Experimental anti-tumor effect of emodin in suspension – in situ hydrogels formed with self-assembling peptide

Abstract Lung cancer is a major cause of cancer-related deaths worldwide. Stimulus-sensitive hydrogels, which can be formed by responding to stimuli in the cancer microenvironment, have been widely studied as controlled-release carriers for hydrophobic anticancer drugs. In this study, self-assembling peptide RADA16-I was used to encapsulate the hydrophobic drug emodin (EM) under magnetic stirring to form a colloidal suspension, and the colloidal suspension (RADA16-I-EM) was introduced into environments with physiological pH/ionic strength to form hydrogels in situ. The results showed that RADA16-I had good cell compatibility and the RADA16-I-EM in situ hydrogels can obviously reduce the toxicity of EM to normal cells. In addition, compared with free EM (in water suspensions without peptide) at equivalent concentrations, RADA16-I-EM in situ hydrogels significantly reduced the survival fraction of LLC lung cancer cells, while increased the uptake of EM by the cells, and it also induced apoptosis and cell cycle arrest in the G2/M phase more significantly and reduced the migration, invasion, and clone abilities of the cells in vitro. The RADA16-I-EM in situ hydrogels also showed better cancer growth inhibition effects in cancer models (mice bearing LLC cells xenograft cancer), which induced cell apoptosis in the cancer tissue and reduced the toxic side effects of EM on normal tissues and organs in vivo compared with the free EM. It was revealed that RADA16-I can be exploited as a promising carrier for hydrophobic anticancer drugs and has the potential to improve the administration of anticancer drugs to treat cancer effectively with enhanced chemotherapy.


