Crosslinkable poloxamer in-situ gel as a submucosal cushion for endoscopic submucosal dissection

Background: Hypertonic saline solution is used for endoscopic submucosal dissection (ESD) as a submucosal uid cushion (SFC). However, the application of SFC is limited by challenges including a short effect duration as well as increased risk of perforation and bleeding. Methods: An in-situ gel crosslinked acrylate/thiol Poloxamer 407 (PA-PSH) can be used as a novel SFC for ESD to avoid perforation, and the feasibility and safety of the PA-PSH for ESD was evaluated in vitro and in vivo . Results: PA-PSH thermal injection led to a longer-lasting elevation with clearer margins as compared with glycerol, with a high mucosal elevation induced by thiol-ene reaction in physiological conditions between acrylate Poloxamer 407 (PA) and thiol-capped Poloxamer 407 (PSH). Precise ESD along the margins of the elevated mucosa was achieved without obvious adverse effects such as bleeding, perforation and tissue damage. Conclusion: The PA-PSH in-situ gel provides an excellent submucosal injection system, which has great potential to improve the ESD technique.

the elevated mucosa was achieved without obvious adverse effects such as bleeding, perforation and tissue damage.
Conclusion: The PA-PSH in-situ gel provides an excellent submucosal injection system, which has great potential to improve the ESD technique.

Background
Endoscopic submucosal dissection (ESD) is a standard treatment modality for gastrointestinal neoplastic lesions. The formation of an adequate submucosal cushion between the lesion and the proper muscle layer can facilitate an en bloc resection and reduce the risk of perforation for ESD. En bloc resections could reduce the risk of local recurrence after the endoscopic resection and provide more accurate histopathological assessments. An ideal submucosal injection solutions could provide a longlasting and su ciently high submucosal cushion without no obvious adverse effects. They also should be inexpensive, readily available and easy to inject [1][2][3] .
Saline solution is usually used for ESD as a submucosal uid cushion (SFC). However, saline solution would be rapidly absorbed by the surrounding tissue thus requires repeated injections, making operation di cult. In addition, it is still a challenge to resect lesions larger than 2 cm in diameter completely 4 . Therefore, diverse agents like glycerol, hyaluronic acid, gelatin and so on are applied for ESD and avoid perforation, however, most of these agents are viscous and not easy for injection. [5][6][7][8][9][10][11][12][13] The in-situ gel after injection such as photosensitive gels 14 or thermosensitive gels [15][16][17] were utilized to avoid the drawbacks. However, there still some limitations. For example, the sol-gel (solution to gel) transformation based on photoinitiated free radical polymerization rely on ultraviolet light, which may be di cult in deep areas in vivo. Meanwhile, although thermogels like PLGA-PEG-PLGA 15 and poloxamers 17 demonstrated good e cacy and safety, they are easy to clog inside long delivery tools under human physical body temperature. Therefore, it is necessary for developing more new submucosal agents for ESD.
Here, we exploited an PA-PSH in-situ gel as a novel submucosal injection substance for ESD. The application of PA-PSH in-situ gel as an SFC in ESD procedure was as shown in Fig. 1. This in-situ gel was low sticky solution ex vivo but could transform into high condensed gel in vivo, which was triggered by thiol-ene reaction and was an ideal material for ESD. Furthermore, it is designed based on poloxamer, a material approved by the US Food and Drug Administration (FDA). The current study assessed the feasibility, durability and tissue biocompatibility of PA-PSH in-situ gel in both resected porcine stomachs and living minipigs.

Creating Submucosal Cushion And Performing ESD In Living Minipigs
The in-situ gel formation test was carried out using minipigs. Minipigs were fasted for 2 days with polyethylene glycol electrolytes powder treatment before operation. ESD was performed in minipigs under general anesthesia, with a endoscope (Olymplus H180, Olymplus Optical Co. Ltd, Japan). All of the injection agents contained a 1% methylene blue (v/v).
To create SFCs, each group (n = 6) of minipigs was submucosally injected with 10 mL 3% sodium pentobarbital solution, through an endoscope accessory channel by a 23-gauge needle. After anesthetizing the animals, 10 mL (1% methylene blue, v/v) PA-PSH or glycerol solution (control) was injected into the femoribus internus subcutaneously within 1 min respectively, observing the SFCs at 0, 15 and 30 min after injection. Besides, ESD procedures were implemented after the formation of SFCs in living minipigs.
In vivo degradation of PA-PSH in-situ gel and histologic examination In vivo degradation of PA-PSH was evaluated endoscopically, comparing with the mucosal elevation on 9th day. All the minipigs were sacri ced after evaluation of the in vivo degradation of PA-PSH and the tissue damage were examined. All the histological sections were stained with hematoxylin and eosin (HE), and observed on a light microscope (Axiovert 200, Zeiss).

