Self-assembled Injectable Peptide Hydrogels ... - ACS Publications

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Self-assembled Injectable Peptide Hydrogels Capable of Triggering Antitumor Immune Response Ruirui Xing, Shukun Li, Ning Zhang, Guizhi Shen, Helmuth Möhwald, and Xuehai Yan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00787 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 19, 2017

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Biomacromolecules

Self-assembled

Injectable

Peptide

Hydrogels

Capable of Triggering Antitumor Immune Response Ruirui Xing1, Shukun Li1,3, Ning Zhang1, Guizhi Shen1, Helmuth Möhwald4, Xuehai Yan1,2,3* 1

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

Academy of Sciences, Beijing 100190, China 2

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

Beijing 100190, China 3

University of Chinese Academy of Sciences, Beijing 100049, China

4

Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, D-14476 Potsdam/Golm,

Germany

ACS Paragon Plus Environment

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ABSTRACT

Self-assembled peptide hydrogels are particularly appealing for drug delivery, tissue engineering, and anti-tumor therapy due to various advantageous features including excellent biocompatibility and biodegradability, defined molecular and higher organized structures and easy availability. However, the poor mechanical and rheological properties of assembled peptide hydrogels cause difficulties in injection, thus limiting further applications. Herein, injectable peptide-based hydrogels with tunable mechanical and rheological properties were obtained by combination with a positively charged poly-peptide (poly-L-lysine, PLL). Electrostatic coupling between PLL and a self-assembling dipeptide (Fmoc-FF) provides a smart switch to enable the fibrous hydrogels to be shear-thinning and self-healing, thus leading to the formation of supramolecular hydrogels with rheological properties suitable for injection. The latter can be flexibly adjusted by merely varying the concentration or the molecular weight of the polypeptide to satisfy a variety of requirements in biological applications. The hydrogels, consisting of helical nanofibers stabilized with disulfide bonds, are prepared and further injected for antitumor therapy. The results demonstrate that the helical fibrous hydrogel, without the addition of antigens, immune regulatory factors, and adjuvants, can activate T cell response and efficiently suppress tumor growth. Therefore, injectable hydrogels self-assembled by a combination of small peptides and bio-macromolecules present a great potential for biomedical applications, especially for development of a new type of immuno-responsive materials towards antitumor therapy.

Keywords: Dipeptide, Rheological properties, Injectable hydrogel, Self-assembly, Antitumor immune response


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INTRODUCTION Self-assembled peptide nanostructures (for example fibers or hydrogels) provide significant advantages in a broad range of applications1-8, such as drug delivery and controlled release9-11, three-dimensional (3D) cell culture12,

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, tissue engineering14, and antitumor therapy15-20.

Injectable hydrogels, especially peptide-based hydrogels are particularly appealing for biomedical

applications,

because

they

provide

various

advantages

including

high

biocompatibility, good biodegradability, simple and distinct structural elements, as well as adjustable mechanical properties21, 22. Peptide-based hydrogels provide numerous benefits such as multivalency, multifunctionality, and specificity23. These hydrogels with high bioactivity provide an ideal platform to perform biological functions, which hold potential for developing therapeutics for critical illnesses as inflammations, cancer, and immunological diseases24. In special supramolecular peptide hydrogels provide routes for the design of immune vaccines and to a certain extent eliminate the need for adjuvants25-28. Injectable hydrogels ask for perfect behavior such as shear-thinning and/or self-healing depending on reversible interactions between biological building blocks. Actually, selfassembled hydrogels, especially peptide-based hydrogels, usually lack tunable interactions between the fibrous structures and thus cannot meet the needs for on-demand applications29-32. Recently, a series of efforts had been spent on the design and construction of hydrogels possessing tunable crosslinking interactions33-38. Most of the commonly used methods are covalent crosslinking approaches initiated by ultraviolet radiation, extreme pH, and high temperature or by chemical agents such as glutaraldehyde. These are regarded as biologically incompatible, and the purification procedures are expensive and tedious, thus, limiting the applications of hydrogels39-41. Self-assembly via non-covalent interactions provides new routes


