Antitumor Photodynamic Therapy Based on Dipeptide Fibrous

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Antitumor Photodynamic Therapy Based on Dipeptide Fibrous Hydrogels with incorporation of Photosensitive Drugs Manzar Abbas, Ruirui Xing, Ning Zhang, Qianli Zou, and Xuehai Yan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00624 • Publication Date (Web): 22 Sep 2017 Downloaded from http://pubs.acs.org on September 24, 2017

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ACS Biomaterials Science & Engineering

Antitumor Photodynamic Therapy Based on Dipeptide Fibrous Hydrogels with Incorporation of Photosensitive Drugs Manzar Abbas,ac Ruirui Xing,a Ning Zhang,a Qianli Zou,a and Xuehai Yan abc* a.

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering,

Chinese Academy of Sciences, No. 1 North 2nd Street, Zhongguancun, Beijing 100190, P. R. China. b.

Center for Mesoscience, Institute of Process Engineering, Chinese Academy of Sciences,

No. 1 North 2nd Street, Zhongguancun, Beijing 100190, P. R. China . c.

University of Chinese Academy of Sciences, Beijing 100049, P. R. China.

ACS Paragon Plus Environment

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ABSTRACT

Hydrogels self-assembled by biologically-based building blocks including peptides and proteins can be used as an ideal drug delivery platform and present great promise for biomedical applications. Herein, photodynamic antitumor therapy based on injectable Fmoc-FF/PLL hydrogels was achieved by encapsulation of the photosensitive drug Chlorin e6 (Ce6), as the hydrogels exhibit multiple favorable therapeutic features, including shear-thinning and selfhealing properties, good biocompatibility, and perfect biodegradability. Such injectable hydrogels are shown to be well suited for local injection and sustained drug delivery at the tumor site, especially towards therapy of superficial tumors. In vivo therapeutic results show, that the tumor growth can be efficiently inhibited, especially under the strategy “once injection, multipletreatments”. During the whole treatment period, no detectable toxicity or damages to normal organs are observed. Therefore, the injectable hydrogels can be applied as a promising delivery platform for therapy of superficial tumors.

Keywords: Dipeptide, hydrogel, self-assembly, injectability, photodynamic therapy


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INTRODUCTION

Photodynamic therapy (PDT) is emerging as an effective antitumor (especially optically accessible tumors) therapeutic modality due to its high selectivity, and non-invasive characteristics.1,2 It is a US food and drug administration approved technique, by which the tumor cells and/or tissues can be damaged through the generation and accumulation of singlet oxygen, a product of photochemical reactions between photosensitizers (PSs) and molecular oxygen stimulated by a specific light wavelength. Therefore, the PDT efficiency to a large extent relies on the effective delivery and release at the tumor lesions. However, the majority of PSs are suffering from the poor solubility, unwanted toxicity and systemic light sensitivity.3,4 To circumvent these issues, a multitude of delivery nanocarriers have been introduced, but many of them are disappointing due to their poor biocompatibility and degradability.5,6

Delivery platforms, especially injectable hydrogels self-assembled from biomolecules such as peptides or proteins, have presented great promise for biomedical applications and attracted increasing interest in recent years.7-16 Their tunable viscoelasticity, gel-sol or sol-gel phase transition, biodegradability and biocompatibility offer broad applications in drug delivery and controlled release.13,17-23 Due to the flexibly designed and well-organized structures, a number of peptides and their derivatives have been used as elegant building blocks for the design of nanoscale ordered hydrogels, especially those made from simple biomolecules such as dipeptides. 24-33

The aromatic dipeptide, diphenylalanine (FF), as the smallest self-assembled peptide

sequence, is derived from the core recognition motif of Alzheimer’s disease β-amyloid polypeptide.34-36 One of the diphenylalanine peptide derivatives N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) has the ability to self-assemble and form ordered fibrous hydrogels