Introduction
Recently, cancer has become one of the major malignant diseases causing human death, and its morbidity and mortality are increasing. Possible treatment strategies include chemotherapy, surgery, radiotherapy, immunotherapy, and gene therapy Wei et al., 2017;Bykov et al., 2018). However, due to surgical resection of cancers and recurrence, the nonspecific accumulation of radiation or chemotherapeutic drugs in healthy tissues and cells limited their applications in clinical treatment for the lack of targeting, unfavorable side effects, and multidrug (Xiong et al., 2015;Ostrom et al., 2017;Mahvi et al., 2018). At the same time, due to the poor water solubility, rapid degradation in the liver and rapid excretion in the kidneys, most anticancer drugs currently used cannot successfully accumulate at targeting cancer sites, resulting in low efficacy. Except the above, the toxic and side effects caused by addition of excipients or solvents also limit the clinical use of many antitumor drugs (Almeida et al., 2014;Khaliq et al., 2017;Cullen et al., 2020). To overcome these limitations, localized drug delivery systems, such as hydrogels (Pan et al., 2018), micelles (Debele et al., 2017;Xiang et al., 2018), liposomes (Park, 2018;Zhu et al., 2020), nanoparticles (Lin et al., 2017), and electrospun fibers (Ding et al., 2019;Feng et al., 2019) are recently investigated to extend circulation time of the cancer drug in body and release the drug slowly, while reducing the toxicity of the drug to normal tissues and enhancing the therapeutic efficacy.
Emodin (EM) is a natural anthraquinone derivative extracted from the rhizome of rhubarb, polygonum cuspidatum, polygonum multiflorum, and other traditional Chinese medicines (shown in Figure 1(A)). Many studies have shown that EM has anti-inflammatory, bacteriostatic, and immunomodulatory effects (Lee et al., 2010;Lu et al., 2011;Alisi et al., 2012). With the further study of the anticancer activity of EM, the researchers also found that EM can treat lung cancer, breast cancer and other cancers by inhibiting cancer cell proliferation, migration, and promoting its apoptosis (Fu et al., 2007;Li et al., 2013). As EM is practically insoluble in water, easily precipitating to form crystals in water solution, and its characteristic of sudden release in a short time, together with its large side effects, EM has limited clinical use. Even in in vivo and in vitro experiments, it is inevitable to use some organic solvents such as dimethyl sulfoxide (DMSO), ethanol, or methanol before they can be used. The use of solvents, such as DMSO, which is toxic to the body, limited the administration concentration of EM (Park et al., 2019;Tu et al., 2019). Therefore, EM's water solubility is crucial to treat cancer, it is necessary to develop a suitable drug delivery system to improve the delivery of EM and other hydrophobic drugs, to improve the efficacy while reducing the side effects of these drugs.
Injectable hydrogels have attracted much interest due to fewer side effects, easy administration, local treatment, and (C) a scheme of RADA16-I self-assembly through hydrophobic interaction and ionic-complementarity; (D) a proposed model of RADA16-I self-assembly to form nanofiber structures (Liu et al., 2011) and schematic representation of the interaction between hydrophobic drugs and RADA16-I. Oxygen atoms are red, nitrogen atoms are blue, carbon atoms are white, and hydrogen atoms are gray. In this conformation, all of the hydrophobic alanine side chains are aligned on one side, and all of the arginine and aspartic acid side chains are aligned on different sides to produce two different faces: the hydrophobic and the hydrophilic. On the polar side, arginine alternates with aspartic acid. The dimensions are about 5 nm in length, 1.3 nm in height, and 0.8 nm in width. controllable drug release. Some studies have reviewed its application in some fields, such as tissue engineering and antibacterial therapy (Li et al., 2018;Zhang et al., 2019;Qiu et al., 2020). Especially, 'smart' hydrogels demonstrate a sol-gel transition responding to external stimuli, such as pH, light, and temperature (Hu et al., 2017;Milcovich et al., 2017;Wei et al., 2020). It has injectable solution state before administration, and it can be transformed into gel state immediately under the stimulation of external conditions (Chu et al., 2013;Morsi et al., 2016;Norouzi et al., 2016). Injectable hydrogels have certain advantages. First, it is usually administered by intra-tumoral and peri-tumoral injection, which helps to increase the concentration of the drug at cancer sites and reduce systemic toxicity. Second, it is simple to make its formulation, which can be prepared without crosslinking agents, organic solvents, and chemical synthesis (Yin et al., 2021). Moreover, it can deliver various therapeutic agents for different models of cancer therapy, such as chemotherapy (He et al., 2015), photo-thermal therapy (Zeng et al., 2019), gene therapy (Saravanan et al., 2019), etc. Ion complementary self-assembling peptides have attracted more and more researchers' attention as drug carrier materials. As they are synthesized from natural amino acids, they have good biosafety (Eskandari et al., 2017;Qiu et al., 2018;Cong et al., 2020;Tarvirdipour et al., 2020). RADA16-I is the most typical self-assembling peptide in the ion complementary self-assembling peptide family. The hydrophilic amino acids in the structure of RADA16-I include positively charged arginine (Arg, R) and negatively charged aspartic acid (Asp, D). The hydrophobic amino acid is nonpolar amino acid alanine (Ala, A) (shown in Figure 1(B)). RADA16-I can be self-assembled into a secondary structure with b-sheets by ion complementarity and hydrophobic interaction in aqueous solution, in which all hydrophobic and hydrophilic amino acids are arranged on different sides, and finally further assembled to form nanofibers (Liu et al., 2011;Shamsi, 2016;Taghavi et al., 2018;Wu et al., 2018) (shown in Figure 1(C)). The hydrophobic regions of its highlevel structure can encapsulate the hydrophobic drugs, while the hydrophilic regions can keep the system stable in aqueous solution (Figure 1(D).
Previous studies have shown that RADA16-I can load hydrophobic anticancer drugs, such as paclitaxel (Liu et al., 2011), 5-fluorouracil (Ashwanikumar et al., 2016), and mangiferin . It can improve the solubility and stability of anticancer drugs in aqueous solution, so to effectively improve the delivery and release of anticancer drugs. Therefore, based on the previous studies on the construction and sustained and controlled release of RADA16-I-EM suspension-in situ hydrogels drug delivery system (Wei et al., , 2021, this study mainly focused on the effects of RADA16-I-emodin hydrogels on lung cancer through the cell experiments in vitro and cancer local drug delivery in vivo of the constructed RADA16-I-EM in situ hydrogels delivery system (Figure 2), and the feasibility of RADA16-I as a hydrophobic drug carrier material was further evaluated. Figure 2. Illustration to develop pH/ion responsive RADA16-I hydrogels loaded with an anti-cancer drug -EM for cancer treatment. The RADA16-I-EM suspension was injected into a cancer bearing mouse model through intra-tumoral injection, while the suspension immediately formed hydrogels in situ under the stimulation of the microenvironment in the cancer. Sustained release of EM from the hydrogels inside or beside the cancer exerts inhibitory effects on the cancer cells.