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for fabricating injectable hydrogels with transient and reversible crosslinks. Numerous attempts have been reported for the self-assembly of functional hydrogels via non-covalent interactions as hydrogen bonding, hydrophobic or electrostatic interactions, polymer-nanoparticle (NP) interactions42 and host-guest interactions. These dynamic and reversible interactions are of great significance for developing hydrogels with flexible mechanics. However, viscoelastic behavior and shear-thinning or self-healing properties for supramolecular hydrogels are facing challenges, as one has to compromise flexibility and stability 22. The poly-peptide used here (poly-L-lysine, PLL) is a well-known cationic bio-macromolecule with excellent biocompatibility. It is commonly used for enhancing penetration through (negatively charged) cell membranes43. Under mild conditions, N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) can be efficiently self-assembled into fibrous hydrogels, but its poor mechanical properties limit the application of injection12. Here, we found that the electrostatic interactions between the negatively charged dipeptide Fmoc-FF and the positively charged PLL can enhance the entanglement and tunability between the self-assembled peptide fibers. This led to the formation of injectable hydrogels with adjustable rheological properties, exhibiting shearthinning and self-healing. It may offer a new alternative for design and fabrication of novel injectable materials with distinct supramolecular organization and perfect viscoelastic behavior. Therefore, an injectable hydrogel based on combination of Fmoc-FF and PLL-SH is constructed for antitumor therapeutic application. The self-assembled nanofibers have a helical structure, to some extent similar to fimbrial antigen in biology44. Introduction of a thiol group can further stabilize the fabricated helical fibrous structures by formation of a disulfide bond. This kind of hydrogel consisting of helical nanofibers can serve as a promising vaccine to activate the T cell response and efficiently suppress tumor growth without the need of antigens, immune regulatory


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factors, and adjuvants. They are able to open the door for immune-regulation and anticancer applications.


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MATERIALS AND METHODS Materials. N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) was a product of Bachem Company. Poly-L-lysine (PLL) with different molecular mass (1000-5000, 4000-15000, 1500030000, 30000-70000) and Truant’s reagent were purchased from Sigma. Dimethyl sulfoxide (DMSO), Na2B4O7·10 H2O, EDTANa2, hydrochloric acid (HCl), Tris-HCl, NaCl, CaCl2, and NaN3 were products of Beijing Chemical Works. 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2H-tetrazolium bromide (MTT) was bought from Alfa Aesar. RPMI 1640, bovine serum, penicillin, streptomycin, pancreatin, proteinase K and PBS were purchased from M&C Gene technology LTD. Formalin, paraffin, hematoxylin and eosin were supplied by the institute of laboratory animal science, Chinese academy of medical sciences. Mouse IFN-gamma Platinum Elisa Kit was a product of eBioscience. Water was prepared by a double-stage Millipore Milli-Q Plus purification system and was replaced every 12 h. All solutions were freshly prepared and stored at 4 oC. Preparation of hydrogels. The Na2B4O7-EDTA buffer solution was prepared first. Na2B4O7·10 H2O (572 mg) and EDTANa2 (3.7 mg) were dissolved in 10 mL Milli-Q water, then the pH of the buffer solution was adjusted to 7.5 with the help of 0.1 M hydrochloric acid (HCl). The powder of N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) (2 mg) was pre-dissolved in 10 uL dimethylsulfoxide (DMSO). Then 485 uL of Milli-Q water was mixed with 5 uL of buffer solution and added into the Fmoc-FF solution above. After sonication for 15 minutes, the mixture solution was aged for 3 days at room temperature. Fmoc-FF hydrogels were thus obtained. Poly-L-lysine (PLL) was dissolved in the above buffer solution with a final concentration of 4 mg mL-1. The Fmoc-FF/PLL hydrogels were obtained after aging for 3 days by mixing 2 mg Fmoc-FF (pre-dissolved in 10 uL DMSO) with 5 uL 4 mg mL-1 PLL buffer