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utilizing a sequential change in pH under physiological conditions.37,38 These hydrogels perform good biocompatibility and biodegradability presenting potential as injectable materials.39,40 Selfassembled dipeptide hydrogels have been manipulated in our previous work to be injectable by balancing the shear-thinning and self-healing properties with a combination of negatively charged Fmoc-FF and oppositely charged polypeptides (Poly-L-Lysine, PLL).41 The hydrogels exhibiting favorable therapeutic features were suited for drug delivery and sustained release. Herein, we have achieved photodynamic antitumor therapy based on injectable Fmoc-FF/PLL hydrogels. A photosensitive drug, Chlorin e6 (Ce6), was encapsulated within the self-assembled dipeptide hydrogels for photodynamic therapy of superficial tumors in vivo (Figure 1). Ce6 has a definite molecular structure, proper absorption coefficient within infrared portion and high efficiency to generate reactive oxygen species, is an ideal agent for photodynamic cancer therapy. Nevertheless, the shortcomings of Ce6 (such as indissoluble, low bioavailability and easily degradable) need to be tackled urgently. The hydrogels loaded with Ce6 exhibit excellent performance as an in vivo injectable platform: 1) The intrinsic biological origin, biocompatibility and biodegradability make the injectable materials biosafe; 2) The shear-thinning and selfhealing performance make the hydrogels injectable; 3) The hydrogels can be injected to the lesions acting as a “depot” system for localized delivery and sustained release; 4) The hydrogels can be provided as a means for therapy against skin disease or superficial tumors. Besides, such a drug delivery platform based on self-assembled fibrous hydrogels can efficiently lower drug dosage and thus reduce undesirable toxicity or damage to normal tissues. The localized delivery, sustained release and consistent laser treatments can enhance the antitumor therapeutic efficacy. Compared with the Ce6 alone, the hydrogel loaded with Ce6 exhibited perfect local preservation and sustained drug release, thus efficiently inhibiting the tumor growth and recurrence. Besides,


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the mouse living state and the vital organs showed no obvious damages. The injectable materials and the antitumor strategy with “once injection, multiple-treatments” were proved to be simple, safe and effective. Therefore, the self-assembled peptide-based hydrogels have broad prospects in biomedical applications. They can be developed as a novel tool for intratumoral delivery of photosensitizers and have promising application in photodynamic antitumor therapy, especially for treatment of superficial tumors.

RESULTS AND DISCUSSION Preparation and characterization of the drug loaded injectable hydrogel. In our previous work, injectable peptide-based hydrogels with tunable mechanical and rheological properties were prepared by combination with positively charged poly peptide (poly-L-lysine, PLL) and negatively charged dipeptide Fmoc-diphenylalanine (Fmoc-FF) (Figure S1).41 Briefly, hydrogels of Fmoc-FF/PLL were prepared through a “step by step” self-assembly method using Tris-HCl solution (pH=7.8). Dissolving Fmoc-FF at a 4 mg mL-1 concentration in 10 mM Tris-HCl buffer resulted in the formation of a homogeneous solution. When an equal amount of PLL (dissolved in Tris-HCl buffer) was added into freshly prepared Fmoc-FF buffer solution, a co-assembled gel was obtained, as shown in Figure S2a. Fmoc-FF/PLL hydrogel self-assembled by a combination of small peptides and bio-macromolecules exhibited excellent biocompatibility, biodegradability, tunable mechanical and perfect rheological properties, suited for drug loading and controlled release. Photodynamic therapy (PDT) has emerged as an efficient treatment for certain malignancieslike skin, head and neck, gastrointestinal and gynecological cancers.1-2 However, one critical issue for PDT is negative side effects of the photosensitizer (PS), for example, the poor


