Novel endoscopic tattooing dye based on polyvinylpyrrolidone-modified polydopamine nanoparticles for labeling gastrointestinal lesions

Yongkang Lai† ^ab, Mengni Jiang†^a, Xinyuan Zhang†^cd, Liang Zhang^a, Zheng Chen^d, Yiqi Du*^e, Shige Wang*^d, Jiulong Zhao*^a and Zhaoshen Li*^a
aDepartment of Gastroenterology, Shanghai Changhai Hospital, Naval Medical University, Shanghai 200433, China. E-mail: jlzhao9@163.com; zhaoshen-li@hotmail.com
^bDepartment of Gastroenterology, Ganzhou People's Hospital, Jiangxi Medical College, Nanchang University, Ganzhou 341000, China
^cSchool of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China
^dSchool of Materials and Chemistry, University of Shanghai for Science and Technology, Shanghai 200093, China. E-mail: sgwang@usst.edu.cn
^eDepartment of Gastroenterology, Shanghai Institute of Pancreatic Diseases, Changhai Hospital; National Key Laboratory of Immunity and Inflammation, Naval Medical University, Shanghai, China. E-mail: duyiqi@hotmail.com

First published on 16th August 2024

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Introduction

With the continuous progress and promotion of endoscopy technology, gastrointestinal endoscopy has become an important tool to diagnose and treat early lesions in the digestive tract.^1,2 Endoscopy can assist oncologists with scheduled follow-up observation of lesions in the digestive tract that cannot be surgically resected for the time being. Additionally, it can also assist surgeons in locating lesions that are difficult to detect by examination or palpation during surgery, such as small or flat tumors, diverticula, arteriovenous malformations, etc.^3–7 However, it is challenging to endoscopically locate and observe or laparoscopically treat lesions at later stages due to visceral movement or changes in the patient's position.^8–10 For example, for the localization of intestinal lesions, due to the high degree of intestinal freedom and stretching, the difference between the tumor site determined by enteroscopy and intraoperative visualization can reach 6.38–21.03%.^10–12 Inaccurate preoperative lesion localization not only increases the operation time and difficulty but also leads to the surgeon resecting the wrong bowel segment, causing irreparable damage to the patient.⁷ Thus, precise preoperative lesion localization has become an urgent need in current clinical treatment.

Endoscopic tattooing (ET) is a method of endoscopic (including ultrasonic endoscopy) marking of gastrointestinal lesions with dyes and can guide subsequent surgical resection of the lesions or regular review of the diagnosis and treatment of the lesions.⁷ It is one of the most commonly used methods for locating gastrointestinal lesions before laparoscopic surgery.¹³ Compared with endoscopic positioning using titanium clips, which is another commonly used method for preoperative lesion localization, ET is less likely to cause inaccurate positioning.¹⁴ Additionally, previous studies have shown that ET can effectively assist surgeons in rapidly and accurately locating lesions, reducing surgical trauma, and shortening surgical time. This benefit is particularly pronounced in early- and mid-stage gastrointestinal tumors where the plasma membrane surface remains intact.^14–18

Currently, the commonly used dyes for ET include India ink, indocyanine green (ICG), and a new sterile carbon compound (SPOT). India ink has a complex composition, and the solvents it contains, such as ethylene glycol, phenol, wormwood, and gelatin, are also prone to locally causing inflammation or allergic reactions.⁷ Furthermore, the complexity of using India ink, which requires strict dilution and sterilization before use, limits its use.¹⁹ ICG is an important dye that was originally used to quantify cardiac output and is safer than India ink. However, ICG provides less durable tattoos and cannot be used in patients who are allergic to iodine.²⁰ SPOT is currently the only FDA-approved drug for this indication, and many studies have demonstrated its safety and durability;^7,21 however, its high price has somewhat prevented its use in clinical patients. Hence, there is still a need for ET dye with cost effectiveness, biosafety, and a long durable tattoo.

