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An injectable hexapeptide hydrogel for localized chemotherapy prevents breast cancer recurrence Yingqiu Qi, Huan Min, Ayeesha Mujeeb, Yinlong Zhang, Xuexiang Han, Xiao Zhao, Greg J Anderson, Ying Zhao, and Guangjun Nie ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19258 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 9, 2018

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ACS Applied Materials & Interfaces

An injectable hexapeptide hydrogel for localized chemotherapy prevents breast cancer recurrence

Yingqiu Qi1,2,3#, Huan Min1,3#, Ayeesha Mujeeb3#, Yinlong Zhang3, Xuexiang Han3, Xiao Zhao3, Greg J Anderson4, Ying Zhao3,5*, Guangjun Nie3,5*

1

College of Science, Northeastern University, Shenyang 110819, China

2

College of Life and Health Sciences, Northeastern University, Shenyang 110819,

China 3

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety , CAS

Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China. 4

QIMR Berghofer Medical Research Institute, Royal Brisbane Hospital, QLD 4029,

Australia 5

University of Chinese Academy of Sciences, Beijing 100049, China

*

Corresponding authors:

Ying Zhao, email:[email protected] Guangjun Nie, email: [email protected] #

These authors contributed equally to this work.

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Abstract Although post-surgical chemotherapy is frequently used for the treatment of breast cancer, tumor recurrence is still a frequent event. Enhancing the efficacy of chemotherapy via localized drug delivery may help to prevent breast cancer recurrence. To achieve this goal, we designed a hydrogel nanocarrier that could be injected at tumor site by co-assembly of tailor-made hexapeptide and doxorubicin (Dox). Evidently based on our findings, the sustained release of drug from the hydrogel led to a reduction in cancer recurrence, including the suppression of primary regrowth and distant metastasis. This localized chemotherapy strategy did not show any obvious side effects in vivo, and represents a promising adjuvant therapeutic strategy for breast cancer recurrence.

Keywords: peptide self-assembly, hydrogel, injectable, localized chemotherapy, cancer recurrence

1. Introduction Breast cancer is the second most frequent cause of cancer-related deaths in women worldwide

1-5

. Surgical resection is the preferred clinical strategy for treating early or

late-stage breast cancer 6. However, post-surgical recurrence, including regrowth at the primary tumor site and distant metastasis mainly caused by residual microtumors remain problematic

7-9

. Although systemic chemotherapy after surgical resection is

frequently used to prevent cancer recurrence, very few chemotherapeutic drugs accumulate at the primary tumor site adverse side effects

10,11

, and systemic chemotherapy often has

12

. Therefore, improved strategies are urgently needed to

overcome these limitations. One approach is to use localized chemotherapy following surgery to achieve high drug concentrations at the tumor site to decrease risk of cancer recurrence and achieve long-term relief

13-16

. The localized delivery of

cytotoxic drugs should also reduce systemic toxicity, thus improving patient compliance 17-20. 2 ACS Paragon Plus Environment

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There has been considerable interest in developing enhanced post-surgical localized chemotherapy systems

21-24,

and injectable hydrogels, with their highly

organized three-dimensional network based on physical or chemical crosslinks have attracted wide attentions

25-28

. Hydrogels mimic the physiological environment of

natural tissues, and their ability to retain water molecules makes them well-suited to drug loading in vitro and release in vivo

29-32

. Previous studies have shown that

injectable hydrogels encapsulating small chemotherapeutic drugs, therapeutic proteins, or microRNA can be successfully used for localized combination therapy to facilitate tumor regression, and consequently cancer recurrence and metastasis were prevented 33-38

. Importantly, with excellent properties of biocompatibility, biodegradability, and

multifunctionality, peptide-based hydrogels in particular have shown considerable potential in various bio-medicinal applications to

date

successful

use

of

39-42

self-assembling

. According to our understanding, peptide

hydrogels

as

local

chemotherapeutics delivery system to prevent cancer recurrence have not yet been published.