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solubility, easy clearance by the body, and the high unwanted toxicity. Here, a photosensitive drug, Chlorin e6 (Ce6), was used as a model for encapsulation within the self-assembled dipeptide hydrogels, as shown in Figure S2b. The introduction of the photosensitive drug Ce6 caused little effect on the fibrous structure (Figure 2 and Figure S3) of the Fmoc-FF/PLL hydrogels. Spindly fibers with a diameter of 50-100 nm arranged in an ordered way and crosslinked into a network structure, and the fibers further intertwined closely to form a stratified structure with folds (Figure S4). The Ce6 can be encapsulated effectively in the hydrogel making the Fmoc-FF/PLL hydrogels a promising responsive drug delivery carrier for photodynamic therapy. The Fmoc-FF/PLL hydrogel before and after loading with Ce6 showed shear-thinning and self-healing properties, which are basic requirements for in vivo injection, as shown in Figure S5. When subjected to shear forces, the electrostatic interaction between the fibers weakened and induced the transmission from gel to sol, indicating shear-thinning. In this state, the photosensitive drug Ce6 can be delivered effectively to the lesion location, as the hydrogel can pass through a 26-gauge (260 µm) needle without clogging. Removing the shear force, the fibers interweaved together and back to the gel state, indicating the self-healing behavior. So the drug Ce6 can be located in situ after the delivery of the hydrogel. As shown in Figure 3, we evaluated the shear-thinning and self-healing properties of the hydrogels loaded with Ce6 by rheological analysis of the elastic modulus (storage modulus, G’) and loss modulus (viscous modulus, G’’). The hydrogels before and after loading with Ce6 both show perfect rheological properties. Strain-dependent oscillatory rheology (Figure 3a) of the hydrogels exhibits a great anti-shear ability. When the strain increases up to 100%, the value of G’ and G’’ intersect at a point and then turn over, indicating the transition from gel to sol state,


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showing good shear-thinning behavior. Besides, the frequency dependent rheology confirms the hydrogel behavior at a fixed strain of 0.1% with a frequency ranging from 0.01 to 50 rad s-1 (Figure 3b). It also shows a broad linear viscoelastic region of the hydrogels. Next, in order to study the self-healing behavior of the hydrogels, alternation of the oscillatory strain between 100% and 0.1% under the same frequency (1 rad s-1) was applied. As shown in Figure 3c, for a high magnitude strain (100%), the hydrogels exhibit shear-thinning, as G’’>G’, indicating a sol state. Besides, when the strain is switched to a low magnitude strain (0.1%), the sol exhibits quick recovery to gel state, indicating a self-healing behavior. In addition this can still be achieved even at the third cycle. In a continuous flow experiment we measured the instantaneous viscosity modulus under a controlled frequency of 0.1 rad s-1 and a wide range of shear rates from 0.01 s-1 to 10 s-1, as shown in Figure 3d. It shows good shear-thinning performance. The instantaneous viscosity modulus of the hydrogel can be nearly restored to its initial value within a recovery period (as the shear rate ranging from 10 s-1 to 0.01 s-1), which exhibits good selfhealing behavior. The rheological moduli (G’ and G’’) of the hydrogels to some extent relate to the loading content of Ce 6. This is because the drug can interact with the dipeptide fiber through non-covalent interactions such as hydrophobic effects and electrostatic forces, which can influence the hydrogen bonding pattern of peptide self-assembly. Therefore, we controlled the loading content within the hydrogels, which does not change the rheological behavior (Figure S6). Summing up the above, the Fmoc-FF/PLL hydrogels are ideal carriers for encapsulation of photosensitive drugs. The Ce6 loaded Fmoc-FF/PLL hydrogels possess perfect mechanical properties (shear-thinning and self-healing), rendering promising applications for in vivo antitumor treatment, especially for the therapy of superficial tumors.


