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In vivo imaging of the stability and sustained cargo release of an injectable amphipathic peptide-based hydrogel Edith Oyen, Charlotte Martin, Vicky Caveliers, Annemieke Madder, Bruno Van Mele, Richard Hoogenboom, Sophie Hernot, and Steven Ballet Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01840 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 14, 2017
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In vivo imaging of the stability and sustained cargo release of an injectable amphipathic peptide-based hydrogel Edith Oyen,1,2,3 Charlotte Martin,1 Vicky Caveliers,4 Annemieke Madder,5 Bruno Van Mele,2 Richard Hoogenboom,3* Sophie Hernot4* and Steven Ballet1* 1
Research Group of Organic Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, 1050
Brussels, Belgium. 2
Research Group of Physical Chemistry and Polymer Science, Vrije Universiteit Brussel,
Pleinlaan 2, 1050 Brussels, Belgium 3
Supramolecular Chemistry Group, Department of Organic and Macromolecular Chemistry,
Ghent University, Krijgslaan 281-S4, 9000 Ghent, Belgium 4
In Vivo Cellular and Molecular Imaging, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090
Brussels, Belgium. 5
Organic and Biomimetic Chemistry Research Group, Department of Organic and
Macromolecular Chemistry, Ghent University, Krijgslaan 281-S4, 9000 Ghent, Belgium
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Abstract
Hydrogels are promising materials for biomedical applications such as tissue engineering and controlled drug release. In the last two decades, the subclass of peptide hydrogels is gaining increasing interest from the scientific community thanks to their numerous advantages, such as biocompatibility, biodegradability and most importantly, injectability. Here, we report on a hydrogel consisting of the amphipathic hexapeptide H-FEFQFK-NH2, which has previously shown promising in vivo properties in terms of releasing morphine. In this study, the release of a small molecule, a peptide and a protein cargo as representatives of the three major drug classes is directly visualized by in vivo fluorescence and nuclear imaging. In addition, the in vivo stability of the peptide hydrogel system is investigated through use of a radiolabeled hydrogelator sequence. Although it is shown that the hydrogel stays present during several days, the largest volume reduction takes place within the first 12 hours after subcutaneous injection, which is also the time frame wherein the cargos are released. Compared to the situation where the cargos are injected in solution, a prolonged release profile is observed up to 12 hours, showing the potential of our hydrogel system as a scaffold for controlled drug delivery. Importantly, this study elucidates the release mechanism of the peptide hydrogel system which seems to be based on erosion of the hydrogel providing a generally applicable controlled-release platform for small molecule, peptide and protein drugs.
Keywords Controlled-release, peptide hydrogels, in vivo imaging, radiolabeling
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1. Introduction Hydrogels – three-dimensional network structures which can retain large amounts of water – have proven to be useful material scaffolds in several biomedical applications such as tissue engineering and sustained drug release.1,2 In the latter application, the hydrogel matrix serves as a drug delivery depot that slowly releases entrapped drugs in a controlled way, rendering the drug concentration in blood plasma more stable over an extended period of time.3 As such, frequent administration and high drug dosages can be avoided, consequently leading to a potential reduction in toxicity and side effects, and thus ultimately favouring the patient’s comfort and compliance.4,5 Moreover, the hydrogel matrix creates an aqueous environment in which the pharmaceuticals are protected from early degradation, which in turn increases the half-life time of the drugs. This asset is of great interest especially for biopharmaceuticals, such as peptide and protein therapeutics, which are commonly short-living due to protein unfolding, fast enzymatic degradation, etc.5,6 Depending on the nature of the cross-links in the gel network, hydrogels can be classified into two major classes: chemically (permanent) or physically (reversible) cross-linked hydrogels. The first are built-up from covalently cross-linked building blocks, while the latter rely on physicochemical (non-covalent) interactions such as hydrophobic interactions, hydrogen bonding, ionic interactions and Van der Waals interactions.1,7,8 Recently, the interest in physically cross-linked hydrogels has increased, especially in the fields of drug delivery and tissue engineering, because these gels offer specific advantages over chemically cross-linked hydrogels.8,9 Physical cross-linking does, for example, not depend on the addition of cross-linking agents and organic solvents, which are often toxic and thus have to be removed from the gel before (clinical) application.8,9 Moreover, due to the highly stable structure of covalent bonds in chemically cross-linked hydrogels, these hydrogels cannot be delivered through the needle of a syringe, requiring either surgical implantation or sometimes