Herein, we designed a new endoscopic tattooing dye based on polydopamine (PDA)/polyvinylpyrrolidone (PVP). PDA is a substance formed by the oxidative self-polymerization of dopamine under specific conditions. Due to its high adhesion and stabilizing properties, PDA is widely used in the surface coating of various materials.^22–24 Therefore, the use of PDA/PVP as the dye for endoscopic tattooing not only maintains a long-lasting localization effect but also high biosafety, which causes no adverse reactions in the human body. Meanwhile, PDA is inexpensive and can be produced in large quantities, which is expected to realize clinical conversion easily. This new dye PDA/PVP thus provides the first candidate option that we know of for endoscopic tattooing with cost effectiveness, biosafety, and long durability (Scheme 1).

Experimental

Materials

Dopamine hydrochloride (98%, for synthesis, 972664) was purchased from J&K Scientific (Beijing, China). PVP (C₆H₉NO)_n, (V900010) was obtained from Sigma-Aldrich (Shanghai, China). Sodium hydroxide (AR, 96%; granular, S111518) was purchased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Cell Counting Kit-8 (CCK-8) (CK04) and live/dead Cell Staining Kit (No. 40747ES76) were purchased from Yeasen Biotechnology (Shanghai, China) Co., Ltd. Ketamine, atropine, azaperone, isoproterenol, and isoflurane inhalation were obtained from Changhai Hospital.

Characterization

The hydrodynamic diameters and zeta potentials of the nanoparticles were measured using a ZetaSizer Nano-ZS 90 (Malvern Instrument, Malvern, UK). X-ray diffraction (XRD) was performed using a Bruker D2 Phaser (Germany) to observe the crystal structure of the samples. The morphological characteristics and elemental mapping images of the nanoparticles were observed using transmission electron microscopy (TEM) (FEI Talos F200X G2, USA, 200 kV). Scanning electron microscopy (SEM) images were taken with a field emission scanning electron microscope (ZEISS Sigma 300, Germany).

Synthesis and characterization of PDA/PVP

Dopamine (90 mg) was dissolved in 40 mL of deionized water, NaOH (1 M, 0.38 mL) was added, and the solution was put into an ice water bath for 4 h. After centrifuging (21000 rpm, 10 min) and washing with PBS 3 times, the supernatant was removed and dispersed again into 5 mL of deionized water. Then, 0.1 g of PVP was added, and the mixture was stirred at room temperature overnight. The prepared PDA/PVP solution was kept at 4 °C for further experiments.

Total antioxidant capacity of the PDA/PVP nanoparticles

The total antioxidant capacity of PDA/PVP was assessed using the ABTS method. In brief, the deep green ABTS solution was generated by reacting ABTS stock solution (7.4 mM) with potassium persulfate (K₂S₂O₈) solution (2.6 mM) in the dark for 12 hours, followed by dilution with anhydrous ethanol. Subsequently, 100 μL of PDA/PVP solution (0, 10, 15, 20, 25, and 30 μg mL⁻¹, in DI water) was mixed with 1 mL of ABTS solution and incubated in the dark at 37 °C for 6 minutes. Finally, the absorbance was monitored using ultraviolet-visible-near-infrared (UV-Vis-NIR) spectroscopy (Shimadzu, Japan) at wavelengths ranging from 400 to 900 nm. The total antioxidant capacity was determined using the equation below:

where A_b is the absorbance of the blank (ABTS + ethanol), and A_n is the absorbance of the experimental group (ABTS + ethanol + PVP/PDA).

Reactive nitrogen species (RNS) and reactive oxygen species (ROS) scavenging abilities

To study the RNS and ROS scavenging abilities, PDA/PVP nitrogen-centered free radicals (DPPH) and oxygen-centered free radicals (PTIO) were used. Briefly, at 37 °C, different concentrations of PDA/PVP (0, 10, 15, 20, 25, and 30 μg mL⁻¹, in DI water) were added to 1 mL of DPPH working solution. After incubating the mixture in the dark for 25 minutes, the absorbance of the supernatant was analyzed using UV-Vis-NIR spectroscopy between 400 and 800 nm. Similarly, different concentrations of PDA/PVP (0, 10, 15, 20, 25, and 30 μg mL⁻¹, in DI water) were co-incubated with PTIO solution in the dark for 30 minutes, and the absorbance of the supernatant was measured using UV-Vis-NIR spectroscopy at 400–800 nm. The antioxidant capacity was determined using the equation below:

where A_b is the absorbance of the blank (DPPH/PTIO + ethanol), and A_n is the absorbance of the experimental group (DPPH/PTIO + ethanol + PVP/PDA).