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Controlled drug release in vivo. Here, we built a breast (MCF-7) xenografted tumor mouse model to assess the utility of Fmoc-FF/PLL co-assembled hydrogels for sustained and controlled drug delivery through an in vivo imaging system at different time points. MCF-7 is a breast cancer cell line and is widely used in building tumor model, especially superficial tumors. The fluorescence intensity of a Ce6 solution decays rapidly and almost vanishes approximately 24 h post intra-tumor injection (Figure 4a and Figure S7). However, the Ce6 encapsulated in the coassembled hydrogel can be preserved in the tumor site and shows localized and sustained delivery within 48 h. The drug was detected even at the 8th day post injection, as shown in Figure S8. So, with the help of the co-assembled dipeptide hydrogel, the photosensitive drug Ce6 can be concentrated at the disease site for a long time, realizing sustained release. These features are important for decreasing the drug dosage and reducing the toxicity of the photosensitive drugs. Therapeutic efficacy against tumors. The Fmoc-FF/PLL hydrogel exhibiting multiple favorable therapeutic features (such as injectable, enhanced rheological properties, good biocompatibility and biodegradability) are well suited for local injection and drug delivery at the tumor site for therapy of superficial tumors. Therefore we evaluated the therapeutic effects for photodynamic therapy of superficial tumors (MCF-7) in vivo. Based on the enhanced drug retention ability of the co-assembled hydrogel as confirmed, the experiments were carried out through the method as “once injection, multiple-treatments”. When the tumor size of tumor-bearing mice reached about 150 mm3 (day 0), the mice were divided into 5 groups. Mice in groups having received an intra-tumor injection of phosphate buffer solution (PBS), Fmoc-FF/PLL hydrogel or co-assembled hydrogel encapsulated with equivalent photo-agent content of 2 mg kg-1 body weight. The mice of experimental groups (III,


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VI, V) received laser treatments for different times post injection of hydrogel encapsulated with Ce6, as shown in Figure S9. Mice in group III received laser treatment (two times) at 4 h and 8 h after injection (day 0). Mice in group VI received the same irradiation at 4 h (day 0), 8 h (day 0), 24 h (day 1) and 48 h (day 2), totally four treatment cycles. The mice of group V received treatments after 4 h (day 0), 8 h (day 0), 24 h (day 1), 48 h (day 2), 72 h (day 3), and 96 h (day 4), totally six treatment cycles. The wavelength of the laser was chosen as 635 nm, the power was set as 100 mW cm-2, and the time of per exposure was 10 min. The mice in control groups (I and II) received the most times of laser treatments (totally six treatment cycles) post injection of PBS or Fmoc-FF/PLL hydrogel. I and II are not further specified, VI is probably IV. As shown in Figure 5a, tumors of mice in group I grew quickly, and the size reached to 1,000 mm3 within two weeks. In group II, the tumor volumes of the mice showed a slight decline when a hydrogel injection was received compared with the mice in group I, but then a rapid growth of from the third day towards a final volume of approximately 1,000 mm3. Mice in these two groups received the most times of laser treatments, but the tumor volumes had not been restrained, indicating the biosafety of laser light under such conditions. Besides, the FmocFF/PLL hydrogel is biocompatible, but is not able to suppress tumor growth. The contrast is obvious, tumors in all other experimental groups (III, IV and V) showed remarkable delays in tumor growth or even tumor regression after two weeks treatments, suggesting that multipletreatment prevented the growth of tumors efficiently. The tumors of mice in group III received two times of treatments showing an obvious recurrence at the 4th day and group IV, receiving four times of treatments, the tumor recurred at the 8th day (Figure 5b). Receiving the most times of treatments, the tumors of mice in group V showed recurrence until 13th day, indicating the “once injection, multiple-treatments” strategy can decrease the risk of recurrence. At the end of