IR and 1 H-NMR
PA-PSH was synthesized as reported ( Fig. S1) 19,20 . The crosslink of PA and PSH was con rmed by FT-IR, which the C = O stretching vibration peak of the acrylate group was appeared at 1732 cm − 1 (Fig. S2).
Furthermore, the chemical structures of PA and PA-PSH conjugates were con rmed by 1 H-NMR. 1 H-NMR spectrum of PA in Fig. 2a,b clearly showed increased signals at 1.2 ppm and 6.0-6.5 ppm comparing with the spectrum of Poloxamer 407 (P407), which represented the protons from acryl and propylene oxide units.
The synthesized PSH was further con rmed by FT-IR (Fig. S2), the C = O stretching vibration of terminal thio-propionyl groups in PSH was appeared at 1736 cm − 1 . For disul de crosslinked P407 (PSS), three peaks appeared on the GPC curve at 21.19, 22.23 and 23.84 min. (Fig. S3). For PSH, the shoulder peak at 21.29 min disappeared, and for PA-PSH mixture, the peak at 24.01 min broadened largely and a remarkable shoulder peak at 25.34 min appeared, which resulted from products with larger molecular weights had formed based on the thiol-ene reaction 20 . 2c), while at 37 °C, pH 7.4, the copolymer solution turned into a transparent gel (Fig. 2d).

Submucosal injection of in-situ gel in resected porcine stomachs
The feasibility of in-situ gel was assessed in fresh resected porcine stomachs in different concentrations, with PBS and glycerol as controls. The results were shown in Fig. 3a. Although adequate mucosal elevations occurred in each group, the in-situ gel created SFCs were more durable than that of PBS or glycerol. Moreover, no signi cant change in size, shape or consistency of SFCs under the submucosa elevated by the in-situ gel was observed over 1 h. In contrast, the elevation created with PBS and glycerol collapsed quickly in 15 min. In short, the ability of mucosal elevation and maintenance exhibited a sequence in-situ gel > glycerol > PBS.

Submucosal injection of in-situ gel in living minipigs
The in vivo e cacy in living minipigs were further evaluated, as shown in Fig. 3b. The mucosa was lifted by different agents immediately after injection. However, the SFCs created by glycerol collapsed obviously after 15 min injection and disappeared at 30 min. In contrast, the SFCs of the in-situ gel remained unchanged in 30 min. Even after 9 days, the elevation of the in-situ gel still presented an apparently visible edge at the injection site without obvious ischemia or ulceration, while the injection sites of the controls was hard to nd (data not shown).

PA-PSH-assisted ESD in living minipigs
As shown in Fig. 4a,b, the procedure of ESD was performed with the assistance of the methylene blue labled in-situ gel, it could be accomplished in early stage with the guidance of the mucosal elevation created by the in-situ gel, with no signi cant side effects like major bleeding and perforation. All of the procedures were successful and all the minipigs were sacri ced on 9th day after the ESD procedure, and its stomach was dissected with an ulcer displayed at early healing stage.

Biosafety of PA-PSH in-situ gel
The in vivo biodegradation of PA-PSH in-situ gel was examined in minipigs (Fig. 4c). An obvious elliptic protrusion appeared at the injection site after injection and the integrity of the mucosal elevation remained at least 9 days. The height and size of the protrusion decreased due to the degradation of the matrix in vivo. The degradation of the in-situ gel was attributed the hydrolysis of ester bonds in polyoxypropylene segments, and the nal degradation products were biocompatible 21 . The gel protrusion was clearly observed at 0th and 9th day, separately (Fig. 4c). Additionally, after injection of insitu gel solution, neither apparent epithelial damage nor in ammation cells were observed in the injection site (Fig. 4d). After 9 days of injection, granulation tissues were appeared on the postoperative ulcer bed and the edge of the ulcer was re-epithelialized (Fig. 4e). All these results con rmed the considerable biocompatibility of the PA-PSH in-situ gel as a submucosal injection agent.