Cell culture

Mouse fibroblasts (L929, passage 10) and RAW264.7 (passage 10) cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Beijing, China). For cell culture, the cells were cultured at 37 °C in a 5% carbon dioxide (CO₂) atmosphere in DMEM (Gibco, CA, USA) supplemented with 10% (v/v) FBS and 1% penicillin and streptomycin (Gibco, Grand Island, USA). To ensure the reliability of our results, this study exclusively utilized cells from the first 10 passages.

Cytotoxicity assay

L929 cells were seeded in a 96-well plate at a density of 1 × 10⁴ cells per well. After overnight cell attachment, the medium was changed to medium containing different concentrations of PDA/PVP, and the cells were incubated for 24 h. Then, the live/dead cell staining assay was performed by replacing the medium with calcein-AM and PI dyes after co-culturing PDA/PVP with cells. After incubation for 30 min at 37 °C, the cells were observed under a fluorescence inverted microscope (Leica, DMIL LED, Germany). Cell viability was assessed using the CCK-8 assay. Briefly, 100 μL of 10% CCK-8 dye solution per well was used to replace the medium, and the water-soluble methotrexate dye was measured at 450 nm by incubation at 37 °C for 2 h using a microplate reader (Molecular Devices SpectraMax® i3, USA). Three replicate wells were used for each group, and cell viability was calculated according to the following formula:

Hemolysis evaluation

Fresh blood was collected from the rats and centrifuged (5000 rpm, 5 min) to remove the supernatant. Then, 2 mL of collected blood was diluted to 50 mL with saline and set aside at 4 °C. Three hundred microliters of diluted blood was taken containing different concentrations of PDA/PVP (0, 500, 1000, 1500, and 2000 ppm). Then, 300 μL of erythrocytes was added to 1.2 mL of saline and deionized water to serve as negative and positive controls, respectively. The mixtures were incubated at 37 °C for 2 h, and the absorbance of the supernatant was measured at 540 nm using a microplate reader (Molecular Devices SpectraMax® i3, USA). The hemolysis ratio was calculated as follows:

Intracellular free radical-scavenging capacity of PDA/PVP

Initially, RAW264.7 cells were seeded at a density of 5 × 10⁵ cells per well in a 6-well culture plate. After overnight adherence, fresh culture medium containing different concentrations of PDA/PVP was added, and H₂O₂ (1 mM) was used for stimulation for 6 hours. Finally, the ROS scavenging capacity of PDA/PVP in RAW264.7 cells was assessed using a flow cytometer.

Flow cytometry

Following the stimulation of RAW264.7 cells, the cells were incubated with DCFH-DA (Beyotime Biotechnology, China) for 20 minutes. Subsequently, the cells were washed three times with PBS by centrifugation. Flow cytometry analysis was performed using a flow cytometer (ACEA NovoCyte). The excitation wavelength was set at 488 nm, and the emission wavelength was set at 525 nm.

Animal studies

All animal procedures in this study were approved by the Ethics Committee of the First Affiliated Hospital of Naval Medical University (Shanghai, China, Approval Number: SYXK(A.E)2020-0033). All animal experiments were conducted in accordance with protocols approved by the Laboratory Animal Center of Changhai Hospital, Naval Medical University and the policies of the Ministry of Health. Kunming mice were purchased from the Shanghai Laboratory Animal Center and kept at the Institute of Pancreatic Diseases, Shanghai Changhai Hospital. The mice were housed in a room with a temperature of 18–22 °C, a 12-hour light–dark cycle, and a relative humidity of approximately 55%.