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the treatment period, the mice were sacrificed and the tumors were excised and weighed (Figure 5c, 5d). The tumors of mice in PBS group and hydrogel group were fourfold heavier and obviously larger than in the other experimental groups. The body weight and organ index of mice are important indicators of the life quality and state of being.42 Some negative factors such as drug toxicity or side effects usually result in rapid weight loss. As shown in Figure 6a, the body weights of the mice in all groups have showed an increasing tendency, regardless of laser irradiation or not, and no matter the irradiation times. Besides, the organ index of mice in experimental groups was kept at a fixed level, when compared with the blank groups, as shown in Figure 6b. Further, we monitored the histological features of the vital organs (heart, liver, lung, and kidney) of the mice at different treatment groups, the results of H&E stained slices are presented in Figure 6c. Hepatocytes in the liver samples were found normal and no hyperplasia was found in fibrous tissues. No pulmonary fibrosis was found in the lung samples. The glomerulus can be observed clearly in kidney sections. The membrane structures of endocardial, epicardial, and myocardial are clear, so there are no abnormal cells in heart samples. Neither obvious damage nor inflammation was observed in the spleen samples. In vivo therapeutic results showed that the tumor growth can be efficiently inhibited, and no detectable toxicity or damages to normal organs are observed, demonstrating the potential applications on non-invasive therapeutics against cancers, especially for superficial tumors.

CONCLUSIONS We have achieved effective antitumor photodynamic therapy based on injectable selfassembled peptide fibrous hydrogels with encapsulation of a photosensitive drug (Ce6). The


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assembled Fmoc-FF/PLL hydrogels can locally deliver the drug Ce6 to the tumor site through intratumoral injection, and sustainably release Ce6 for multiple laser-illumination treatments. In vivo photodynamic therapy results show, that the tumor growth is inhibited and the recurrence rate is decreased obviously, especially under the strategy “once injection, multiple-treatments”. Besides, no acute toxicity and negative side effects were found during the whole treatment period. The organ index remains normal and no obvious damage is caused to vital organs. Such self-assembled peptide fibrous hydrogels could be developed as an effective and robust tool for localized delivery and sustained release of therapeutic agents, with the advantages of lowering the drug toxicity and improving the bioavailability, and have a promising application on antitumor therapy, especially for treatments of superficial tumors.


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EXPERIMENTAL METHODS Materials. N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) was laboratory reagent grade purchased from Bachem Company. Chlorin e6 (Ce6) were purchased from Frontier Science Company. All other reagents were purchased from Sigma Aldrich Company. Cell culture media (RPMI 1640) and fetal bovine serum (FBS) were purchased from Invitrogen. Dulbecco’s phosphate buffered saline (PBS), trypsin-EDTA (0.5% trypsin, 5.3 mM EDTA tetrasodium), and the antibiotic agents penicillin-streptomycin (100 U mL-1) were supplied by M&C Gene Technology Ltd (Beijing, China). Water was prepared in a double-stage Milipore Milli-Q Plus purification system. All solutions were freshly prepared for immediate use in each experiment. Synthesis of the injectable hydrogels. The powder of poly-peptide (PLL, Mw: 15-30 KDa) was dissolved in Milli-Q water with a final concentration of 0.5 mM. Tris-HCl buffer solution (10 mM) was prepared and the pH of the buffer was adjusted to 7.8 with the help of 1 M HCl. Fmoc-FF powder (2 mg) was dissolved in the buffer solution above to a concentration of 4 mg mL-1. Then 5 uL of PLL solution was added to the Fmoc-FF buffer solution. The mixture solution was blended and aged for three days to form a stable hydrogel. For drug loading, 0.2 mg Ce6 was dissolved in 20 uL DMSO and then mixed with the hydrogel with a final concentration of 0.4 mg mL-1. Morphology characterizations of the hydrogels. The hydrogel samples were freeze dried using a vacuum freeze dryer before visualization by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM measurements were performed with a Hitachi S4800 electron microscope. The imaging of samples was performed at a 15 k voltage. TEM


