Hemopressin-based pH-sensitive hydrogel - ACS Publications

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Hemopressin-based pH-sensitive hydrogel: a potential bioactive platform for drug delivery Huy Dao, Jun Chen, Bernabe S Tucker, Vinoy Thomas, Ho-Wook Jun, Xing-Cong Li, and Seongbong Jo ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b00423 • Publication Date (Web): 11 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018

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ACS Biomaterials Science & Engineering

Hemopressin-based pH-sensitive hydrogel: a potential bioactive platform for drug delivery

Huy Minh Daoa, Jun Chenb, Bernabe S. Tuckerc, Vinoy Thomasc, Ho-Wook Junb, Xing-Cong Lid, Seongbong Joa,* a

Department of Pharmaceutics and Drug Delivery, University of Mississippi, MS 38677,

USA. b

Department of Biomedical Engineering, University of Alabama at Birmingham, AL 35294,

USA. c

Department of Materials Science and Engineering, University of Alabama at Birmingham,

AL 35294, USA. d

National Center for Natural Products Research, University of Mississippi, MS 38677, USA.

*Corresponding

author:

Seongbong Jo Department of Pharmaceutics and Drug Delivery, University of Mississippi, MS 38677, USA E-mail address: [email protected] Tel: +1 662 915 5166 Fax: +1 662 915 1177

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Abstract Peptides with proper sequences are capable of self-assembling into well-defined nanostructures, which can subsequently grow and entangle into three-dimensional nanomatrices. In this study, hemopressin – a cannabinoid receptor modulating peptide derived from α-chain of hemoglobin known to self-assemble into nanofibrils – was examined for its potential applicability as a gelator. The results indicated that hemopressin’s gel formation was dependent on pH and salt concentration. Although hemopressin’s macroscopic states showed differences, its microscopic structure remained largely unchanged, in which it consisted mainly of the anti-parallel β-sheet conformation as confirmed by FTIR (C=O stretch peaks at 1630 cm1

and 1695 cm-1) and CD (β-sheet peak at 195 nm). The major difference between the gel and

sol states was displayed in the fibrils length, in which the gelation at pH 7.4 resulted in 4-μm fibril, whereas the solution at pH 5.0 showed an 800-nm fibril. The pH-dependent sol-gel phase transition property was then utilized for the investigation of the pH-responsive release of FITCdextran (4 to 40 kDa) from hemopressin fibrillary gel. Finally, the biocompatibility of the peptide was demonstrated by proliferation assay of cultured bone marrow mesenchymal stem cells (hMSCs). Altogether, the results suggested that hemopressin is a potentially promising candidate as therapeutically active platform for drug delivery.

Keywords: Hemopressin, β-sheet, Self-assembly, Peptide hydrogel, pH-sensitive


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1.

Introduction Peptide-based self-assembling systems have recently emerged as a novel versatile material

with expanding applications in various fields including drug delivery 1–5 and tissue engineering scaffolds 6–11. It is recognized that the secondary structure of peptide fibril that forms from selfassembly mainly consists of α-helix, β-sheet, β-hairpin, and random coil. These fibrils, given proper conditions, can grow and entangle into a three-dimensional hydrogel network. The forces that drive such self-assembly process include π- π stacking, hydrogen bonding, hydrophobic interaction, and opposing forces such as electrostatic repulsion 12–16. Peptidic hydrogel systems possess several attractive traits that can be potentially advantageous in drug delivery applications. First, facile synthesis of peptides via solid-phase methods allow specific sequence modifications to achieve various desired properties 17–20. For example, the suppression of hemopressin’s aggregation tendency was effectively displayed by analogs with conservative sequence changes 21. In another example, multidomain-peptide fibers were used in conjunction with liposomes to form a unique hydrogel system that could release bioactive agents in a controlled-manner at multiple time-points 22. As exemplified by previous studies, such peptides with varied sequences could assemble into various types of ordered structures at nano-scale through hierarchical self-assembly processes 15,23,24. Second, most peptide-based hydrogels are highlighted by their formations in ambient conditions that mimic physiological states 18,25. For example, RADA16 peptide exhibits sol-gel transformation upon exposure to neutral pH 26,27. Similarly, a wide variety of di-phenylalanine self-assembly structures - ranging from nanotube, vesicle, fibril, to hydrogel - could be obtained by simple adjustments of physical parameters such as solvent type, pH, or concentration

6,28

.