In vivo biocompatibility

Fifteen Kunming mice were randomly divided into 5 groups of three mice each; one group was not subjected to any treatment, and the remaining four groups were given subcutaneous injections of 300 μL of different concentrations of PDA/PVP (500, 1000, 1500, and 2000 ppm) once each. Then, they were kept normally for 2 weeks. After that, the heart, liver, spleen, lungs, and kidneys of the mice were collected for H&E staining. Blood was collected from the mouse eyes for routine blood tests, and serum was obtained for liver and kidney function tests. Blood counts were measured by a Myriad Veterinary Automatic Blood Cell Analyzer (BC-2800vet, China). Liver and kidney function were measured by an automatic dry biochemistry analyzer (Celercare M5, China).

In vivo tattoo

Three Bama pigs (male, 25–30 kg) were purchased from Laiboorganism Biotechnology Co., Ltd (Jiangxi, China) and raised at the animal experimental center of Naval Medical University. All pigs underwent endoscopic tattooing with PDA/PVP under anesthesia in the esophagus, stomach and rectum. Briefly, ketamine (25 mg kg⁻¹), atropine (0.04 mg kg⁻¹), and azaperone (4 mg kg⁻¹) were given intramuscularly before anesthesia. Then, anesthesia was induced by isoproterenol (1.66 mg kg⁻¹), which was maintained via inhalation of 1–2.5% isoflurane. After general anesthesia, the pigs were kept in the lateral position. Then, 0.5 mL of saline was injected into the submucosa using an endoscope (GIF-Q260J; Olympus Co., Ltd, Tokyo, Japan)-affiliated needle at a 45° angle into the submucosa at the mid-esophagus, the greater curvature of the stomach, and the rectum 3 cm from the anus of the three pigs to elevate the mucosa. Subsequently, 0.1 mL of PDA/PVP solution was injected before the injection needle was withdrawn. Endoscopy was performed again on days 15 and 60 after endoscopic tattooing to observe the tattooing status. Pigs were sacrificed after the last endoscopic test, and the esophagus, stomach, and rectum markers were evaluated via H&E staining. Additionally, the heart, liver, spleen, lungs, and kidneys of the pigs were collected for H&E staining to evaluate the in vivo safety.

In vivo wound healing evaluation

Circular full-thickness skin lesions with a diameter of approximately 6 mm were created on the backs of eighteen Kunming mice. Subsequently, the mice were randomly divided into three groups (n = 6) and the wound were coated with PBS (control), PVP, and PDA/PVP, respectively, and fixed with gauze. Images of the wounds were captured with a digital camera on days 0, 3, 7, 10, and 14 to monitor the wound healing process. The formula for calculating the wound healing rate was as follows: wound closure rate = (wound area on day 0 − wound area on a specific day)/wound area on day 0. On days 7 and 14, three mice from each group were euthanized, and histological investigations were conducted using H&E and Masson staining following the manufacturer's instructions.

Statistical analysis

All the data are expressed as the mean ± standard deviation. All images were drawn with Origin 2021 and GraphPad Prism 9.5.

Results and discussion

Characterization of PDA/PVP

In this study, the novel PVP-modified PDA nanoparticle was prepared by combining PVP with PDA and was developed as a durable and stable endoscopic tattooing stain for endoscopic tattooing. PDA is the main pigment of natural melanin.²⁴ PDA is readily deposited on almost all types of inorganic and organic matrices.^24–26 PVP can enhance the biocompatibility and colloidal stability of PDA under physiological conditions. In this study, we utilized the adhesion properties of PDA, and combined it with PVP to enhance its stability, thereby creating an endoscopic tattooing dye.

