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Enzymatic biodegradation of hydrogels for protein delivery targeted to the small intestine Jennifer M Knipe, Frances M Chen, and Nicholas A Peppas Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm501871a • Publication Date (Web): 12 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
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Biomacromolecules
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Enzymatic biodegradation of hydrogels for protein 8
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delivery targeted to the small intestine 12 13 14 16
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Jennifer M. Knipe a, Frances Chen a, Nicholas A. Peppas a,b,c* 17 18 20
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a Department of Chemical Engineering, C0400, The University of Texas at Austin, Austin, TX 2
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78712, USA 23 24 25
b Department of Biomedical Engineering, C0800, The University of Texas at Austin, Austin, TX 26 28
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78712, USA 29 31
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c College of Pharmacy, C0400, The University of Texas at Austin, Austin, TX 78712, USA 32 3 34 35 37
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KEYWORDS hydrogel, biodegradation, trypsin, peptide, intestinal delivery 38 39 41
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Multi-responsive poly(methacrylic acid-co-N-vinyl pyrrolidone) hydrogels were synthesized 43
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with biodegradable, oligopeptide crosslinks. The oligopeptide crosslinks were incorporated using 4 45
EDC-NHS zero-length links between the carboxylic acid groups of the polymer and free primary 46 48
47
amines on the peptide. The reaction of the peptide was confirmed by primary amine assay and IR 50
49
spectroscopy. The microgels exhibited pH-responsive swelling as well as enzyme-catalyzed 51 53
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degradation targeted by trypsin present in the small intestine, as demonstrated upon incubation 5
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with gastrointestinal fluids from rats. Relative turbidity was used to evaluate enzyme-catalyzed 56 57 58 59 60 ACS Paragon Plus Environment
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degradation as a function of time, and initial trypsin concentration controlled both the 5
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degradation mechanism as well as the extent of degradation. Trypsin activity was effectively 6 8
7
extinguished by incubation at 70°C, and both the microgels and degradation products posed no 10
9
cytotoxic effect to two different cell lines. The microgels demonstrated pH-dependent loading of 1 12
the protein insulin for oral delivery to the small intestine. 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment
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Introduction 4 6
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Environmentally-responsive hydrogels, or hydrophilic, crosslinked polymer networks that 7 9
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undergo physicochemical changes in response to one or more environmental stimuli, offer the 1
10
specificity of highly tunable materials combined with excellent biocompatibility 1-4. As the next 12 13
generation of biomaterials, these “intelligent” networks are able to respond to or mimic 14 15
biological environments and processes such as vascularization 5, 6, tumor physiology 7, 8, 18
17
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endosomal compartments 9-11, or the extracellular matrix 12, 13. This capability could be 19 20
instrumental in achieving various biomedical advances, including tissue regeneration and 21 23
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controlled delivery of biological therapeutics 14, 15. 24 26
25
Hydrogels with pH-responsive behavior are among the most widely utilized “intelligent” 27 28
hydrogel systems for drug delivery applications 16. Polyanionic hydrogels such as 31
30
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poly(methacrylic acid) (PMAA) exhibit complexation via hydrogen bonding at low pH 32 3
conditions, such as that of gastric fluid, and undergo increased swelling due to ionization of the 34 35
carboxylic groups at neutral pH conditions, such as that of the intestine 17. Thus, PMAA 38
37
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copolymers have been widely utilized as oral drug delivery carriers or coatings for their ability 39 40
to protect a loaded therapeutic from denaturation and proteolytic degradation as it travels through 41 43
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gastric conditions yet swell and release the therapeutic at the site of absorption in the small 45
4
intestine 18-23. 46 47 49
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Biodegradation is another possible environmental response of hydrogels designed as drug 51
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delivery applications 14, 24. Polymers that degrade by hydrolysis, such as polyanhydrides 25, 26, 53
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poly(orthoesters) 2, 27, poly(caprolactone), and poly(lactic acid) and poly(glycolic acid) 28, 29 are 54 56
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widely used for drug delivery. However, polymers that undergo enzyme-catalyzed degradation 57 58 59 60 ACS Paragon Plus Environment