In this study, we generated an oligopeptide (FEFFFK) using natural amino acids (without chemical functional groups), which self-assembled in the presence of chemotherapeutic drugs such as doxorubicin (Dox) to form well-organized nanofibers with an anti-parallel β-sheet structure. When injected around the site of 4T1 or MDA-MB-231 tumors in mice, the drug-loaded hydrogel was able to suppress cancer regrowth at the primary tumor site and reduce distant metastasis. Given its ease of preparation, injectability and local drug delivery capacity, this strategy for the co-assembly of multifunctional peptide hydrogels shows great potential to prevent breast cancer recurrence.

2. Materials and methods 2.1 Hydrogel formation and characterization. 3 ACS Paragon Plus Environment


FEFFFK peptide was synthesized according to a standard solid-phase peptide synthesis technique. Based on peptide self-assembly, Dox-free hydrogel (peptide hydrogel) and Dox-loaded hydrogel (peptide hydrogel + Dox) were prepared. Briefly, peptide solutions were prepared by dissolving the powder at concentrations ranging from 10 to 60 mg/mL in distilled water. The peptide solutions were vortexed to obtain a homogeneous mixture, heated to 70 °C for 1 h to ensure complete dissolution of peptides, and then cooled at room temperature. To adjust the pH of the mixture (above pH 6), 15 mM NaOH was used. Gelation occurred in less than 5 mins. The samples were stored at room temperature. To prepare the Dox-loaded hydrogels (Sigma, Beijing, China), the peptide powder (30 mg/ml) was directly dissolved in water containing Dox (0-160 µg). The concentration of peptide and Dox were varied to provide solutions of defined Dox: peptide ratios. The mixture was prepared following described above. For hydrogel characterization studies, a peptide concentration of 30 mg/mL was used. The morphology and width of fibers within the gel matrices were investigated using transmission electron microscopy (TEM, JEM-200CX, Jeol Ltd., Japan) and atomic force microscopy (AFM, dimension ICON, Brokers American). To characterize the molecular structure within the hydrogels (α-helix, β-sheets or random coils), Fourier transform infrared (FTIR) spectrometry (Thermo Nicolet 5700) and circular dichroism (CD) spectrophotometry (Jasco CO, Japan) were used. The viscoelastic properties of the hydrogels were investigated on a Bohlin C-VOR 200 digital rheometer with 20 mm parallel plate geometry. A solvent trap was used to keep the hydrogel hydrated and the temperature was maintained at 25°C. The experiments were repeated 3 times (each time on a new sample) to ensure reproducibility. Results are presented as the mean ± standard deviation from three independent experiments.

2.2 In vitro cell cytotoxicity studies. The cell lines MDA-MB-231 and 4T1 were obtained from National Platform of Experimental Cell Resources for Science-Technology (Beijing, China). Cells were 4 ACS Paragon Plus Environment

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cultured in DMEM medium and RPMI 1640 medium (Wisent, Beijing China) respectively, supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (WRSENT Beijing, China). Cells were seeded at a density of 1 × 104 cells/well in a 96-well plate. After 24 h, varied concentration of Dox were added respectively. Cell viability was investigated by using Cell Counting Kit 8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. For the cytotoxicity assay in vitro, MDA-MB-231 and 4T1 cells were seeded into 24-well plates at a density of 2 × 104 cells per well and incubated with free Dox, Dox-free hydrogel and Dox-loaded hydrogel (added into the inserts). Cell viability was tested using the CCK-8 assay after 72 h of culture. For the 11 days cell culture period, 2 × 104 cells/well were seeded into 24-well plates. After 24 h, 100 µL of Dox-loaded hydrogel (30 mg/mL) containing 40 µg Dox were added into the up inserts (0.4 µm pore size, Corning, USA). Free Dox was added to the control wells to assess the difference between Dox released from the hydrogels and free Dox, respectively. At day 3, medium was changed and 1 × 104 cells were reseeded. Cell viability was investigated using CCK-8 assay up to 11 days.