Preparation of RADA16-I solution and RADA16-I-EM colloidal suspension
Took appropriate amount of RADA16-I powder, added sterilized ddH 2 O, and ultrasonically made it completely dissolve to obtain RDA16-I solution. The final concentration of RADA16-I used in the experiment was 5 mg/mL; took appropriate amount of EM powder and added it to 5 mg/mL RADA16-I solution, magnetically stirred for 48 h to obtain RADA16-I-EM colloidal suspension.

Cell lines and cell culture
LLC mouse lung cancer cell and 4T1 mouse breast cancer cell were purchased from Shanghai Fuheng Biotechnology Co., Ltd. (Shanghai, China). MLE12 normal mouse lung cells and HC11 normal mouse breast cells were purchased from Nanjing Saihongrui Biotechnology Co., Ltd. (Nanjing, China). These cells were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA), 100 U/mL penicillin, and 100 lg/mL streptomycin at 37 C in a humidified atmosphere with 5% CO 2 .

MTT assay
LLC, 4T1, MLE12, and HC11 cells in logarithmic growth phase were collected by trypsinization, and the complete culture medium was re-suspended into single cell suspension. The cells were inoculated in 96-well plate (4000 cells per well) and incubated overnight. Then incubated with RADA16-I solution (0.5, 1.0, 2.0, 4.0, and 6.0 mg/mL) for 24 h, 48 h, and 72 h to evaluate the cell cytotoxicity. In the cell proliferation inhibition experiment, the cells were treated by free EM (EM water suspension (EMS), at the concentrations of EM as 60, 80, 100, 120, 140, 160, 180, and 200 lM) and RADA16-I-EM in situ hydrogels (in which the concentration of RADA16-I was 5.0 mg/mL, and concentration of EM were 60, 80, 100, 120, 140, 160, 180, and 200 lM) and cultured for 24 h and 72 h. Ten microliters MTT (5 mg/mL, Sigma, St. Louis, MO) was added into each well. After incubation at 37 C for 4 h, the supernatant was removed and DMSO (Sigma, St. Louis, MO) was added into each well. A microplate reader (Bio-Rad Laboratories, Hercules, CA) was used to evaluate the cells viability at a wavelength of 490 nm.
where A1 refers to the absorbance of cells treated with drugs at 490 nm; A2 stands for the absorbance of cells in nontreated group and A0 is the absorbance of cells in noncell group.

Colony formation assay
LLC cells were incubated with free EM and RADA16-I-EM in situ hydrogels (the final concentration of EM was 120, 160, and 200 lM) for 24 hours. The cells were collected by trypsinization. The drug treated and control cells were placed in a six-well plates at a density of 800 cells/well and maintained in medium containing 10% FBS. The culture medium was replaced every three days during colony growth. After 10 days, the cell colonies were washed with ice-cold PBS, fixed with pre-cooled methanol for 10 minutes, and stained with crystal violet for 15 minutes. Colonies were examined and calculated.
2.6. Cellular uptake assay Cellular uptake of EM (self-green fluorescence) in RADA16-I-EM in situ hydrogels was evaluated in LLC cells. The cells were inoculated in a six-well plate at 37 C for 24 h, then free EM and RADA16-I-EM in situ hydrogels (the final concentration of EM was 120, 160, and 200 lM) were added. After incubation in 37 C for 24 h, the cells were washed with cold PBS for three times, digested with trypsinization, suspended in 300 lL of PBS, and analyzed by flow cytometry (Becton-Dickinson, San Jose, CA). For qualitative analysis, the cells were seeded on a confocal dish and cultured for 24 h, then incubated with free EM and RADA16-I-EM in situ hydrogels (the final concentration of EM was 120, 160, and 200 lM) for 24 h, then washed with cold PBS for three times and fixed with 4% paraformaldehyde for 30 min, and stained with DAPI for 20 min. The cells were then captured by confocal laser scanning microscope (Leica, Wetzlar, Germany).

Transwell migration/invasion assays
After incubating LLC cells with free EM and RADA16-I-EM in situ hydrogels (final concentration of EM was 120, 160, and 200 lM) for 24 h, the cells were collected by trypsinization for migration and invasion analysis. In the Transwell cell culture plate, 1 Â 10 5 cells were added to the upper chamber of the device at a serum-free concentration of 200 lL, and the lower chamber was filled with 700 lL culture medium with 20% FBS. After 15 h of incubation at 37 C, the filters were fixed in methanol for 15 min, stained with crystal violet for 15 min and then the non-migration cells were removed from the upper surface of the membrane with a cotton swab. Nine random microscopic fields (Â200) were counted per well, and calculate the average value. For the Transwell invasion experiment, the cell membrane of the upper chamber was pre-coated with 100 lL of 2.5 mg/mL Matrigel (BD Biosciences, Bedford, MA).