Discussion
ESD is an effective procedure in the treatment of gastrointestinal tumors, with low trauma. However, because of the using of electrocautery in ESD procedure, the incidence rates of bleeding and perforation could reach to 16% and 10%, respectively 22 . To reduce the incidence of side effects especially perforation, submucosal agent is used to guide the ESD procedure by creating an enclosed cushion. Good submucosal agent should possess features, including longlasting, biocompatible, economically viable, safe and easy to inject and form SFCs, etc. Saline solution is a commonly used submucosal agent, however, it is easy to diffuse at the injection site. Hypertonic saline solution could present better lesionlifting ability and longer duration than saline solution, for the reason of its high osmotic pressure, it still cause visible tissue damage and in ammation 23 . Besides, photo-crosslinked chitosan hydrogel could form SFCs in-situ, however, it should be irradiated with UV light for 5 min to form an elevated mucosa, which was not feasible in vivo experiments 24 . Additionally, the high viscosity of normal gel like Cook Medical's submucosal lifting gel and ORISE™ Gel Submucosal Lifting Agent also limits its application in ESD 25,26 .
In this study, the PA-PSH in-situ gel was a low viscous sol at room temperatuere (RT) and transformed into hydrogel under physiological conditions, which could easily be injected by a 23-gauge disposable catheter injection needle (Fig. 2c,d). During the ESD procedure, it is vital for the mucosal solution to maintenance a longlasting and high enough mucosal elevation. Although the two control groups formed a SFCs after injection, the protrusions disappeared quickly. In contrast, the PA-PSH in-situ gel remained a satisfactory mucosal lifting at all examined time points (Fig. 3). Besides, the submucosal elevation formed by the in-situ gel maintained shape in 9 days (Fig. 3b, Fig. 4c). There was no obvious tissue damage at the injection site. Moreover, the mucosal elevation created by the in-situ gel made the ESD procedure more simple. As the guidance of the longlasting mucosal elevation, an en bloc resection of the lesion could be operated quickly and successfully without bleeding and peforation, and only one single injection of the PA-PSH solution is needed (Fig. 4a,b). In addition, photographs of HE-stained tissue sections further demonstrated the safety of the in-situ gel without damage in muscularis propria (Fig. 4d,e). It is worth noting that, only healthy animals were used in this study. The e cacy of the PA-PSH in-situ gel in the ESD of the animals with gastric lesions might be examined in a future study.

Conclusions
In conclusion, the developed PA-PSH in-situ gel was a low viscous aqueous solution at RT and could quickly transform into a semi-gel under human normal physiological environment ( 37 °C, pH7.4). After submucosal injection into resected pig stomach or in living minipigs, it could provide a long-lasting and high submucosal cushion, assisting and guiding the operation of ESD procedures with no perforation and bleeding. Moreover, the in-situ gel solution had good injectability and good biocompatibility, which can be developed as an ideal submucosal injection solution.
Abbreviations ESD, endoscopic submucosal dissection; SFC, submucosal uid cushion; NS, saline solution; FDA, Food and Drug Administration; IR, infrared; GPC, gel permeation chromatography; Mw, molecular weights; THF, tetrahydrofuran; IT, insulation-tripped. Declarations SG and XW conceived and designed the project. ZT and YL participated in collecting the data, YG, CH and JC collected, analyzed and interpreted the data as well as drafted the manuscript. All authors read and approved the nal manuscript.

Funding
This study is supported by grants from National Natural Science Foundation of China [81972392,81672545].

Availability of data and materials
The data and materials used in the current study are available from the corresponding author upon reasonable request.
Ethics approval and consent to participate All experiments were performed according to guidelines established by the Second Military Medical University.

Consent for publication
Not applicable. Figure 1 Schematic diagram illustrating the application of PA-PSH in-situ gel as an SFC in ESD procedure.   (a) Change in mucosal elevation as a function of time after injection of indicated 6 samples into a resected porcine stomach. The methylene blue was mixed as the color agent; (b) endoscopic changes of mucosal elevation shapes as a function of time after injection of PA-PSH in-situ gel (PA-PSH-1, 17 wt% ; PA-PSH-2, 14 wt%) and control. The methylene blue was mixed as the color agent. Figure 4 (a,b) Photographs of ESD produce, the methylene blue was added as the color agent; (i) the mucosal elevation created with the PA-PSH in-situ gel (PA-PSH-1, 17% w/w; PA-PSH-2, 14% w/w); (ii,iii) circumferential mucosal cutting using an IT knife; (iv) a submucosal cavity formed via suction of the insitu gel underneath mucosa after completion of the circumferential mucosal cutting; (v) lesion resected by ESD; (c) an in vivo endoscopic observation 9 days after creation of a submucosal cushion with the PA-PSH in-situ gel (PA-PSH-1, 17% w/w; PA-PSH-2, 14% w/w) and control), the methylene blue was added as the color agent; (d,e) histological section made immediately after mucosal lifting produced with the insitu gel (d) and histological section made 9 days after mucosal lifting induced with the PA-PSH-1 (e).

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