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images were obtained by a Zeiss EM 912 Omega transmission electron microscope operated at 120 kV, for which samples were carefully placed onto the carbon-coated copper grids. Rheological experiments. Dynamic rheological experiments were performed using an Anton Paar Physica MCR 302 rheometer at 25 oC. The measurements of the shear moduli (storage modulus, G’ and loss modulus, G’’) were recorded and analyzed using a TA instruments TRIOS software. The gel samples (0.5 mL) were prepared in a plastic tube with a flat base directly. The gels should be aged for three days before measurements. The gel was taken out from the tube remaining whole and placed on the middle of flat slab carefully. Dynamic oscillatory strain amplitude sweep measurements were carried out at a frequency of 10 rad s-1 with a strain ranging from 0.01% to 100%. Dynamic oscillatory frequency sweep measurements were conducted at 1% strain amplitude with the angular frequency ranging from 0.1 to 100 rad s-1. In order to test the recovery behavior of the hydrogels, a larger strain of 100% was applied on the gels for 200 s and then a small strain of 0.1% was applied on the gels for 100 s, this procedure was repeated for three times. The instantaneous viscosity modulus was measured with a wide range of shear rate from 0.01 s-1 to 10 s-1 under a controlled frequency of 1 rad s-1. Besides, in order to prevent of evaporation of water from the hydrogels, a lid was needed. Subcutaneous mouse model. All animal experiments were conducted in accordance with the guidelines of the Animal Experimentation Ethics Committee of Institute of Process Engineering， Chinese Academy of Science for animal care, handling and termination. Female BALB/c nude mice (5-6 week, 16 g) were purchased from Beijing HFK Bioscience Company and housed in a specific pathogen free (SPF) facility with clean water and enough food. After acclimatization for three days, the mice were subcutaneously injected 100 uL of human breast adenocarcinoma


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(MCF-7) cell suspension with a concentration of 5.0×107 cells mL-1 at the right hips. After a week of keeping, the MCF-7 xenoimplanted tumor model was well established. Tumor accumulation and tissue distribution. When the tumor size reached approximately 120 mm3, mice were divided to two groups (3 mice per group). The mice received an intra-tumor injection of photo-agent solution (Ce6) or Fmoc-FF/PLL hydrogel with equivalent Ce6 content of 2 mg kg-1 body weight. Optical fluorescence Imaging was conducted on a KODAK in vivo imaging system (FX Pro imaging system) after drug injection at different time points (0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h). Fluorescent images were collected and analyzed through the KODAK software. In vivo antitumor experiments. The mice were segregated into five groups (four or five mice per group) when the tumor size reached approximately 150 mm3. Mice in Group I received an intra-tumor PBS injection and mice in Group II obtained an intra-tumor injection with FmocFF/PLL hydrogel. All the mice in treatment groups (Group III-V) were given an intra-tumor injection with equivalent photo-agent content at 2 mg kg-1 body weight followed by laser treatment at different time points, Group III: 4 h and 8 h after the injection (total two exposure); Group VI: 4 h, 8 h, 24 h, and 48 h after the injection (total four exposures); Group V: 4 h, 8 h, 24 h, 48 h, 72 h, and 96 h after the injection (total six exposures). The wavelength of the laser was chosen as 635 nm, and the power set as 100 mW cm-2, the time per exposure is 10 min. The length and width of the mice tumor was recorded daily by using a digital caliper, and the volume of tumors was calculated by the following formula: Tumor Volume=(Width)2×Length/2

(1)


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The body weight changes were recorded and the tumors of mice were photographed using a digital camera every day. After two weeks treatment, the mice were sacrificed and the main organs (heart, liver, lung, and kidney) were taken out. The histopathological tests were performed according to a standard procedure: first, the tissue samples were washed with normal saline (0.9%) for three times in order to remove the remaining blood; second, the samples were fixed in a 4% formalin solution, and then they were embedded in paraffin blocks, sectioned into 5 µm slices, and mounted onto the glass slides; third, standard hematoxylineosin (H&E) staining was conducted, and the photos of sections were taken using a 100× optical microscope; last, the results were delivered to a professional pathologist. Statistical analysis. Statistical analysis was conducted using SPSS version 13.0 software. All analyses were carried out in triplicate and the data were expressed as means and standard deviations (SD). For statistical analyses Analysis of variance (ANOVA) was performed in in vivo experiments. Values of P

Antitumor Photodynamic Therapy Based on Dipeptide Fibrous

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