Cell apoptosis analysis
Annexin V-Alexa Fluor 647/PI dual staining was used to analyze apoptosis. LLC cells were inoculated in a six-well plate.
After 24 hours of culture, the cells were treated with RADA16-I-EM in situ hydrogels and free drug EM (in which the final concentration of RADA16-I was 5.0 mg/mL, and EM were 120, 160, and 200 lM) for 24 h. The cells were collected, washed with cold PBS for three times, suspended in 300 lL binding buffer and stained with Annexin V-Alexa Fluor 647/ PI. Finally, the cells were analyzed by flow cytometry (Becton-Dickinson, San Jose, CA).

Cell cycle analysis
The proportion of cells in the G0/G1, S, and G2/M phases was detected by flow cytometry. Briefly, LLC cells were incubated with free EM and RADA16-I-EM in situ hydrogels with EM concentration of 120, 160, and 200 lM for 24 h, then the cells were harvested by trypsinization and then fixed overnight with 75% ethanol at À20 C. The fixed cells were centrifuged at 1000 rpm for five minutes and re-suspended in 300 lL staining buffer before detection, 10 lL propidium iodide (PI, 50 lg/mL) was added, and the suspension was incubated at 37 C for 30 minutes in the dark at room temperature. Finally, the cells were detected by flow cytometry (Becton-Dickinson, San Jose, CA).

In vivo anti-lung cancer efficacy
Male C57 mice were purchased from Speyford Biotechnology Co., Ltd. (Beijing, China). The mice were kept in pathogenfree conditions. The mice were injected subcutaneously into the right flanks with 1 Â 10 6 cells/mL (100 lL) of LLC lung cancer cells. Ten days after inoculation, and the cancer volumes were calculated using the equation, V ¼ 0.5 Â length Â width 2 . After the cancer volume reached 150-250 mm 3 , LLC lung cancer-bearing C57 mice were randomly divided into four groups (eight mice per group) with these treatments: PBS þ 20% PEG400 (control), RADA16-I, free EM, and RADA16-I-EM in situ hydrogels. Fifty microliters of RADA16-I (5.0 mg/mL), free EM and RADA16-I-EM in situ hydrogels at an EM dose of 10 mg/kg every two days were injected into the cancers of mice in situ. The body weight and tumor size of mice were measured every two days after treatment. After treatment for 12 days, the animals were humanely sacrificed, and the main organs (heart, liver, spleen, lung, and kidneys) were collected and fixed with 10% paraformaldehyde for 48 h for histopathological examination to evaluate the adverse reactions. The cancer tissues were collected for HE staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), and Ki-67 antibody staining to detect the subcutaneous growth of the cancer. All animal care and experiments were conducted in accordance with National and Institutional Policies on Animal Health and Well-Being. The protocol was approved by the Institutional Animal Care and Use Committee of Zunyi Medical University (approval number: ZMUER2014-2-069).

Statistical analysis
All data were obtained from at least three independent experiments and all statistical analyses were performed using SPSS18.0 statistical software (SPSS Inc., Chicago, IL) or GraphPad Prism 5.0 software (GraphPad). All the obtained data are expressed as mean ± standard deviation (SD). Oneway analysis of variance (ANOVA) with the LSD-t or the Dunnett-t-test was used for multi-group comparison, and Student's t-test was used for comparison between two groups. Differences were considered statistically significant when the p value was less than .05.

Toxicity of RADA16-I in situ hydrogels
To assess the cytotoxicity of RADA16-I in situ hydrogels in vitro, the viability of four cell lines (LLC, 4T1, MLE12, and HC11 cells) incubated with the RADA16-I at the concentration range of 0.5-6.0 mg/mL, was evaluated using MTT assay in the time and concentration dependent manner. As shown in Figure 3, in four cell lines, at the same time, the cytotoxicity increased slowly with the increase of the concentration of RADA16-I solution; furthermore, at the same concentration, the cytotoxicity also increased slightly with the prolonged of the time. However, the final survival rate of cells is close to or greater than 85% in the range of 0.5-6.0 mg/mL, indicating that the peptide RADA16-I has low toxicity to cells and self-assembling peptide RADA16-I has good cell compatibility as a new drug carrier.

