyophilized to create a porous structure that when initially mixed with Lipiodol™, would uptake the oil into part of the bead matrix. This has been used to impart radiopacity to the bead but we also surmised that the surface properties would also be rendered more hydrophobic in nature and hence more compatible in an subsequent oil:water emulsion. Note that the lyophilization process was shown not to alter the drug release kinetics of the beads (data not shown). The second approach was to employ the more hydrophobic polymer used to make DC Bead LUMI™ in which the DEBs are modified with hydrophobic triiodobenzyl groups in order to make them permanently radiopaque. Our testing of the lyophilized DEBs using AFM force-distanceamplitude measurements demonstrated that there was a greater pull-off force for the non-iodinated bead compared to the two iodinated beads tested. Since the AFM probe tip is hydrophilic and has a greater attractive force for more hydrophilic surfaces, the non-iodinated bead was determined to have a comparatively less hydrophobic surface than the iodinated beads; and similarly the 33 mg iodine/mL iodinated bead, had a greater pull of force compared to the 155 mg iodine/mL iodinated bead. Hence, the greater the iodine content, the greater the hydrophobicity of the surface and the lower the pull-off force measured.
An increased or comparable rate of drug loading was observed in DEB emulsions compared to DEBs loaded by the standard pharmacy methods. For the non-iodinated DEB emulsions there was a marked incease in loading rate with >98% Dox loaded within 2 mins compared to 30 mins for DC Bead™. This effect is due to a higher surface area to volume ratio of DEBs to Dox maintained in the water phase in which they both reside once the emulsion is formed. The addition of iodine to the structure of the iodinated DEBs increases surface hydrophobicity, causing DEBs to partition into the oil phase, thus slowing the loading rate by restricting access to the drug that is partitioned in the aqueous compartment of the emulsion. Loading rates for Dox in this instance are comparable between the iodinated DEB emulsion and standard methods used for drug loading DC Bead LUMI™. It is interesting that in clinical practice at high volume centres or where there is a need to rapidly prepare another drug loaded vial of DEB for immediate administration, the non-iodinated DEB emulsion formulation offers an almost instantaneous process of loading the DEBs with high levels of Dox without the need to wait.
Microscopic analysis of the various DEB emulsions revealed that it was possible to formulate the two DEB types in order to enhance preference for DEBs to reside at the oil:water interface and stabilise the droplets. This is achieved however, in a quite different manner for the two DEB types as the non-iodinated DEB has a preference for the aqueous phase, where as the iodinated DEB has a preference for the Lipiodol™ oil phase. This is despite the fact that the non-iodinated DEB is mixed with Lipiodol™ in the first instance in order to absorb it into its porous structure, prior to mixing with the aqueous component. This means the surface of the non-iodinated DEB remains more hydrophilic in character. [26]. Particles can be used to stabilize emulsions (Pickering emulsions) but these are typically formed using small particulates in the range 200 nm-1µm, as small particle sizes give high packing efficiency at the oil:water interface [27]. Here DEBs in the size range 40-150 µm have been used in the creation of various emulsion formulations, that whilst not adding to the stability of emulsions relative to the non-DEB containing emulsion, are sufficiently resistant to separation to allow their practical use in a TACE procedure. The more hydrophobic radiopaque DEBs appear to provide a far superior stability compared to the non-radiopaque DEBs, suggesting their presence within the oil phase and at the oil:water interface, is more efficient in helping to retard the separation of the phases.
The stability of an emulsion directly relates to its handling and administration through a microcatheter and hence suitability for use in TACE procedures. Unstable emulsions form a clear separation of phases within a short timeframe (<5mins) at which point delivery becomes difficult to achieve. The visual appearance of the emulsions can also indicate its type, (w/o, o/w) under dye assisted light microscopy and whether there has been an inversion of the phases, which is important from a drug delivery perspective if the aqueous drug is no longer confined within the continuous oil phase. In a study by de Baere et al. [28], an in vitro comparison using a variety of characterisation methods was conducted under shear and pressure rates representative of those experienced in large human arteries [28]. As part of this investigation de Baere et al. utilised an in vitro "hepatic arterial tree" composed of an 8 mm bifurcating silicon tube with a continuous phase of glycerol at 45%, for characterisation of droplet mixing and emulsion type [21]. An evolution of this platform termed the Vascular Flow Model (VFM) was used in this study for the characterisation of DEB emulsions under dynamic flow conditions. DEB emulsion stability under flow was consistently higher in iodinated DEB emulsions compared to the non-iodinated DEB versions, although stability decreased markedly for both formulations as the content of the aqueous component was increased. This is believed to be due to the tendency for the o/w emulsion to invert to a w/o state, increasing the surface area of the aqueous compartment and diluting the DEB stabilizing effect, which increases dispersion and breakdown of the emulsion within the flowing aqueous continuous phase. The DEB emulsions were obviously capable of being trapped at the channel outlet by the filter and accumulated at the distal end causing a drop in flow rate in the channel and a subsequent increase in the hydrostatic pressure in the system due to this occlusion. The non-radiopaque DEB emulsions were more effective in blocking the flow as these beads are somewhat softer than the iodinated counterparts as we have previously shown [29] and pack together at the distal end more effectively by reducing the space between the particulates, whereas the iodinated DEBs being very stiff, accumulate but remain as spherical spheres with significant inter-sphere spaces present to allow some residual flow and relief of the pressure ( Figure 6B).
