Preparation of Polyethylene Glycol Monomethyl Ether Chitosan-diethylenetriamine Pentaacetic Acid and Its Effect on SrCl2 Excretion and Radiation Protection in the Digestive Tract of Rats

Feng Zeng Third Military Medical University Southwest Hospital Yao Xiao Third Military Medical University Southwest Hospital Weilin Fu Third Military Medical University Southwest Hospital Xiangyu Chen Third Military Medical University Southwest Hospital Hao Zeng Koppers Chemical Industry Corp. Ltd Yi Zhang Third Military Medical University Southwest Hospital Chuanchuan Liu Third Military Medical University Southwest Hospital Linghu Cai Third Military Medical University Southwest Hospital Minghua Liu (  mhliuswhcq@126.com ) Third Military Medical University Southwest Hospital https://orcid.org/0000-0002-4701-5547


Introduction
After the severe nuclear power plant accidents at Chernobyl on 26 April 1986, Fukushima in March, 2011, and atomic bombs at Hiroshima and Nagasaki, high levels (large amount) of diverse ssion products have been discharged into the environment [1][2][3]. The environment of nuclear exposure sites is highly complex, and the radionuclides may be ingested into the blood through intestinal mucosa leading to cumulative damage to the human body, causing serious diseases and death [4][5][6]3]. Importantly, the tissue and organ damage can be avoided by preventing the absorption of radionuclides through the digestive tract when nuclear accidents happened. Moreover, the broad-spectrum contaminative radionuclide chelating pharmaceuticals such as DTPA and EDTA have revealed side effects [7], and there are no xed dosages for the digestive tract. Hence, early nuclear decontamination of the digestive tract is not suitable during nuclear exposure.
Diethylenetriamine pentaacetic acid (DTPA) is a broad-spectrum chelating agent with certain side effects [7]. However, the dosage form of DTPA or its structure can be modi ed to reduce its side effects, improve chelating ability, reduce dosage, increase usage, and extend its treatment time [8,9]. Chitosan (CS) has nontoxicity, biodegradability, good biocompatibility, and can be absorbed by organisms [10][11][12]. Furthermore, some experiments have shown that chitosan has radioprotective effects [13]. Additionally, it may scavenge oxygen free radicals and exhibit anti-in ammatory properties [14].
Polyethylene glycol (PEG) is a type of linear, noncharged, nontoxic, nonantigenic, nonimmunogenic, biocompatible, highly soluble, and biodegradable polymer material, which can be dissolved in aqueous solution as well as in organic solvents [15]. PEG is a commonly used drug for clinical application in the gastrointestinal tract, and is approved by the FDA for use in food and medicine [16]. Polyethylene glycol monomethyl ether (mPEG), exhibiting both hydrophilicity and exibility, is used to increase the water solubility of macromolecules and in grafting polymers [17].
In the present study, mPEGCS-DTPA was synthesized from diethylenetriamine pentaacetic acid, chitosan, and polyethylene glycol by organic synthesis and chemical cross-linking, and an oral solution was prepared. We assessed the effect of mPEGCS-DTPA on the excretion and radioprotective effects of radionuclide 89 strontium in the digestive tract of rats.

Preparation of iodinated polyethylene glycol monomethyl ether (mPEGI)
In this experiment, 20 g (10 mmol) MPEG was added into a 2 250 mL three-neck ask, thereafter, 8 mL of triphenylphosphine (30 mmol) and 4.3 mL (30 mmol) of methyl iodide were added and reacted at 130 ℃ for 6 h under nitrogen protection. The solution was allowed to cool at 20~30℃, and then toluene was added. Next, the solution was stirred to dissolve the crude product, and then poured into anhydrous ether to obtain a white precipitate. The lter residue was washed thrice with anhydrous ether and the light yellow mPEGI solid powder was obtained by vacuum drying.

Preparation of phthalic anhydride chitosan
Here, 4.967 g (31 mmol-NH 2 ) chitosan, 13.797 g phthalic anhydride (93 mmol) and 200 mL Ndimethylformamide (DMF) were added into a 250 mL ask, protected by nitrogen, and reacted at 130 ℃ for 7 h. The reaction was cooled at 20~30℃, ltered, and the ltrate was poured into ice water to precipitate a dark-yellow solid, which was washed with ethanol and dried under vacuum to obtain darkyellow phthalic anhydride chitosan solid.