TEM observation revealed that the PDA/PVP nanoparticles were spherical (Fig. 1A). The result of XRD showed that these nanoparticles had broad characteristic peaks at 2θ = 16.9°, 26.9°, and 36.3°, confirming its non-crystalline nature (Fig. 1B). SEM also showed that the PDA/PVP nanoparticles exhibited a regular spherical structure (Fig. 1C). The above results indicate that the PDA/PVP particles prepared in this study are spherical nanoparticles. The zeta potential values of PDA and PDA/PVP were −12.5 ± 0.37 mV and −35 ± 0.53 mV, respectively (Fig. 1D). PDA/PVP particles with such a high negative zeta potential cannot be easily taken up or removed by cells and thus remain well stabilized in the tissue mucosa. Next, we explored the stability of PDA/PVP by detecting its particle size in different solutions at different times. The PDI (polydispersity index) of PDA/PVP nanoparticles in DMEM on day 0, day 1, day 3, and day 7 were 0.161, 0.177, 0.186, and 0.194, respectively. In simulated body fluid, the PDI values on day 0, day 1, day 3, and day 7 were 0.132, 0.144, 0.153, and 0.159. In saline, the PDI values on day 0, day 1, day 3, and day 7 were 0.156, 0.157, 0.177, and 0.179. In 5% glucose solution, the PDI values on day 0, day 1, day 3, and day 7 were 0.154, 0.162, 0.168, and 0.174. As shown in Fig. 2A, the PDA/PVP nanoparticles exhibited good stability in DMEM. Similarly, we also explored the nanoparticle size variation of PDA/PVP in simulated body fluid (Fig. 2B), saline (Fig. 2C) and 5% glucose solution (Fig. 2D), and the results indicated that PDA/PVP nanoparticles exhibited good stability in different solutions. Besides, the results showed that after culture in the above media, the PDA/PVP nanoparticles exhibited a significant Tyndall effect. This indicates that PDA/PVP can be uniformly dispersed in the studied solutions, which further proves the stability of PDA/PVP. The above results indicate that PDA/PVP with stable performance was successfully synthesized. The PDA/PVP solution (dye solution) was black and stable for a long time in different solutions.

In vitro free radical scavenging activity

Oxidative stress is an imbalance between the oxidant and antioxidant systems of cells and tissues, resulting from the excessive generation of free radicals.²⁷ The excessive accumulation of free radicals can lead to cellular dysfunction and is closely associated with the development of inflammatory diseases and wound injuries.^28,29 Therefore, effectively clearing free radicals at sites of inflammation or injury can prevent further progression of inflammation and promote wound healing. The phenolic hydroxyl groups of PDA can undergo redox reactions with ROS, converting ROS into more stable molecules, such as water and oxygen.³⁰ Thus, we hypothesize that PDA/PVP has good free radical scavenging ability.

We first evaluated the total antioxidant capacity of PDA/PVP using the ABTS assay. As shown in Fig. 3A, with increasing concentrations of PVP/PDA, the clearance efficiency of ABTS gradually improved. By monitoring the characteristic absorption peak of PDA/PVP at 752 nm in the presence of ABTS we observed a gradual decrease in the characteristic absorption peak of ABTS with increasing PDA/PVP concentration. Optical images also showed that, compared to that of the control group, the solution color gradually faded with increasing PDA/PVP concentration. These results confirm the excellent antioxidant capacity of PVP/PDA.

Subsequently, we used the DPPH assay (Fig. 3B) and PTIO assay (Fig. 3C) to separately evaluate the ability of PDA/PVP to clear RNS and ROS. Like total antioxidant capacity study results, with increasing concentrations of PVP/PDA, the characteristic absorption peaks of DPPH at 517 nm and PTIO at 557 nm gradually decreased. Optical images also showed gradual fading of the solution color, indicating the good clearance ability of PDA/PVP for RNS and ROS. Quantitative analysis revealed that when the concentration of PDA/PVP reached 30 μg mL⁻¹, the PDA/PVP ABTS, DPPH, and PTIO removal rates were 34%, 51%, and 41%, respectively (Fig. 3D). In summary, PDA/PVP has excellent free radical scavenging ability at low concentrations, suggesting its potential anti-inflammatory and wound healing capabilities.