Cytotoxicity of RADA16-I-EM in situ hydrogel
The cytotoxicity of EM in different formulations against the cancer cells or normal cells was investigated via an MTT assay. The obtained results are shown in Figure 4(A), the inhibitory effect of different EM formulations on cell proliferation was concentration-dependent and time-dependent. After incubation with free EM and RADA16-I-EM in situ hydrogels for 24 h and 72 h, much higher proliferation inhibition rate was observed in cells treated with RADA16-I-EM in situ hydrogels than free EM, which indicated that RADA16-I-EM in situ hydrogels could promote the release of EM. It can be concluded that RADA16-I can improve the solubility and increase the dissolution of EM to improve the inhibitory effect of EM on the proliferation of cancer cells. Interestingly, as shown in Figure 4(B), with the increase of drug EM concentration and incubation time, both RADA16-I-EM in situ hydrogels and free EM had certain cytotoxicity to normal cells, but the cytotoxicity of RADA16-I-EM in situ hydrogels was lower than free EM. This may be due to the slow release of EM into cells by RADA16-I-EM in situ hydrogels, which did not immediately reach the minimum toxic concentration of the drug, so its toxicity to normal cells was low.

Rada16-I-EM in situ hydrogels decreases cell proliferation in LLC cancer cells
Cell clone formation assay is an important index to evaluate cell population dependence. Only cancer cells or cells with the tendency of malignant lesions can form colonies, and the number of colonies formed by a single cell clone is greater than or equal to 30 cells as the standard, which can reflect the clone formation ability of a single cell and the ability of malignant transformation (Mao, 2014). Therefore, in order to further evaluate the proliferation of LLC cells treated with RADA16-I-EM in situ hydrogels, colony formation tests  were carried out to examine the long-term effects caused by drug-loaded in situ hydrogels. As shown in the staining plates, RADA16-I in situ hydrogels had no significant effect on cell proliferation compared with control, while RADA16-I-EM in situ hydrogels remarkably reduced the quantity and colony size of LLC cells compared with free EM (p<.05, Figure 5).

Cellular uptake of EM in RADA16-I-EM in situ hydrogels
In order to achieve efficient cytotoxicity, the in situ hydrogel loaded with EM should be internalized into the cell to ensure the intracellular EM concentration. As shown in Figure 6, after 24 h incubation, RADA16-I-EM in situ hydrogels-treated cells exhibited relatively high EM intensity in the cytoplasm, while the EM uptake was significantly lower in the cells incubated with free EM. Later, the quantitatively cellular uptake of EM in different formulations was investigated via flow cytometry. After 24 h of incubation, RADA16-I-EM in situ hydrogels exhibited a higher cellular uptake than free EM (p<.01), suggesting that EM loading into RADA16-I could help to enhance the intracellular drug concentration, and the conclusion was the same as the confocal result. It is demonstrated that RADA16-I-EM in situ hydrogels can be effectively absorbed by cells and EM can be transferred to the cytoplasm.