Preparation of mPEG chitosan (mPEGCS)
In general, 2.967 g AgNO 3 was dissolved in 10 mL water. Next, 1.032 g KOH was dissolved in 5 mL water, then dripped into the dissolved AgNO 3 , and the gray precipitate was obtained via ltration. Thereafter, 0.77 g (3.3 mmol) gray precipitate was added into a 250 mL ask, and 4.571 g (2 mmol) mPEGI, 0.582 g (2 mmol) phthaloyl chitosan, and 40 mL DMF were added and re uxed at 60 ℃ for 16 h. Later, 30 mL 85% hydrazine hydrate and 60 mL water were added, after which the temperature was increased to 90 ℃, and then reacted for 15 h. Furthermore, the solution was cooled, ltered, and then 50 mL double-deionized (DD) water was added. Next, the water and unreacted hydrazine hydrate were removed by rotary evaporation. The process was repeated thrice. The ltrate was decompressed and evaporated to remove the solvent to stickiness, and then transferred to a dialysis bag, dialyzed with DD water for 3 days, and freeze-dried; later, 2.3 g mPEGCS white solid was obtained.

Fourier transform infrared spectroscopy (FT-IR)
The appropriate amounts of samples were mixed with KBr powder at a mass ratio of 1:200, and then tested after ne pressing; the scanning range of approximately 4000-400 cm −1 (FT-IR800) was tested with mPEGCS-DTPA.

Nuclear magnetic hydrogen spectroscopy analysis ( 1 H NMR)
The superconducting nuclear magnetic resonance instrument was used to scan the nuclear magnetic hydrogen spectrum (BrukerAV-400). Chitosan, mPEGCS and mPEGCS-DTPA polymer was dissolved in D 2 O solution. Scanning condition: frequency, 400 MHz, range, approximately 0-14 ppm.

Cytotoxicity test of mPEGCS-DTPA
The cytotoxicity of mPEGCS-DTPA on cells was evaluated via CCK-8 assay, according to the manufacturer's instructions. Brie y, ICE-6 cells were cultured in 96-well plates at a density of 5 × 10 3 cells per well and treated with mPEDCS-DTPA at different concentrations (0, 0.01, 0.05, 0.1, and 0.5 mg/mL) for 2 days at 37°C. The absorbance was measured using a Thermo Scienti c microplate reader at 450 nm. . After a 7-days adaption period, the rats (180-220 g) with good health were selected for use.
Thereafter, they were randomly divided into three groups (n = 6 per group): NS ( 89 SrCl 2 + NS, 1 mL of normal saline was administered by oral gavage 30 min after 89 SrCl 2 ), DTPA (60 mg/kg of DTPA dissolved in 1 mL of normal saline was administered via oral gavage 30 min after 89 SrCl 2 ), and mPEGCS-DTPA (60 mg/kg of mPEGCS-DTPA dissolved in 1 mL of normal saline was administered via oral gavage 30 min after 89 SrCl 2 ). Rats in the experimental groups were placed in metabolic cages individually, thereafter, feces and urine were collected daily for future measurement. The venous blood (100 µL) of rats was collected by cutting their tail before and 2, 8, and 24 h after oral administration of 89 SrCl 2 ; the blood sample was then added to the known amount of nitric acid, and the β-radioactivity was detected. At 24 h after oral gavage of 89SrCl 2 , 1% pentobarbital sodium was administered to anesthetize the rats. The metacarpal bones were removed aseptically and stored in a refrigerator at 4 ℃ for follow-up and βradioactivity detection. At 48 h, all rats were sacri ced, and then left femur tissues were collected and stored at 4 ℃ for further radiochemical analysis,the small intestine tissues were collected and xed in 4% paraformaldehyde for 48 h for further evaluation. The metacarpal bones and femoral bones were ashed at 750 ℃ for 4 h, dissolved in HNO 3 , and neutralized to PH 4-6 with 1 mmol/L NaOH. Thereafter, the small intestinal tissue was xed with paraformaldehyde for 48 h, followed by routine para n embedding.
Sectioning and HE staining were used to observe the degree of histopathological injury, and the images of pathological sections were further captured. Feces were homogenized and subjected to the same procedure as for the bones. Urine was collected at the bottom of the metabolic cage with a known volume of HNO 3 . Radioactivity of these samples was measured in a scintillation cocktail (AQUASOL-2, NEW England Nuclear) by a liquid scintillation spectrometer (LKB, Racbeta 1219). The measurements were performed for 1 min with corrections for chemiluminescence and chemical quenching [18, 19].