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unreliable in situ gelling methods.7,10 Hence, physically cross-linked hydrogels offer a major advantage because they are held together by weak, non-covalent interactions, which can be perturbed by the application of shear stress. Indeed, passage of the gel through the needle of a syringe causes a temporary rupture of the physicochemical interactions by which they exhibit viscous flow. After cessation of shear, a restructuration phase takes place during which the non-covalent interactions are restored, resulting in a reassembly of the gel structure.10 One particular subclass of these physically cross-linked materials encompasses peptidebased hydrogels. The use of peptides as building blocks is truly attractive since the resulting hydrogels are among others biodegradable (i.e. naturally degradable by proteolytic enzymes), they have a high chance of being biocompatible (i.e. not provoking an immune response) and cytocompatible (i.e. non-cytotoxic and allowing cells to adhere and proliferate), they are easy to synthesize and – as explained above – injectable.10,11 This work covers the in vivo evaluation of an injectable peptide hydrogel composed of amphipathic peptides, wherein hydrophobic amino acids are alternated with hydrophilic amino acids. It is conceivable that these peptides form β-sheets and β-sheet bilayers, and selfassemble into fibers. Entanglement of the fibers further ensures formation of the hydrogel network (Figure 1).12-14 The investigated system comprises the previously reported amphipathic hexapeptide H-FEFQFK-NH2 1 (Figure 1), which showed promising results for the in vitro release of model drugs and in vivo release of morphine.15,16 In the present work, the stability of this peptide hydrogel as well as its controlled cargo release capacity is monitored in vivo over time using non-invasive imaging techniques. Herein, both nuclear and fluorescence imaging modalities were investigated. To this end, the release of representative cargos of the three most important drug classes (i.e. a small molecule with MW < 500 Da, a peptide with MW of ca. 2 kDa and an antibody-derived fragment (nanobody) of ca. 15 kDa) was evaluated. To our knowledge, we are the first to address the in vivo behaviour of these
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types of amphipathic peptide-based hydrogels and to screen the in vivo controlled-release of different types of cargos from the same peptide gel system. Also, this study is important for the elucidation of the release mechanism of this peptide hydrogel system.
-strand
hydrophobic collapse
-strand alignment -sheet
= hydrophobic
-sheet bilayer
= hydrophilic
O
H N
H3N
N H
O
1
O
O
H N
O N H
O H2N
H N
fiber
hydrogel network
O NH2
O
O H3N
Figure 1. Schematic representation of the self-assembly process of amphipathic hexapeptide H-FEFQFK-NH2 1.
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2. Materials and methods 2.1. Synthesis and 111In-labeling of the peptide hydrogelator Peptide synthesis - Peptide hydrogelator 1 and its DOTA-labeled analogue 6 were synthesized by standard Fmoc-strategy solid phase peptide synthesis (SPPS) on Rink Amide resin (ChemImpex, polystyrene matrix, 100-200 mesh, 0.47 mmol g-1). Amino acid (3 eq.) activation
was
carried
out
with
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HBTU, 3 eq.) and N,N-diisopropylethylamine (DIPEA, 4 eq.) in dimethylformamide (DMF). Coupling of amino acids lasted for 40 min at room temperature (rt). Fmoc-deprotection was carried out using 4-methylpiperidine (20 v/v% solution in DMF), while washing steps were performed with DMF and dichloromethane (DCM). For the synthesis of N-terminally derived DOTA-peptides, DOTA-tris(tert-butyl)-ester was used (2 eq.), and coupled by an activation with 2 eq. HBTU and 3 eq. DIPEA, and a coupling time of 1 h. Cleavage and side chain deprotection was executed using a trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/water (95:2.5:2.5, v/v/v) mixture at rt for 1.5 h. After vacuum evaporation, crude peptide was precipitated in diethyl ether and lyophilized. After dissolution in acetonitrile/water (ca. 50:50, v/v), purification was carried out by preparative reversedphase high-performance liquid chromatography (RP-HPLC). Collected fractions were lyophilized to retrieve the purified peptide (> 98 % purity according to HPLC analysis). 111
In-labeling of DOTA-peptide -
111
InCl3 (5 µl, ± 4 MBq) (Mallinckrodt) was added to
200 µL 1 mg/mL DOTA-peptide 6, dissolved in 0.2 M ammonium acetate buffer (NH4OAc) pH 5, and incubated for 30 min at 50°C. Radiochemical purity (> 95 %) was verified by ITLC using 0.1 M citrate buffer pH 5 as mobile phase. 200 µL of
111
In-peptide hydrogelator 6 was
diluted up to 1 mL (c(6) = 154 µM) with phosphate buffered saline (PBS, 10 mM) and incorporated within the hydrogel as described below. 2.2. Synthesis and 111In-labeling of the different cargos
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Fluorescent cargo - IRDye800CW N-hydroxysuccinimide (NHS) ester (Li-COR Biosciences) was solubilized at 4 mg/L in PBS. The solution was stirred for 2 h in order to hydrolyze the NHS ester (completion of the reaction verified by HPLC), resulting in compound 2 as fluorescent cargo (MW = 1068 g/mol). The dye solution was either used as such, or incorporated within the hydrogel as described below. Small molecule cargo - DOTA-4-amino-2-cyclohexylmethyl-indolo[3,4-c]azepin-2-on 3 was prepared in three standard deprotection-coupling steps from its t-butyloxycarbonylprecursor (Boc-precursor). Synthetic details can be found in ESI. Peptide cargo - DOTA-peptide cargo 4 was synthesized analogous to DOTA-peptide 6 (see above). Nanobody® (Nb) cargo - The conjugation of p-SCN-Bn-CHX-A’’-DTPA chelator ([(R)-2amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid, Macrocyclics) to the nanobody cAbVCAM1-517 was performed as described previously.18-21 Briefly, twenty-fold molar excess of chelator was incubated with 1 mg/mL (68 µM) of Nb dissolved in 0.05 M sodium carbonate buffer pH 8.7 for 2 h at rt, for conjugation to the primary ε-amine lysine side chain groups of the Nb.21 The conjugated Nb 5 was subsequently purified by size-exclusion chromatography on a Superdex 75 10/300 GL column (GE Healthcare) in 0.1 M NH4OAc pH 7 (elution rate 0.5 mL/min). 111
In-labeling of different cargos -
111
InCl3 (10-20 µL, ± 9 MBq) was added to 1 mg of
DOTA-compound 3, 1 mg of DOTA-peptide 4 or 4.5 nmol of DTPA-Nb 5 (supplemented up to 1 mg with unconjugated Nb) dissolved in 200 µL 0.2 M NH4OAc pH 5, and incubated for 30 min at 50°C. Radiochemical purity (> 97 %) was verified by ITLC using 0.1 M citrate buffer pH 5 as mobile phase.