2.3 Dox release and degradation studies in vitro and in vivo. To investigate the release of Dox from Dox-loaded hydrogels, 100 µL Dox-containing hydrogels were transferred into inserts in 24-well plates and 1 mL PBS was added to the wells carefully at 37 °C. Dox released from Dox-loaded peptide hydrogels was monitored at different time intervals according to the standard curve. For the assessment of degradation, hydrogel samples were immersed in PBS (1 mL) at pH 7.4 and incubated at 37°C. The solution was changed every other day for 20 days. The percentage weight loss (% WL) of the hydrogel was determined every other day during the incubation period. The following formula was used % WL = [(Wi−Wf)/Wi] × 100%, where Wi and Wf are the initial weight of the hydrogels before incubation in PBS and the weight of the hydrogels after incubation in PBS, 5 ACS Paragon Plus Environment


respectively. To assess the concentration of Dox at the tumor site and plasma following injections of Dox-loaded hydrogel or intravenous injections of a similar Dox dose in vivo. Mammary tumor model was established using 4T1 breast cancer cells. Briefly, cells were grown in vitro culture, harvested then resuspended in PBS at a density of 4 × 107 cells/mL. The resulting suspension (50 µL) was mixed with 50 µL of matrigel and the preparation was subcutaneously implanted into the breast of nude mice. When the tumor size reached 100 mm3, tumor-bearing mice were subcutaneously injected with 200 µL Dox-loaded hydrogel or intravenous free Dox (5 mg/kg). The tumor and the vein plasma were collected at 1, 4, 7, 10, 15, and 20 days. Tumors were homogenized in RIPA buffer (Solarbio, China). Liquid−liquid extraction, external standardization, and HPLC were used to measure. Briefly, tumor homogenates in RIPA buffer were extracted with acetonitrile (ACN), and the extract was transferred to a different tube, evaporated to dry in all CAN 43. Blood samples (300 µL) were treated with 1 mL of isopropanol : water (9:1, v/v) containing 0.075 N hydrogen chloride for 24 h at 4°C to extract Dox and then centrifuged at 1,000 g for 20 min 44. Mobile phase was added to each tube, an aliquot was injected into the HPLC (Shimadzu, Kyoto, Japan), and the compounds were separated using a symmetry C18 reverse phase column (5 µm; 4.6 × 150 mm, Thermo Scientific). The flow rate was 1.0 mL/min, and the eluent was monitored at 485 nm for Dox. The sum concentration of Dox was used to evaluate the drug quantity in the tumors. All separations were performed at room temperature.

2.4 Local cancer recurrence murine model. BALB/c mice (female, 6 weeks age, 16-18 g body weight) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). All animal protocols were approved by the Institutional Animal Care and Use Committee. A murine model of local cancer recurrence in vivo was established as described previously 45. Briefly, luciferase-tagged 4T1 cells (50 µL of the resulting suspension including 1 ×107 6 ACS Paragon Plus Environment

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cells/mL and 50 µL of matrigel) were injected into mammary fat pads of the mice. When tumors reached 200 mm3 after approximately one week, they were resected. To mimic the presence of residual microtumors on the surgical bed, approximately 1% of the original tumor mass was left in situ 45. Mice were subsequently randomized into one of the following four treatment groups: 1) saline; 2) Dox-free hydrogel; 3) intravenously (i.v) Dox (2 mg/kg 14,40); or 4) equivalent dose Dox-loaded hydrogel (n = 10 per group) implanted at the resection site. Mice were monitored for local breast cancer recurrence (local recurrence was defined as tumor(s) present anywhere on the back of animals under investigation). Tumor presence was monitored via the bioluminescence of 4T1 cells by inject a substrate to get a readout. The volume of any tumors detected was determined using the formula tumor volume = length × width2 × 0.5. Micro-metastases of the lungs were also tested. Animals were euthanized when they exhibited signs of impaired health or when the volume of the tumor exceeded 2,000 mm3.