Statistical analysis
All data are presented as the mean ± standard deviation (SD). Statistical signi cance was determined by one-way ANOVA followed by LSD post hoc analysis and repeated-measurement data were analyzed by repeated-measurement analysis of variance (pairwise comparison using the least signi cant difference test) (SPSS statistics 25, IBM Inc.). P-values <0.05 were considered statistically signi cant.

Preparation and characterization of mPEGCS-DTPA
To synthesize mPEGCS-DTPA, mPEG was initially grafted with chitosan 6-OH, thereafter, the hydrophilic compound mPEGCS was synthesized [20,21]. Then, mPEGCS-DTPA was constructed by a chemical bond between -NH 2 and -COOH under EDC/NHS condition. MPEGCS-DTPA showed good solubility in water, which is convenient for the follow-up preparation of the oral dosage form for the digestive tract [22,23].
The absorption peak of the carboxyl group -COOH appeared near 1639.38 cm −1 (Fig. 1B), that of secondary amide appeared at 1395.34 cm −1 (Fig. 1B), and that of the ether bond appeared at 1108.99 cm −1 (Fig. 1B). The results indicated that MPEG and DTPA were grafted on chitosan.
The 1 H NMR of chitosan, mPEGCS, mPEGCS and mPEGCS-DTPA are illustrated in Figure 1C. The single peaks near the PEGCS δ 2.5, 3.6, and 3.7-4.0 ppm are the proton peak on the mPEG side chain -OCH 3 , -OCH 2 CH 2 and the protons on the chitosan C (3-6) and chitosan C (1) acetal, respectively, indicating that the polyethylene glycol monomethyl ether branch is attached to the chitosan. Moreover, the proton peak of chitosan-NH 2 in mPEGCS around δ 2.6-3.0 ppm (Fig. 1C) disappeared in mPEGCS-DTPA. Thus, DTPA was successfully grafted onto mPEGCS.

3.3.
Effect of mPEGCS-DTPA on the excretion of radioactive strontium in rats 3.3.1 Radioactive level of strontium in rat bones after oral gavage of drugs The rats were administered by oral gavage of 89 SrCl 2 for 30 min. The β-radioactivity count in the metacarpal bone of the mPEGCS-DTPA group was lower than that of the NS group at 24 h ( Fig. 3A; P < 0.01). The β-radioactivity count in the femur measured at 48 h in the mPEGCS-DTPA group was lower than that in the NS group ( Fig. 3B; P < 0.01).

3.3.2
The β-radioactivity counting in blood of rats at different time after oral gavage of 89 SrCl 2 .
The β-radioactivity counting of each group was measured before, 2, 8, and 24 h after oral administration of 89 SrCl 2 (Fig. 4). The changing trend of blood radioactivity count in the DTPA group and mPEGCS-DTPA group was similar as that in the NS group, and β-radioactivity counting in rats indicates rapid absorption with a radioactive blood peak observed 2h after ingestion [24]. However, the radioactivity count in the mPEGCS-DTPA group and the DTPA group was lower than that in the NS group at 2 h (P < 0.05), that in the mPEGCS-DTPA group was lower than that in the NS group at 8 h (P < 0.01), and that in the DTPA group was lower than that in the NS group at 8 h (P < 0.05). No signi cant difference was observed in the blood radioactivity between mPEGCS-DTPA and DTPA groups at 2 and 8 h phase point.

Comparison of fecal excretion after oral gavage of drugs
Feces were collected daily before and after oral gavage of 89 SrCl 2 in rats for measuring the daily radioactivity count. Under normal feeding conditions, the total fecal radioactivity count of rats in each group was at the basal level, at 24h after oral gavage of 89 SrCl 2 (Fig. 5A), the β-radioactivity counting in mPEGCS-DTPA group was higher than that in the NS group on Day 1(P < 0.01), whereas that (Fig. 5B) in the DTPA group was higher than that in the NS group on Day 2 (P < 0.01).

Comparison of urine excretion after oral gavage of drugs
Urine was collected to measure the daily radioactivity count of urine before and after oral gavage of 89 SrCl 2 in rats. Under normal feeding conditions, the urinary radioactivity count of rats in each group was at the basal level, and the urine was collected daily after oral gavage of 89 SrCl 2 . The total urine radioactivity count of rats after oral gavage of 89 SrCl 2 at 24 h (Fig. 6A) in the mPEGCS-DTPA group was higher than that in the NS group (P < 0.05) and the DTPA group (P < 0.01) on Day 1, whereas the count in the mPEGCSDTPA group (Fig. 6B) was higher than that in the NS and DTPA groups (P < 0.001).