In vitro biocompatibility and the ability to protect cells from ROS-induced damage

Good biosafety and hemocompatibility are key to helping nanomaterials transition from bench to bedside.³¹ The viability of the L929 cells was assessed by live/dead cell staining and CCK-8 assays after 24 h of incubation with different concentrations of PDA/PVP. The L929 cells observed under inverted phase contrast microscopy were mainly live cells (green fluorescence, Fig. 4A) after treatment with different concentrations of PDA/PVP, which indicated that PDA/PVP had a good cellular safety profile. Furthermore, the CCK-8 assay also showed that L929 cell activity remained above 90% after cultured with PDA/PVP at a concentration ranging from 500–2000 ppm (Fig. 4B). In addition, we evaluated the hemocompatibility of PDA/PVP, which is another important indicator of nanomaterial biocompatibility. As shown in Fig. 4C, the hemolysis rates were all lower than 5% when the PDA/PVP concentrations were 500, 1000, 1500, and 2000 ppm, indicating that PDA/PVP has good hemocompatibility.

Previously, we demonstrated via in vitro experiments that low concentrations of PDA/PVP nanoparticles can clear ROS and RNS. To investigate whether PDA/PVP has a protective effect against free radical-induced cellular damage, ROS, as a representative, was used to treat RAW264.7 cells to establish a cellular inflammation model. After H₂O₂ treatment, the cells were stained with the ROS-sensitive fluorescent dye 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and flow cytometry was used to measure the intracellular ROS levels (Fig. 4D and E). The results indicate that free PVP basically had no scavenging effect on intracellular ROS in RAW264.7 cells, while PDA/PVP achieves a clearance rate of 28.54%, suggesting that PDA/PVP possesses ROS clearance capabilities, thereby protecting the cells.

In vivo biocompatibility

In vivo biocompatibility is also an important test for assessing whether nanoparticles can be safely applied for clinical translation.³² Kunming mice were injected with different concentrations of PDA/PVP nanoparticles subcutaneously and then kept them normally. After two weeks, the heart, liver, spleen, lungs, and kidneys of Kunming mice were removed for H&E staining. As shown in Fig. 5A, compared with those in the control group, none of the different concentrations of PDA/PVP had any effect on the vital organs of the mice. In addition, we collected serum from the mice for routine blood tests as well as liver and kidney function tests. As shown in Fig. 5B–E, the routine blood test results revealed no significant changes in the percentage of red blood cells, white blood cells, platelets, or lymphocytes in the mice after subcutaneous injection of PDA/PVP nanoparticles. Fig. 5F–I showed the liver and kidney functions of the mice, which were also not affected by the injection of different concentrations of PDA/PVP into the skin compared to those of the control group. The above results indicated that PDA/PVP nanoparticles have good in vivo biosafety.

In vivo wound healing in a mouse model

Endoscopic tattooing dyes can lead to unavoidable damage and adverse reactions at the tattoo sites.⁷ Therefore, an endoscopic tattooing dye with ROS-scavenging and wound healing-promoting effects will significantly reduce local inflammation and adverse reactions. In vitro experiments have shown that PDA/PVP possesses excellent ROS and RNS clearance capabilities; hence, we speculate that PDA/PVP also has a healing-promoting function on wounds. As shown in Fig. 6A, the wound area on the backs of the mice decreased with increasing postoperative time. Compared to the control group, the wound healing rate of the PDA/PVP group was faster. The wound healing rate of the PVP group was not significantly different from that of the control group, indicating that PDA plays a primary role in promoting wound healing, consistent with previous findings.^33,34 The wounds in the PDA/PVP group were closed after 14 days, while those in the control group did not fully heal. Quantitative analysis of the wounds revealed a healing rate of 99% ± 1.12% in the PDA/PVP treatment group, which was significantly different from that in the control group (89% ± 1%) (p < 0.05) (Fig. 6B).

Then, H&E and Masson staining were performed on the wounds to determine the extent of granulation tissue, epidermal thickness, and blood vessel quantity while describing the regenerated skin layers. As shown in Fig. 6C, wound length was assessed on the 7th and 14th days. The wounds in the PDA/PVP group were shorter than those in the control group, while the wounds in the PVP group were not significantly different from those in the control group. These results indicate that wounds treated with PDA/PVP healed well, with a faster healing rate and collagen synthesis.