2.5 Anti-tumor model in vivo. Mammary tumor model was established using MDA-MB-231 breast cancer cells. Briefly, cells were grown in vitro culture, harvested then resuspended in PBS at a density of 4 × 107 cells/mL. The resulting suspension (50 µL) was mixed with 50 µL of matrigel and the preparation was subcutaneously implanted into the breast of nude mice. Treatment was started when the tumor size reached 60 mm3 (approximately 2 weeks after injection). Mice (n = 5) were divided into four treatment groups; (1) 100 µL saline (negative control group); (2) 100 µL free Dox (40 µg/100 µL) (injected i.v. positive control group); (3) 100 µL of Dox-free hydrogel (30 mg/mL, injected around the tumor site); and (4) 100 µL Dox-loaded hydrogel (30 mg/mL, containing 40 µg Dox, injected around the tumor site). The antitumor effect was quantified according to tumor weight and volume (calculated as described above). The body weight of mice was also recorded. Histology samples at week 5 were fixed in buffered formalin, dehydrated, and processed for paraffin embedding. Samples were sectioned and 7 ACS Paragon Plus Environment


stained with H&E.

2.6 Surgical observation of Dox-loaded hydrogel post-treatment in vivo. To access the wound healing in hydrogel-treated groups. After 4T1 tumors up to 100 mm3, the tumors were resected. Photographs were taken at different day to investigate gradual wound healing in mice. To assess the degradation of Dox-loaded hydrogel in vivo, female BALB/c mice were injected subcutaneously with 4T1 cells (1 ×107 cells/mL). When the tumor volume reached approximately 100 mm3 (usually around 5 days), Dox-loaded hydrogel were injected around the tumor site. Mice were scarified at various times thereafter and photographs were taken, and relatively quantified were examined. To assess the biocompatibility of Dox-loaded hydrogel in vivo, female BALB/c mice were injected subcutaneously with Dox-loaded hydrogel, Dox-free hydrogel or free Dox. Normal mice with no injections served as the control group. After 3 weeks, mice were scarified. Samples of heart, liver, spleen, kidney, and lungs were collected, fixed in paraformaldehyde and blocked for histology. The tissue samples were subsequently sectioned and stained with hematoxylin-eosin (H&E) and examined microscopically for signs of tissue damage.

2.7 Statistical analysis. Analysis was performed by SPSS17.0 statistical analysis software. Statistical analysis was conducted by Students t test for comparison of two groups, followed by Newman–Keuls test. A p value of less than 0.05 was considered statistically significant, *p< 0.05, **p< 0.01, ***p< 0.001. Kaplan-Meier curves were used to analyze the survival of mice with different treatment groups, and the log-rank test was used to obtain a P-value for the significance of Kaplan-Meier curves’ divergence, *p< 0.05, **p< 0.01, ***p< 0.001.

3. Results and discussion 3.1 Self-assembled construction of hydrogel for localized chemotherapy. 8 ACS Paragon Plus Environment

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The hydrogel drug delivery system we designed is based on the hexapeptide (FEFFFK). This peptide contains four phenylalanine (F) residues containing aromatic groups, a negatively charged glutamic acid (E) residue, and a positively charged lysine (K) residue. The molecular self-assembly process was driven by strong hydrophobic interactions (π-π stacking interactions) and favorable electrostatic attractions that greatly contributed to the stability of the hydrogel (Figure 1a). This peptide was shown to form a stable and self-supporting hydrogel above pH 6 at a concentration of 30 mg/mL in a 15 mM NaOH solution (Figure S1a). For Dox encapsulation, the hydrogel was heated to 70°C, then cooled in the presence of Dox (at 40 µg/mL) to form a stable drug-loaded hydrogel (Figure S1b&c). To optimize the drug-loading efficacy, different Dox to peptide ratios were evaluated. Stable hydrogels formed at Dox concentrations up to 160 µg/mL, thus suggesting a high Dox-loading capacity of the hydrogel (Figure S1c). To assess the viscosity of the Dox-loaded hydrogel (peptide hydrogel + Dox), which cannot be too high as the materials must be injected, we passed the material through a 29 G needle into a fresh 96-well plate in vitro (Figure S1c). The hydrogel underwent shear thinning upon injection, but re-formed a stable hydrogel in less than 5 minutes, thus indicating that it is an excellent candidate for in vivo studies. TEM and AFM photomicrographs revealed nanofibers morphologies within the hydrogel matrix with an average nanofiber size of 3 nm or 6 nm in the absence or presence of Dox), respectively (Figure 1b&c). The secondary structure by the FTIR results also suggested that Dox had no effect on the molecular arrangement of the peptide hydrogel system, leading to the formation of an antiparallel β-sheet structure (Figure S2). The results of the CD spectra revealed that the β-sheet band enhanced and showed red-shift. These results suggest that hydrophobic side chains (π-π stacking) contributed to the stability of β-sheet structures, and π-π stacking will tighten β-sheet structures and not affect the formation of hydrogen bond in hydrogels