Pathological changes of small intestine injury induced by radiation in rats
The intestinal tissue structure was abnormal and there was pathological damage of the small intestine of rats, including defect of mucous membranes, loss of mucous layer, accumulation and in ltration of in ammatory cells, exfoliation and necrosis of villi, and loss of glands, in the NS and DTPA groups, there were less observed in the mPEGCS-DTPA group.

Discussion
The development of effective methods and drugs to mitigate the nuclides absorption through intestinal mucus is an important area when nuclear accidents or terrorism happens.
PEG is commonly used in hospitals when colonoscopy is needed, due to its cathartic effect [16,25]. DTPA is used as chelating agent, and chitosan has a protective effect on radiation [13]. mPEGCS-DTPA was synthesized by chemical synthesis and cross-linking; it is a hydrophilic compound and cannot pass through the phospholipid bilayer of the cell membrane. mPEGCS-DTPA chelates heavy metal ion Sr, and can be quickly excreted from the body through the gastrointestinal tract [16], which can effectively reduce the absorption of heavy metal ions into the blood through the intestinal tract and prevent serious secondary damage to the body [26].
In this study, rats were orally gavaged with 89 SrCl 2 (48.8 × 10 4 Bq/mL, 1 mL ). At 48 h after oral gavage of 89 SrCl 2 , the rats were sacri ced. The radioactivity count of the heart, liver, spleen, kidney and small intestine were close to the basal level, and a high amount of the radionuclide strontium was deposited in the bone tissue [4,27,5,6].
The newly synthesized mPEGCS-DTPA was co-cultured with rat ICE-6 cells. CCK8 assay revealed that mPEGCS-DTPA was compatible with rat ICE-6 cells at different concentrations [28]. The deposition of radionuclides in metacarpus and femur in mPEGCS-DTPA group was lower than that in NS group, indicating that mPEGCS-DTPA has certain chelating and promoting excretion ability. Previous studies have reported that the peak of strontium absorption into the blood through the digestive tract occurs at 60-120 min, which is consistent with our experimental results. The radioactivity count in blood at 24 h was close to the basal level, and we speculated that strontium was absorbed through the digestive tract mainly within 24 h after intake [5,29]. The count of blood radioactivity in the mPEGCS-DTPA group and DTPA group was lower than that in the NS group at 2 and 8 h, and the lowest was observed in the mPEGCS-DTPA group at 8 h, which may be related to the massive excretion from the digestive tract. To nd out the reasons for the differences in radioactivity count between different groups of bone tissue and blood, pathways of drug metabolism were also exposed. The results of fecal excretion revealed that the radioactive count of feces in the mPEGCS-DTPA group was higher than that in the other two groups on the rst day after ingestion of 89 SrCl 2 , and the total radioactive deposition in feces in the DTPA group was higher than that in the NS group on the second day after intake of strontium, which may be related to the slow excretion of DTPA chelating metal ions through the digestive tract [24,15,16]. However, no signi cant difference was observed in the chelation inhibition effect in the digestive tract compared with the traditional drug DTPA. Chitosan ingested through the digestive tract can be detected in the kidney in a short time [30,10,12]. In this study, the total radioactivity count in urine from the mPEGCS-DTPA group was signi cantly higher than that of the other two groups on the 1st and 2nd days after ingestion of 89 SrCl 2 . We speculated that few 89 SrCl 2 was absorbed into the blood and chelated by mPEGCS-DTPA, and which might be excreted through urine [31,30,32].
The small intestinal mucosal injury caused by 89 SrCl 2 represents radiation injury; in the mPEGCS-DTPA group, this injury was less severe than that in the other two groups as observed by HE staining after ingestion of 89 SrCl 2 for 48 h. Moreover, chitosan has a certain radiation-protective effect on α and β rays, which may cause ionizing radiation damage [13]. Combined with the previous research results of HE staining of rat small intestine, we speculate that the newly synthesized mPEGCS-DTPA has a certain radiation-protective effect on α and β rays, which may cause electric radiation damage to the digestive tract mucosa.
Thus, the results indicate that mPEGCS-DTPA exhibits good cytocompatibility, increases the excretion of the radionuclide strontium, and exerts radiation-protective effect against same in the digestive tract. Although the effect of the excretion of mPEGCS-DTPA still needs to be further explored and improved, it increases the oral dosage form of DTPA used in the digestive tract, has good ionizing radiation-protective effect, and can be used for clinical application.    Radioactivity count in blood at different time points after oral administration of 89SrCl2 in rats (*p < 0.05, and **p < 0.01).