RADA16-I-EM in situ hydrogels inhibits the migration and invasion of LLC cells
The migration and invasion ability of LLC cells were then examined using Transwell assays. In terms of the migration and invasion ability after 15 h as shown by the test assays, we found that LLC cells treated with in situ hydrogels without drug RADA16-I had no statistically significant difference  compared with control cells. But the migration and invasion ability of the cells treated with free EM or RADA16-I-EM in situ hydrogels was significantly decreased, and after RADA16-I-EM in situ hydrogels treatment, the migration and invasion ability of LLC cells was significantly lower than that of cells treated with free EM (Figure 7).
3.6. Effect of RADA16-I-EM in situ hydrogel on cell apoptosis of LLC cells In order to further evaluate the effect of RADA16-I-EM in situ hydrogels on the apoptosis of LLC cells, the apoptosis of LLC cells was detected by flow cytometry. As shown in Figure 8, RADA16-I in situ hydrogels without drug loading had no significant effect on LLC cells, while free EM and RADA16-I-EM in situ hydrogels could induce cell apoptosis and necrosis, among which RADA16-I-EM in situ hydrogels induced LLC cell apoptosis in the early and late stages with the highest rate of 49.7%, compared with 38.3% induced by free EM. These results indicated that RADA16-I could significantly enhance EM induced apoptosis of LLC cells, which might be due to the fact that RADA16-I could stabilize the hydrophobic drug EM, so to increase its solubility, and successfully release EM into cancer cells, thus improving cancer cells uptake efficiency and promoting cancer cells apoptosis.
3.7. Effect of RADA16-I-EM in situ hydrogels on cell cycle progression of LLC cells As cell cycle progression plays an important role in determining cell growth, we then aimed to explore whether the insoluble drug EM packaged in RADA16-I could promote the cell cycle progression of LLC cells after forming hydrogels in situ.
The results of the cell cycle experiment are shown in Figure 9. We found that the cell cycle distribution was not significantly changed in LLC cells after 24 h of incubation  with RADA16-I in situ hydrogels, indicating that RADA16-I did not affect the cell cycle progression. However, a decrease in G0/G1 and S phase and an increase in G2/M phase were observed in LLC cells treated with free EM and RADA16-I-EM in situ hydrogels compared with control cells. The RADA16-I-EM in situ hydrogels significantly arrest LLC cells at the G2/M phase compared with the free EM group, indicating that RADA16-I-EM has a strong ability to inhibit the division and proliferation of cancer cell.
3.8. In vivo cancer suppression evaluation and systemic toxicity evaluation EM in different formulations was injected respectively into LLC cancer-bearing mice and the anticancer effect of RADA16-I-EM in situ hydrogels was monitored by measuring the cancer volume of each group. Meanwhile, the systemic toxicity of mice was determined by measuring the body weight and the injury of main organs (heart, liver, spleen, lung, and kidneys). The results are shown in Figure 10(A).  There was no significant decrease in body weight in each group during treatment. LLC cancers grew extremely rapidly in the control and RADA16-I groups, whereas EM in different formulations had obvious inhibitory effect on cancer growth, especially in the RADA16-I-EM in situ hydrogels group ( Figure  10(B)). Moreover, RADA16-I-EM in situ hydrogels exhibited a better anticancer effect than free EM due to the efficient cellular uptake in vivo. As shown in Figure 10(C), harvested cancers of the RADA16-I-EM in situ hydrogels group were the smallest. RADA16-I-EM in situ hydrogels-treated mice had the highest degree of cancer inhibition with significantly decreased cancer weights compared with other groups (Figure 10(D), p< .01). The cancer slices were then stained. The RADA16-I-EM in situ hydrogels group had largest necrotic areas and the most significant apoptosis in cancer tissue. It also has the lowest number of Ki-67-positive cells compared with other groups, as evidenced by HE, TUNEL, and Ki-67 (Figure 10(E)). EM water suspension, without selected accumulation in cancer tissues, exhibited a poor anticancer effect. As shown in Figure 11, during the treatment period, hepatocytes in all groups showed varying degrees of vacuolar degeneration and inflammatory cell infiltration, of which in EMS group hepatocyte vacuolar degeneration was the severest, followed by RADA16-I-EM in situ hydrogel group, and mild in PBS and RADA16-I groups. At the same time, there was inflammatory cell infiltration in the pulmonary interstitial in all groups, and the pulmonary interstitial was widened in EMS and RADA16-I-EM groups, which may be due to the greater effect of LLC cancer metastasis or drug toxicity on the liver and lung of animals. But in a word, compared with control group, the toxic effect of RADA16-I-EM in situ hydrogels on organs was lower than free EM. The above experimental results showed that RADA16-I could improve the solubility of insoluble drug EM and increase the local concentration of EM in cancer tissue, which could not only enhance the therapeutic effect of drug on cancer, but also significantly reduce the toxicity of drug to normal tissue.

Conclusions
In this study, based on the poor solubility of the hydrophobic drug EM in use, we chose the nanomaterial of self-assembling peptide RADA16-I as a carrier to prepare an EM-loaded RADA16 colloidal suspension (RADA16-I-EM). The solution could immediately form hydrogels in situ under physiological conditions and could be used as a preparation for local administration to improve the low solubility of EM. The drug was accumulated in cancer tissue through the sustained release effect of in situ hydrogels, and then absorbed by cells, thus enhancing the anti-cancer effect. In vitro experiments showed that RADA16-I-EM in situ hydrogels had the best anti-proliferation activity compared with control and free drugs, and in vivo studies in LLC lung cancer-bearing mice showed that the drug-loaded in situ hydrogels could effectively inhibit cancer growth with good biosafety. In conclusion, RADA16-I-EM in situ hydrogels had the behavior of Figure 11. H&E staining of major organs from differently treated groups at day 12 (scale bar: 50 lm). '%' represents the toxic site of the organs tissues during treatment.
slow-release drugs, could provide a promising strategy for anticancer drugs in vivo to control drug release.