46,47

. The fiber

dimensions are in good agreement with previous studies using drug-triggered and cross-linked self-assembling nanofiber hydrogels, self-assembling peptide nanofiber 9 ACS Paragon Plus Environment


scaffolds for controlled release and nanofibrous scaffold from self-assembly of beta-sheet peptides containing phenylalanine for controlled release 48-50. To further understand the stability of the hydrogels, rheological studies were conducted (Figure S3). The average elastic modulus (G') recorded for the drug-free hydrogel was 0.2 kPa, and for the Dox-loaded hydrogel 1 kPa. The higher G' value of the Dox-loaded hydrogel is consistent with strong electrostatic and π–π interactions between Dox and the phenyl groups of the peptide, a characteristic favorable for the aggregation and reinforcement of nanofiber networks. Changes in mechanical stiffness were also associated with differences in nanofibers assembly as describe above, and these, in turn, affected the corresponding hydrogel properties. In vitro drug release profiles were studied (in phosphate buffered saline (PBS), at pH 7.4). As shown in Figure S4a, approximately 85% of the Dox was released from the hydrogel after three weeks in vitro. Furthermore, the hydrogel degraded over time (Figure S4b), with the residual weights of the hydrogels (either with or without Dox) reaching less than 20% of the initial value after three weeks. These data suggest that there was a sustained release of Dox over time that paralleled the degradation of the hydrogel.

3.2 Inhibition of tumor cell regrowth for localized chemotherapy. Our objective at this stage was primarily to examine the cytotoxicity of Dox on MDA-MB-231 and 4T1 breast cancer cells in vitro and selected a dose 40 µg/mL (which provided a 50% reduction in cell viability) for further studies (Figure S5). We then examined the effects of the hydrogels on these cell lines. After three days treatment, the Dox-free hydrogels showed no cytotoxic effects on MDA-MB-231 or 4T1 cells, and cell viability remained at 100%. In contrast, the Dox-loaded hydrogel (with a Dox dose of 40 µg/mL) reduced cell viability to less than 20%, a value similar to that of free Dox (Figure 2a). To simulate regrowth of tumor cells within the primary site after surgical resection, cell medium was replaced in the well plates and fresh tumor cells were re-seeded after three days treatment. 10 ACS Paragon Plus Environment

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After reinoculation with tumor cells, greater cytotoxicity was observed in the Dox-loaded hydrogel group rather than in the free Dox group (Figure 2b). Consequently, we have shown that our Dox-loaded hydrogel have an anti-tumor efficacy potential due to sustained drug release in vitro, suggesting its use for further applications in vivo.

3.3 Pharmacokinetics of Dox released from hydrogels. The successful application of a localized drug delivery system requires the system to target the site of interest (in this case the tumor site), followed by progressive degradation to facilitate sustained drug release. To investigate this, we implanted 4T1 tumors into BALB/c mice and studied them after tumors approximately reached 100 mm3. At that time, the animals were treated by administering free Dox intravenously or by implanting Dox-loaded hydrogel at the site of the primary tumor. We subsequently measured the drug concentrations in both the circulation and in cancer tissue 1, 4, 7, 10, 15, and 20 days after drug treatment (Figure 3). In primary tumors, the Dox levels achieved following hydrogel administration were 1.6 and 1.8 fold higher that the levels achieved using free Dox after 1 and 4 days, respectively (1013.29 ± 136.56 ng/g, and 618.53 ± 60.06 ng/g at day 1; 844.72 ± 115.45 ng/g, and 466.25 ± 32.75 ng/g at day 4). In addition, plasma Dox levels were 2.3 fold higher in the hydrogel-treated group than those in the free Dox group after 1 day (140.13 ± 23.78 ng/mL; 61.85 ± 6.01 ng/mL). Following Dox-loaded hydrogel administration, both the tumor and plasma, Dox levels declined gradually over time and were still detectable 20 days after treatment in the tumor, and up to 15 days after treatment in the plasma. We hypothesized this may have occurred due to the sustained drug release of Dox-loaded hydrogel. In contrast, the Dox injected systemically was nearly undetectable in tumor tissues after 7 days and in the plasma beyond 1 day. The persistence of Dox at the tumor site following hydrogel administration highlights the potential of this system for reducing or preventing the long-term recurrence of tumors.