In vivo tattoo duration in the porcine model

To further verify its practical effect as an endoscopic tattooing dye for marking, we performed endoscopic tattooing tests using PDA/PVP in the esophagus, gastric body, and rectum of pigs. Moreover, we conducted follow-up endoscopy observations of the labeled sites on the 15th and 60th days after tattooing. As shown in Fig. 7A, after injecting PDA/PVP into the submucosa of the digestive tract via endoscopy, the injection site was stained black (red arrow). After 15 days of tattooing, obvious labeling could still be observed through endoscopy. After 60 days of tattooing, the black color of the tattoo markings slightly faded; however, tattooing was still detectable through the endoscope, demonstrating the stability of PDA/PVP as a tattooing dye. Notably, no microscopic inflammatory changes in the mucosa were found during routine follow-up endoscopy, suggesting that the use of PDA/PVP dyes causes no major adverse reactions in the porcine digestive tract.

At the end of the 60 days of endoscopic follow-up observation, the pigs were sacrificed, and the endoscopic labeled sites were removed for H&E staining. As shown in Fig. 7B, black tattooing was deposited under the mucosa of the esophagus, stomach, and rectum, which demonstrated that PDA/PVP still existed under the mucosa of the labeled sites 60 days after staining. Furthermore, H&E staining was performed on the main organs of the pigs to observe the damage caused by PDA/PVP. As shown in Fig. 7C, after circulating in pigs for 60 days, PDA/PVP did not significantly damage major organs (heart, liver, spleen, lungs, or kidneys). The above experimental results indicated that PDA/PVP has good stability and can remain stable under the mucous membrane of the digestive tract for up to 60 days without causing adverse reactions in pigs. Therefore, PDA/PVP has good biological safety as an endoscopic tattooing dye.

Conclusion

With the increased detection of early-stage cancer and other lesions in the digestive tract, the use of minimally invasive laparoscopic or gastrointestinal endoscopic treatments is becoming increasingly widespread. As a result, the need for precise preoperative localization of lesions is increasing. Endoscopic tattooing is an effective method for determining the exact location of small lesions in the gastrointestinal tract. However, the current dye commonly used for endoscopic tattooing has a short duration of tattooing, a high cost, and many adverse reactions. In this study, we developed a novel endoscopic tattoo dye by combining PDA and PVP. In vitro tests revealed that PDA/PVP has good stability and free radical-scavenging abilities. Biological tests demonstrated that PDA/PVP has good biosafety both in vitro and in vivo. In a porcine model, positive tattooing effects lasted for at least 60 days in endoscopic tattooing tests in the esophagus, gastric body, and rectum (3 cm from the anus) of pigs. Additionally, our research results indicate that PDA/PVP nanoparticles can promote wound healing and accelerate the process of wound closure. Overall, PDA/PVP is anticipated to be a novel endoscopic tattooing dye with high safety, long-lasting tattoo effects, and low cost.

Abbreviations

PDA	Prepared polydopamine
PVP	Polyvinylpyrrolidone
ROS	Reactive oxygen species
RNS	Reactive nitrogen species
ET	Endoscopic tattooing
ICG	Indocyanine green
SPOT	Sterile carbon compound
CCK-8	Cell counting kit-8
XRD	X-ray diffraction
TEM	Transmission electron microscopy
SEM	Scanning electron microscopy
CO2	Carbon dioxide

Author contributions

Yongkang Lai collected the data, analyzed the relevant information, and drafted the manuscript; Mengni Jiang and Xinyuan Zhang designed the study and collected the data; Liang Zhang and Zheng Chen analyzed the data. Yiqi Du, Shige Wang, Jiulong Zhao, and Zhaoshen Li designed the article and approved the final submission.

Data availability

Data for this article are available at [original database of endoscopic tattooing dye] [dataset] at [Dryad. https://doi.org/10.5061/dryad.hqbzkh1r9].

Conflicts of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Acknowledgements

We would like to thank Home for Researchers for their help with the manuscript writing. We also thank the support of the National Natural Science Foundation of China (82073386).

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Footnote

† Yongkang Lai, Mengni Jiang and Xinyuan Zhang contributed equally to this paper.

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