As the Dox was shown to prefentially load from the aqueous componment of the mixture into the DEBs in the formulation, the DEB emulsions clearly demonstrated a similar controlled Dox release and low "systemic" burst of drug that is synonymous with DEB-TACE compared to cTACE [8,30]. The more stable iodined DEB low aqueous formulation produced the slowest release profile, which is expected if the drug has to diffuse out of the DEB and cross the oil boundary before being released into the flowing continuous phase and vice versa for the less stable emulsions with higher aqueous content. The pseudopharmacokinetic profile generated using the VFM was a reasonable prediction approximation to the actual PK profile measured in the rabbits in terms of general overall shape, peak drug times both occurring early on in the first few minutes, and both reporting similar trends in drug release from the three emulsions investigated. A major limitation of the VX2 model is the reduced vasculature dimensions when compared to that of human hepatic vasculature [31]. This means that administered doses are scaled according to body weight and vascular access, resulting in scaled representations of the dosimetry experienced in a procedure [31]. This was observed to be particularly impactful in the delivery of emulsions, where difficulties administering the full emulsion dose calculated as part of the agreed protocol were experienced. In these cases it was recorded and included as feedback for emulsion evaluation in terms of handling and overall grade. This further highlights the importance of channel geometry and size for in vitro profiling experiments. This should ideally always be performed using flow conditions that closely replicate those present within the intended treatment indication.
The results generated here demonstrate the benefit of in vitro devices in the evaluation of novel emulsions. Whilst the VFM does not emulate the same level of flow dynamic complexity as the in vivo situation, it does provide a profiling step that enabled selection of favourable iodinated and non-iodinated DEB emulsion formulations suitable to carry forward for in vivo testing. Visualization of flow of all of the emulsions tested using fluoroscopy was clear, as all emulsions either contained Lipiodol™, contrast agent or radiopaque DEBs, all of which are radiodense and will be visible under fluoroscopic X-ray. Radiopaque DEB emulsions could be delivered as descrete imageable droplets, whereas the non-radipaque DEB formulations seemed to occlude the arteries more proximally which could be related to poor emulsion instability as noted in vitro. Lilienberg et al. reported similar conclusions in their swine study, where they observed that the physical embolic effect of emulsions were directly related to frequency of mixing and the stability under flow [24]. After the treatment of the rabbit VX2 tumours, the animals were survived for seven days before imaging. This allows for any trapped contrast agent to be washed out of the vessels [32] and for Lipiodol™ in healthy parts of the liver to be cleared by phagocytosis in the Kupffer cells [33]. CBCT showed more compact densification of the tumour in the cTACE and iodinated DEB emulsion cases indicative of improved penetration into the tumour vessels. The radiopacity in the DEB emulsion case could be due to the presence of both radiopaque beads and Lipiodol™ in the tumour but it is not possible to distinguish between the two in this instance as CBCT is not sensitive enough to detect at single bead resolution. The non-iodinated DEB emulsion was less well-filled and several more proximal feeding vessels obviously contained what is likely to be beads, indicating potential separation of the formulation during delivery.
To our knowledge, this is the first time that novel DEBs have been prepared that are capable of being formulated into stable DEB emulsions with Lipiodol™ and Dox that are suitable for application in TACE procedures. These emulsions therefore potentially combine the advantages of portal vein filling observed with Lipiodol™-based TACE with the beneficial safety profile of the controlled and sustained drug delivery obtained using DEB-TACE. In vitro profiling supports adequate stability for handling and administration and in vivo evaluation confirms low peak drug plasma levels and prolonged radiopacity of the treated area showing the bead+Lipiodol™ location. Further refinement and in vitro evaluation combined with efficicacy testing in vivo of this promising combined technology platform is warranted.