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3.4 Anti-recurrence activity of hydrogel localized chemotherapy. To investigate the post-surgical chemotherapeutic efficacy of Dox-loaded hydrogel for local tumor recurrence. A mammary tumor xenograft resection model was established to mimic the post-surgical recurrence. In detail, 4T1 cancer cells were injected into mammary fat pads of the mice. When tumor volume had reached approximately 200 mm3, 99% of the primary tumor was surgically resected 45. Two days later, the mice were randomly divided into four groups, and each group was treated with saline, Dox-loaded hydrogel for localized chemotherapy (2 mg/kg Dox hydrogel or free Dox (2 mg/kg, intravenous

14,40

), Dox free

14,40

) (Figure 4a). Tumor regrowth was

evaluated using bioluminescence imaging of luciferase activity at 9, 18 and 27 days post-treatment. Using area-under-curve analysis of luciferase activity, total bioluminescence from the mice treated with the Dox-loaded hydrogel was significantly lower in comparison to the other groups (Figure 4b and c). Free Dox treated mice showed only a slight reduction in tumor regrowth. Similar results were obtained when the tumor volume was measured (Figure 4d). H&E staining and PCNA staining images of the recurrence tumors were shown in Figure S6. Compared with control, free Dox and Dox-free hydrogel group, Dox-loaded hydrogel group processes less positive area of PCNA. The results also indicate that the sustained release of Dox in hydrogel can remarkably reduce the number of breast cancer cells at the primary tumor sites after resection. Based on the previous studies

51

, we

speculated that the sustained release of low-dose anti-cancer drugs at the sites of tumor resection would be significantly more effective at preventing tumor recurrence in vivo than a ‘‘burst release’’ system that rapidly exhausts. Meanwhile, long-term and slow drug release also lead to minimized systemic toxicity. Then, we examined metastasis formation in the lungs of BABL/c mice. The lungs are a preferential site of metastasis for 4T1 tumors. The lungs were examined macroscopically, and microscopically following H&E staining. Necropsy indicated that the Dox-loaded hydrogel significantly decreased the number of metastatic foci in the lungs, whereas apparent metastases were observed in other groups, (saline, 21 foci; 12 ACS Paragon Plus Environment

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Dox-free hydrogel, 16 foci; free Dox for systemic chemotherapy, 13 foci; Dox-loaded hydrogel for localized chemotherapy, 4 foci; Figure 5a-c). The reduced number of metastases following Dox-loaded hydrogel treatment likely reflects the inhibition of regrowth at the primary tumor site. Consistent with these findings, therapy with the Dox-loaded hydrogel for localized chemotherapy prolonged the survival of 4T1 tumor-bearing mice (Figure 5d). Thus, 70% of mice treated with Dox-loaded hydrogel showed long-term survival, whereas none of the mice in the other treatment groups survived beyond 40 days after therapy commenced. Herein, our data clearly demonstrates that the injectable Dox-loaded hydrogels possess sustained drug release ability with improved local drug concentrations at the primary tumor sites compared to free Dox for systemic chemotherapy. Also, our designed system efficiently suppressed tumor regrowth and distant metastasis, indicating that hexapeptide-based hydrogels may be a suitable carrier for the inhibition of post-operation locoregional tumor recurrence.