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solution and 485 uL Milli-Q water. Truant’s reagent (0.5 mg) was added into 4 mg mL-1 PLL buffer solution with a concentration of 0.25 mg mL-1. Then the mixture solution reacted in dark for 4 h. The product was dialyzed three times in order to eliminate raw materials and intermediate products of the reaction using buffer solution. Then the mixture was degassed and flushed with N2 at room temperature for 4 h to obtain the PLL-SH solution. Then PLL-SH was desalted using a spephadex G-25 column and a concentration of 4 mg mL-1 was established. The hydrogel of Fmoc-FF/PLL-SH was prepared utilizing the same method as for the Fmoc-FF/PLL hydrogel. Proper ultrasonic treatment and three days aging were also needed to obtain the hydrogels. Morphological characterization. The hydrogel samples were freeze dried before visualization by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The dried hydrogel samples were gently dropped into a drop of Milli-Q water on the surface of carefully washed and dried freshly cleaved mica. Then mica samples were aired-dried prior to imaging by AFM. The AFM test was carried out using a Bruker (USA) dimension operated in tapping mode with a Scan Asyst-Air cantilever under ambient conditions. SEM measurements were performed by a Hitachi S-4800 SEM working at 15 kV or a Philips XL 30 electron microscope at an acceleration voltage of 3 kV after sputtering a layer of gold/Pt. A thin layer of hydrogel was spread on a carbon-coated copper grid. After drying in a desiccator, the transmission electron microscopy (TEM) images were taken with a JEOL JEM-1011 transmission electron microscope, operated at an acceleration voltage of 200 KV. Rheological characterization. Dynamic rheology experiments were performed at room temperature with an Anton Paar MCR302 rheometer. Dynamic oscillatory frequency sweep measurements were conducted at 1% strain amplitude, and oscillatory strain amplitude sweep


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measurements were carried out at a frequency of 10 rad s-1. Storage (G’) and loss modulus (G’’) were determined and analysed using a TA instruments TRIOS software. In order to test the recovery properties of the hydrogels, a larger shear of 500% and a smaller shear of 1% were applied regularly on the hydrogels. The instantaneous viscosity modulus was measured for a controlled frequency of 1 rad s−1 and a wide range of strain from 0.01% to 10%. The experiments were performed using a parallel plate of 15 mm diameter with a proper gap. A lid was prepared in order to induce the evaporation of water existing in the hydrogels. Circular dichroism spectroscopy (CD). In order to study the secondary structure of the hydrogels, circular dichroism spectra were measured using a JASCO J-815 spectrometer at room temperature. The hydrogels were freshly diluted by water or buffer solution before the experiments. The wavelength was set from 185 to 260 nm and a quartz cell with a path length of 1 mm was selected. Each spectrum is the average of three measurements in order to guarantee the experimental accuracy and reproducibility. Cytotoxicity experiments. The cytotoxicity of the hydrogels was assessed by the methyl thiazolyl tetrazolium (MTT) viability assay against B16 cells. The cells were seeded in 96-well plates at 10,000 cells per well in 100 µL of RPMI 1640 containing 10% bovine serum, supplemented with 50 IU/mL penicillin and 50 IU/mL streptomycin, and incubated at 37 oC in 5% CO2 atmosphere for 24 h, followed by adding gel at different concentrations. In order to avoid the effect of water contained in the hydrogels, they were freeze-dried using a vacuum freeze dryer, and then an equivalent of complete RPMI 1640 was used to replace water. After incubation for another 24 h, the cells were treated with 0.1% MTT and incubated for 4 h for dye incorporation. Afterwards, the blue formazans were eluted in DMSO, and the absorbance of