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In-labeled molecules were diluted up to 1 mL with PBS (the corresponding molar
concentrations are: c(3) = 1434 µM, c(4) = 446 µM, c(5) = 68 µM) and either used as such, or incorporated within the hydrogel as described below. 2.3. Hydrogel preparation For the preparation of 111
111
In-labeled hydrogel and hydrogels loaded with fluorescent dye or
In-labeled cargos, the above described 1 mL PBS-containing solutions were added to
20 mg of TFA salt of peptide 1. After sequential vortexing and sonication, hydrogels (2 w/v%) were formed almost immediately (gelation within 5 min or shorter) and let to rest overnight. 2.4. In vivo monitoring of cargo release with fluorescence imaging Animal study protocols were approved by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel (also for section 2.5.). After subcutaneous injection of 150 µL of hydrogel containing 0.5 nmol of IRDye 800CW in athymic nude mice (Charles River, n = 3 per group), fluorescence reflectance images of the posterior side of the mice were acquired over 24 h using the Fluobeam800 (Fluoptics) under 2.5 % isoflurane anesthesia (IsoVet, Eurovet NV/SA). Fluorescence images were overlaid with white light images for anatomical localization. Regions of interest (ROI) were drawn around the site of injection and total fluorescence signal was measured within these ROIs using ImageJ software. 2.5. In vivo monitoring of stability and cargo release with SPECT/CT C57Bl/6J mice were purchased by Charles River (n=3 per group) and all experimental procedures were performed under 2.5 % isoflurane anesthesia (Iso-vet). 150 µL (0.518 ± 0.037 MBq) of the
111
In-labeled hydrogel (stability experiment) or 150 µL (1.702 ± 0.777
MBq) of hydrogels loaded with 111In-labeled cargos (cargo release experiment) were injected subcutaneously on the hind limb. For comparison, 150 µL (1.517 ± 0.335 MBq) of
111
In-
labeled cargos not loaded into a hydrogel were also subcutaneously injected. The specific
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activity at the time of injection was 3 MBq/mg for from 8 to 15 MBq/mg for the
111
111
In-labeled hydrogelator 6 and ranged
In-labeled cargos 3, 4 and 5. Immediately after injection, at
1h30, 3h, 6h, 12h, 24h, 48h and 72h, animals were subjected to a SPECT/CT scan. SPECT images were acquired on an e.cam180 system (Siemens) equipped with two triple-pinhole collimators designed for
111
In (1.5 mm pinhole opening, 250 mm focal length, 47 mm radius
of rotation).21 Images were acquired over 360° in 64 projections of 30 s into a 128 x 128 matrix and reconstructed using an iterative algorithm correcting for radioactive decay and allowing automatic fusion with CT images (based on six
57
Co fiducial markers). Micro-CT
imaging was performed using a dual-source CT scanner (Skyscan 1178, Bruker) with 60 kV and 615 mA at a resolution of 83 µm and a scan time of 2 min. Images were reconstructed using a filtered back-projection algorithm. Maximum intensity projections (MIPs) were generated in Osirix Medical Software. Images were quantified using the software AMIDE (sourceforge.net). For the stability experiments, the size of the gel was determined by automatic delineation*, for the cargo release experiments, the total radioactivity within a ROI drawn on the hind limb was calculated. Results were expressed as percentage of injected dose (%ID/cc) remaining in function of time and images were scaled to the same level. Repeated measures (mixed model) ANOVA was used for the comparison between gel and control groups at different time points (Graphpad Prism 5, significance level was set at 0.05 (*p