3.5 Expanding anti-tumor application of hydrogel localized chemotherapy. To examine the effects of the Dox-loaded hydrogel on primary tumor growth, we established a mammary tumor xenograft model (MDA-MB-231 breast cancer cells injected into BALB/c nude mice). After the tumors reached a size of 60 mm3, the tumor-bearing mice were randomly divided into 4 groups and treated with either saline, free Dox, Dox-free hydrogel or Dox-loaded hydrogel as described above (Figure 6a). Dox-free hydrogel and free Dox failed to suppress tumor growth, unlike Dox-loaded hydrogel where the tumors showed minimal growth (based on the tumor growth curves and final tumor weights shown in Figure 6b-d). In addition, the mice body weight had no obvious change in any treatment groups (Figure 6e). After the treatments, the tumors were sectioned and examined following H&E staining (Figure 6f). The tumor on saline, free Dox and Dox-free hydrogel treated mice showed almost no histological change in images, but, the morphology of tumors from Dox-loaded hydrogel treated mice were substantially different, exhibited large areas of karyolysis, 13 ACS Paragon Plus Environment


suggesting obvious necrosis and apoptosis. These results indicate that our injectable hydrogel system may also be used as a therapeutic strategy to treat primary tumors, especially ductal adenocarcinoma breast cancers.

3.6 Biocompatibility of hydrogel localized chemotherapy. We examined wound healing of mice with tumor resection. Figure S7 showed that the tissue wounds in hydrogel-treated groups completely healed in mice 5 days after post-surgical operation, suggesting the excellent biocompatibility of hydrogel. The H&E images of the tissues from free Dox, Dox-free hydrogel and Dox-loaded hydrogel injection sites at day 5 were shown that free Dox group had obvious inflammation; Dox-loaded hydrogel group showed slight inflammation, while Dox-free hydrogel group had almost no inflammation (Figure S8). The black arrows represent inflammatory cells and dash lines represent severe inflammatory region. These results also indicated that our peptide hydrogel reduced inflammation and had high biocompatibility. To observe the degradation of Dox-loaded hydrogel in vivo, optical images of skin tissues were obtained at days 1, 5, 10, and 20 after subcutaneous administration of Dox-loaded hydrogel. Visually, Figure S9 revealed the hydrogels gradual degradation after injection. Partial degradation of the hydrogel was observed after 10 days of injection, and complete degradation by 20 days. Mice injected with Dox-loaded hydrogel showed no overt adverse effects. Similarly, histological examination of the major organs (heart, liver, spleen, kidney, and lungs) following H&E staining (Figure S10) failed to show any differences in tissue morphology between the Dox-loaded hydrogel treated group and saline treated mice. This may be due to the administration of single and low drug dose in mice. Thus, the Dox-loaded hydrogel had major effect on tumor growth, both with or without surgical resection, without apparent adverse effects in vivo.

4. Conclusions In conclusion, considering the post-surgical recurrence of breast cancer, we have 14 ACS Paragon Plus Environment

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successfully designed a hexapeptide-based hydrogel system that is able to deliver drugs for localized chemotherapy. The hydrogel developed was biocompatible and biodegradable in vivo, showing the injectable property and allowing a sustained release of encapsulated Dox at the tumor site. The injectable hydrogel achieved high therapeutic efficacy against tumor recurrence and demonstrated low toxicity to normal tissues, thus showing great potential for the improvement of therapy following tumor resection. Due to the ease of preparation, injectability and local drug delivery capacity, the peptide based hydrogels for localized chemotherapy may serve as a powerful candidate for the improvement of current clinical therapeutics.

Supporting information available The Supporting Information is available free of charge on the ACS Publications website at DOI: The Supporting Information showing the results about the formation of hydrogels, secondary structure and rheological characteristics of peptide based hydrogel, sustained release and degradation in vitro, the cell viability of MDA-MB-231 and 4T1 after incubation with free Dox at various concentration for 24 h, the H&E staining and PCNA staining images of tumor of peptide hydrogel injection site at different groups representative images of mice wound healing after surgery, representative images of mice wound healing after surgery, The H&E images of the tissues from free Dox, Dox-loaded hydrogel and Dox-free hydrogel injection sites at day 5, visual representation of Dox-loaded hydrogel in vivo and biocompatibility evaluation by H&E staining.

Competing financial interests The authors declare no competing financial interests.