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MTT was measured at 490 nm using a fluorescence microplate reader. The cell viability (%) was calculated by comparison with the control wells. All experiments were performed in triplicate. Degradation of hydrogels in vitro and in vivo. The degradation medium was prepared as follows: 5.0 units/mL proteinase K, 10 mM CaCl2 and 0.2 wt% NaN3 was added into the TrisHCl buffer (0.05 M, pH 7.4), and then the mixture media were placed in a water bath at 37 oC for 4 h in order to activate the proteinase K. As a control, another Tris-HCl buffer (0.05 M, pH 7.4) was prepared. Fmoc-FF/PLL-SH hybrid hydrogels with an approximate diameter of 16 mm and the same dry weight were dropped into degradation media or control buffer, respectively. The gels were incubated at 37 oC, and the dry weight was recorded versus time. The degradation media and control buffer were replaced every 12 h. The Fmoc-FF/PLL-SH hydrogel (100 uL) was injected subcutaneously (s.c.) into the C57/BL6 mice. At day 1 and day 7, the mice were sacrificed and the remaining hydrogels were analysed around the position of the B16 tumours. Subcutaneous mouse model. At the right hind hip of female C57/BL6 mice (about 16 g body weight, 4-6 weeks), purchased from Vital Laboratory Animal Centre (Beijing, China), 100 uL of B16 cell suspension with a concentration of 2.0×107 cells mL-1 was implanted. In accordance with the guidelines of international animal care, the mice were raised in a specific pathogen free (SPF) facility. Enough clean food and water was provided and the growth of tumours was observed every day. After a week of feeding, the B16 subcutaneous mouse model was well built. Antitumor experiment in vivo. The mice were segregated into two groups, when the size of the tumour reached about 200 mm3: the mice of the treatment group were infused subcutaneously (s.c.) with Fmoc-FF/PLL-SH hydrogel (1 uL/mm3 of tumour) once every other day; the mice of the control group were subcutaneously (s.c.) injected with the same volume of NaCl (0.9%). The tumour volume and the body weight were monitored every day. The tumour volume was


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calculated using the following equation: Tumour volume = (Length × Width2)/2. Mice were sacrificed after 12 days of treatment, and the tumours and main organs (heart, lung, liver and kidney) were obtained. The organ index was calculated. The tissues were washed twice using PBS, and then fixed with 4% formalin solution and embedded in paraffin. The dried paraffin blocks were chopped into slices with 5 mm side length and transferred onto clean glass slides. Then standard hematoxylin-eosin (H&E) staining was used. Later, the slides were observed through an optical microscope and photos were taken at × 100 magnification. Flow cytometry. When the tumours grew to about 200 mm3, the mice were divided into four groups: normal mice without hydrogel treatment (Norm Con), normal mice with hydrogel treatment (Norm IM), B16 mice without hydrogel treatment (Model Con) and B16 mice with hydrogel treatment (Model IM). The Fmoc-FF/PLL-SH hydrogel was subcutaneously (s.c.) injected once every other day. After 12 days treatment, the spleen was taken out and washed by PBS. Then, single cell suspension was prepared from fresh hepatocellular carcinoma tissue in DHanks medium. Then the cell suspension was passed through a 200 mesh filter and centrifugation for ten minutes with a rotation speed of 1000 unit. The supernatant liquid was discarded, and the precipitate was washed by 0.83% PDDA twice. Last, the single cell suspension of lymphocyte obtained was stained with Abs against various cell surface markers. The following panel of commercially available and directly fluorochrome conjugated anti-mouse monoclonal antibodies were used in the study: PE anti-mouse CD3, FITC anti-mouse CD4, PE anti-mouse CD8, PerCP anti-mouse CD8, were purchased from eBioscience. Samples were run on a Beckman flow cytometer and data was analysed using Flow Jow 6.0 software. Statistical analysis. Statistical analysis was undertaken using the SPSS version 13.0 software. Data were expressed as means and standard deviations (± SD). T test analysis was used in cell


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experiments. One-way analysis of variance (ANOVA) was used to compare groups with FmocFF/PLL-SH hydrogel treated or not in the animal studies. Values of P

Self-assembled Injectable Peptide Hydrogels ... - ACS Publications

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