The fact that such hydrogel formations could occurs at physiological conditions with high internal hydration renders both the system’s biocompatibility, as toxic cross-linker were not



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associated, and capability to encapsulate and release bioactive molecules without altering their secondary and tertiary structures 1,27,29. Finally, various peptides are recognized for notable therapeutic activities, which allow them to become self-deliverable or produce synergistic effects when used as drug carriers. For example, lanreotide – a synthetic octapeptide which acts as a growth hormone inhibitor – is capable of forming gel at 10% w/w, and has been approved in several countries including U.S. and U.K. for acromegaly treatment via subcutaneous long-acting implants 30. Hemopressin is an oligopeptide with nine-amino-acid residues (PVNFKFLSH; Pro1-Val2Asn3-Phe4-Lys5-Phe6-Leu7-Ser8-His9) originally derived from the α-chain of hemoglobin. This peptide is known to have specific binding to cannabinoid 1 (CB1) receptors

31–34

, to act as an

inverse agonist 32. It exerts anti-nociceptive effect on carrageenan-induced hyperalgesia in rats 35

and reduces food intake in mice without causing any noticeable side effects 36. Microstructure

of hemopressin was investigated by several authors, where it was reported to be related to βsheet fibril formation under physiologically relevant conditions 37. Furthermore, the chirality of Val2, the side chain of Asn3, Leu7, and C-terminal carboxylic acid are considered the major contributions toward peptide aggregation, where substitution in any of the four positions could suppress aggregation. Moreover, positive charge lysine residue (Lys5) flanked by aromatic residues phenylalanine (Phe4 and Phe6) was also suggested to be important in facilitating peptide aggregation 21. Herein, we report pH-sensitive hemopressin hydrogel system as a potential candidate for bioactive platform for drug delivery. In light of previous findings on the peptide, we hypothesized that hemopressin β-sheet fibrils have the potential to grow, entangle, and ultimately form a reversible hydrogel network under appropriate conditions. Based on the pKa of His9 (6.04) and Lys5 (10.67) residues, we expected pH-sensitive gelation of hemopressin. Considering the peptide was previously suggested to adopt random coil conformation in water


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or aqueous solution at pH 5.4

31

, we predicted that the combined effects of two positively

charged residues would effectively disfavor fibril lengthening in acidic condition and thereby remain in solution form. At sufficiently elevated pH, however, the lone protonation of Lys5 leads to reduced electrostatic repulsion between individual hemopressin molecules, and consequently favor the fibril lengthening to the level where gelation could occur. Additionally, the electrostatic repulsion between individual peptide molecules can be reduced by the shielding effect caused by negatively charged ions. Hence, beside increased pH, increased buffer concentration was also thought to be the factor that favors gelation. In this study, we have simulated the conditions that could induce hemopressin’s gelation in a pH-dependent manner and demonstrated its potential applicability as drug delivery platform to achieve synergistic therapeutic efficacy.



2.

Experimental section

2.1.

Materials

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Hemopressin (molecular weight 1088, purity 97.5%) was obtained from NovoPep limited (Shanghai, China). Pierce BCA protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA). Fluorescein isothiocyanate dextran (FITC-dextran) of molecular weights 4, 10 and 40 kDa, and FITC modified bovine serum albumin (FITC-BSA) were purchased from Sigma (St. Louis, MO). Cyquant cell proliferation assay kit (Invitrogen) was purchased from Thermal Fisher Scientific (Waltham, MA). Bone marrow mesenchymal stem cells (hMSCs) were purchased from Lonza, Inc (Walkersville, MD). All other chemicals were obtained from Fisher Scientific (Hanover Park, IL) and used without further purification. 2.2.

Hemopressin fibril size and zeta potential The size and zeta potential of hemopressin fibril were measured using a Zetasizer Nano

(Malvern Instruments, UK). Hemopressin solutions of 0.4 mg/ml concentration were prepared in 10 and 100 mM phosphate-buffered saline (PBS) of pHs 5.0 and 7.4, acidified and alkalized water. All measurements were done at 173-degree scattering angle at 25 oC, using disposable polystyrene cuvettes, and folded capillary cells. 2.3.

Gel formation visual observation PBS solutions at different pH level (5.0 to 11.0), and concentration (10 to 400 mM) were

prepared by dissolving PBS tablets into an appropriate amount of water, and pH was adjusted with 0.1 N sodium hydroxide or hydrochloric acid solution. In a glass vial, 40 μl of PBS solution was mixed with 10 µl hemopressin stock solution of a concentration of 4 mg/100 µl. The mixtures were kept for 2 h at room temperature and subsequently observed for gel formation by inverting the tubes. 2.4.

Rheology study


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All samples were prepared as cylindrical shape of 9 mm in diameter and 6 mm in height. Hemopressin was dissolved in PBS solutions at different pH and salt concentrations. The samples were incubated for 2 h at 37 oC before measurements. Rheometry measurements were conducted using an AR-2000 rheometer (TA Instruments) equipped with an environmental control furnace and aluminum parallel plate. The top plate had a diameter of 12 mm. The bottom plate with ring shaped depression, in a diameter of 25 mm, functioned as the solvent trap. Viscoelasticity of hemopressin hydrogel was tested by oscillatory frequency sweeps in a range of 0.01 to 10 Hz, using a controlled strain at 0.1%. Strain sweep measurements were conducted at 37 oC with a conditioning step of 2 min equilibration time in a strain range of 0.05 to 100% at a constant frequency of 1 Hz. 2.5.