Acknowledgments This work was supported by the Excellent Young Scientists Fund (31722021), the 15 ACS Paragon Plus Environment


National Natural Science Foundation of China (21373067 and 51673051), Beijing Nova Program (Z171100001117010), Beijing Natural Science Foundation (7172164), Youth Innovation Promotion Association CAS (2017056), the National Distinguished Young Scientists program (31325010), the Innovation Research Group of the National Natural Science Foundation (11621505), the Key Research Project of Frontier science of the Chinese Academy of Sciences (QYZDJ-SSW-SLH022), and Beijing Municipal Science & Technology Commission (Z161100000116035).


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Figure 1. Schematic illustration of the peptide hydrogel and its characterization. (a) Molecular structure of the peptide FEFFFK that self-assembled into an anti-parallel β-sheet arrangement. Dox influenced the aggregation of nanofibers within the hydrogel matrix. TEM (b) and AFM (c) photomicrographs revealed the 23 ACS Paragon Plus Environment


elongated nanofibers morphology within the peptide hydrogel (left) and peptide hydrogel + Dox (right). The hydrogels were prepared at a peptide concentration of 30 mg/mL; Dox was at a concentration of 0.04 mg/mL. Scale bar, 500 nm.


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Figure 2. Assessing cell regrowth and recurrence of breast cancer cells in vitro. (a) Metabolic activity of MDA-MB-231 and 4T1 tumor cells after treatment with saline, free Dox, peptide hydrogel and peptide hydrogel + Dox (at day 3 in culture). (b) Influence of free Dox and peptide hydrogel + Dox on the viability of MDA-MB-231 and 4T1 tumor cells up to 11 days in culture. Cells were re-seeded at day 3 to mimic the recurrence in vitro. Error bars represent the mean ± standard deviation (***p< 0.001, n = 5).



Figure 3. Pharmacokinetics of Dox released from hydrogels. The concentration of Dox measured via HPLC within the tumor and plasma following injection of peptide hydrogel + Dox at the tumor site or intravenous injection of a similar free Dox dose. (a) Around the tumor site, Dox was detected for up to 20 days, when peptide hydrogel + Dox was administered, but only up to 4 days following free Dox administration. In addition, Dox levels detected around the tumor were relatively higher following hydrogel administration. (b) In the plasma, Dox was detected for up to 15 days following peptide hydrogel + Dox administration, but only at day 1 following free Dox administration.


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Figure 4. The Dox-loaded hydrogel reduced recurrence of murine breast tumors. (a) Experimental design of the use of peptide hydrogel + Dox in an incomplete surgery tumor model. (b) In vivo bioluminescence imaging of 4T1 tumors after removal of the primary tumor. (c) Quantified bioluminescence for tumors from the different treatment groups. (d) Tumor size measured for up to 31 days after inoculation. Error bars represent the mean ± standard deviation (*p< 0.05, **p< 0.01, ***p< 0.001, n = 10).



Figure 5. The Dox-loaded hydrogel reduced metastasis of murine breast tumors. (a) Representative whole lung photographs and (b) H&E-stained lung sections collected from the mice after the indicated treatments. Metastatic tumors in the lungs are marked with a ‘T’. The scale bar represents 200 µm. (c) Number of lung metastatic foci after different treatments (control group: 21 ± 2, peptide hydrogel group: 16 ± 3, free Dox group: 13 ± 2, peptide hydrogel + Dox group: 4 ± 3, n = 5). (d) Kaplan-Meier of survival curves for different treatments (n = 10).


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Figure 6. Anti-tumor efficiency using hydrogel localized chemotherapy for murine breast cancer model. (a) Experimental design of the use of peptide hydrogel + Dox in an incomplete tumor model. Changes in tumor volume (b) and mice body weight (c) during various treatments. (d) Tumor weight at the end of each treatment. (e) Photographs of excised MDA-MB-231 solid tumors from different treatment groups at the end of the treatment. (f) H&E staining of tumor sections after treatment. (Scale bars represent 100 µm) (*p< 0.05, **p< 0.01). 29 ACS Paragon Plus Environment


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