Fourier transformed infrared spectroscopy Fourier-transform infrared (FTIR) spectra were collected using an Agilent Cary 660 FTIR

spectrophotometer. Hemopressin was dissolved in water, 0.1 M PBS solution of pHs 5.0, 7.5 and 8.5 at a concentration of 10 mg/ml. The samples were kept for 1 h at room temperature, flash-frozen using dry ice and subsequently dried under vacuum using a freeze dryer (Labconco, USA). Dry hydrogel powder was analyzed by attenuated total reflectance (ATR) FTIR using 64 scans at a resolution of 1 cm-1, wavenumber ranging from 1000 to 2000 cm-1. 2.6.

Circular Dichroism spectroscopy Circular dichroism (CD) spectra were collected using an OLIS DSM 20 CD

spectrophotometer. Hemopressin solutions were prepared at a concentration of 100 μM in different media including water, phosphate buffers of pHs 5.0 and 7.4 at concentrations 20 and 50 mM; and 10 mM phosphate buffer containing 50 mM NaCl. All spectra were acquired at 20 o

C using a quartz cuvette with a light path of 1 mm. The number of scan was 5 at a scanning

speed of 50 nm/min. A blank spectrum was acquired from the corresponding media and was subtracted from each spectrum.



2.7.

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pH-sensitivity and drug release study Hemopressin was dissolved in 40 μl pure water to yield a clear peptide solution at

concentration of 2 mg/40 μl. FITC-dextran and FITC-BSA were dissolved in 60 μl 100 mM PBS and each solution was added to the hemopressin solution to result in a hemopressin concentration of 2 mg/100 μl. The final solution was kept for 2 h at room temperature to allow gel formation. Prepared hemopressin hydrogels were then covered with 1000 μl PBS of pHs 5.0, 7.5, and 8.5. Ionic strength of the solution was either 0.01 or 0.1 M. Test samples were incubated at 37±0.5 ºC in an orbital shaker. At predetermined time points, 200 μl of the supernatant was withdrawn and, then diluted using PBS of same pH. The diluted samples were then centrifuged at 5000 rpm for 5 minutes before analyzing concentration of dextran or BSA. Two hundred microliters of fresh solution was subsequently added to test samples to make up for the volume of withdrawn samples. The amounts of released FITC-dextran and FITC-BSA were analyzed using a Perkin Elmer fluorescence spectrophotometer (LC500), at an excitation wavelength of 490 nm and an emission wavelength of 525 nm. Amounts of released hemopressin from a blank hydrogel were also determined by Pierce BCA protein assay to characterize pH-sensitivity and degradation property of the hydrogel. The process was based on manufacturer’s protocol, in which, 100 μl peptide samples were incubated with 2.0 ml working reagents at 37 ºC for 30 minutes and subsequently measured absorbance at 562 nm. 2.8.

Human mesenchymal stem cells viability study The effects of hemopressin on cell viability and proliferation were investigated using cell

viability kit and Cyquant cell proliferation assay. Specifically, human bone marrow mesenchymal stem cells (hMSCs) within passage numbers 5 were used for the experiment. Cells were grown with MSCGM™ Mesenchymal Stem Cell Growth medium kit from Lonza. Inc. The hMSCs were seeded onto a 48 well plate at a cell density of 9000 cells per well. After


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the cells were attached to the well, hemopressin was added to the well at concentrations ranging from 0.0001 mg/ml to 1mg/ml. CyQuant cell proliferation assay and live and dead cell (viability/cytotoxicity) imaging were both triplicated. Cells in the culture plates were maintained under standard culture conditions (37 °C, 95% relative humidity, and 5% CO2). Cell viability was measured at days 1 and 4 after the addition of hemopressin into the well. The images of live and dead cells were captured using Nikon fluorescence microscope. After live and dead cell assay imaging, the samples were collected and stored at -80 °C for CyQuant proliferation assay based on the manufacturer’s protocol. In the process of CyQuant cell proliferation assay, the fluorescence intensity of samples was measured using a microplate reader (EL×800, BIO-TEK Instrument, VT) at 480 nm (excitation) and 520 nm (emission). Finally, the fluorescence intensity was converted to the cell number according to a cell number standard calibration curve. 2.9.

Statistical analysis Statistical analysis was performed using RStudio

38

. All values are expressed as mean

values (n=3) with standard deviation (SD). After the normality was checked, one-way analysis of variance (ANOVA) for multiple comparisons was employed with post hoc test Tukey. Statistical significance was accepted at p

Hemopressin-based pH-sensitive hydrogel - ACS Publications

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