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are becoming attractive as site-specific delivery vehicles due to the localized concentration of 5
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enzymes throughout the body as well as the specificity of enzymatic attack 30, 31. A relatively 6 8
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small number of researchers have investigated the use of hydrogels with enzyme-degradable 10
9
peptide components for the purpose of drug delivery to the small intestine 30, 32, 33, where 12
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enzymes such as trypsin, chymotrypsin, and cathepsin-β are prevalent 34. In particular, peptide 13 15
14
crosslinks are an appealing route to achieve enzyme-specific degradation of the network and 17
16
subsequent drug delivery 30, 32, 33, 35, 36. For example, oligopeptide crosslinks of different lengths 18 19
were incorporated into poly(N-(2-hydroxypropyl)- methacrylamide) (PHPMA) hydrogels, and 20 2
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the release of macromolecules from the gels upon exposure to chymotrypsin or liver enzymes 24
23
was determined to be a function of both peptide length and structure 33. Wanakule et al. 35 25 27
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showed the rapid drug release from peptide-crosslinked PLGA microspheres triggered by trypsin 29
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reached a plateau after only hours, compared to hydrolysis and diffusion mediated release that 31
30
occurred over days. Both the control and timescale of the peptide degradation are attractive 32 34
3
characteristics for drug delivery, particularly via an oral route. 35 37
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Here, poly(methacrylic acid-co-N-vinylpyrrolidone) (P(MAA-co-NVP)) polymer chains 38 39
were used to impart hydrophilic and pH-responsive behavior that either inhibited or allowed 40 42
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diffusion of enzymes into the polymer network due to pH-responsive complexation. The polymer 4
43
chains were crosslinked by a facile bioconjugation reaction with an oligopeptide rich in arginine 45 46
and lysine groups targeted for degradation specifically by the enzyme trypsin. Synthesis, 47 49
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degradation, cytocompatibility, and therapeutic loading and release using the pH-responsive 51
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P(MAA-co-NVP) crosslinked by biodegradable peptide are detailed herein. 52 53 5
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Experimental 56 57 58 59 60 ACS Paragon Plus Environment
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Materials 4 6
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Methacrylic acid (MAA), N-vinyl-2-pyrrolidone (NVP), Irgacure 184® (1-hydroxy7 9
8
cyclohexyl-phenylketone), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride 1
10
(EDC), purified pepsin from porcine gastric mucosa (≥2500 U/mg) and pancreatin from porcine 12 13
pancreas (4x USP specifications), trypsin-EDTA solution (1X) and Nα-benzoyl-L-arginine ethyl 14 16
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ester hydrochloride (BAEE) trypsin substrate, and recombinant human insulin (≥27.5 IU/mg) 18
17
were purchased from Sigma-Aldrich (St. Louis, MO). N-hydroxysuccinimide (NHS) was 19 20
purchased from Pierce Biotechnology, Inc. (Rockford, IL). The custom sequence oligopeptide 21 23
2
GRRRGK was synthesized by CHI Scientific (Maynard, MA). Fluorescamine was purchased 25
24
from Acros Organics (Geel, Belgium). All reagents were used as received. All other solvents and 26 28
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buffers were purchased from Fisher Scientific (Waltham, MA). 29 31
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Methods 32 3 34
Synthesis and Purification 35 36 38
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P(MAA-co-NVP) linear polymer was synthesized by photoinitiated, free-radical 40
39
polymerization. MAA and NVP were added at a 1:1 molar ratio to a 1:1 (w/w) deionized water 41 42
and ethanol solution to yield a 1:3 (w/w) total monomer to solvent ratio. Photoinitiator Irgacure 43 45
4
184® was added at 1 wt% with respect to total monomer weight. The mixture was homogenized 47
46
by sonication then the flask was sealed with a rubber septum. The solution was purged with 48 50
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nitrogen for 20 minutes, then the polymerization was initiated with a Dymax BlueWave® 200 52
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UV point source (Dymax, Torrington, CT) at 100mW/cm2 intensity and allowed to polymerize 54
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for 30 minutes while stirring. 5 56 57 58 59 60 ACS Paragon Plus Environment
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Following polymerization, the linear polymer was purified from unreacted monomer by 5
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addition of 1 N hydrochloric acid (HCl) to precipitate polymer, centrifugation, and resuspension 6 8
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in deionized water. After three wash cycles, the polymer solution was neutralized, frozen in 10
9
liquid nitrogen, and lyophilized. 1 12 13
To synthesize the peptide-crosslinked hydrogels, linear P(MAA-co-NVP) was dissolved in a 14 16
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1:1 (v/v) water:ethanol solution at a concentration of 50 mg/ml. EDC was dissolved in ethanol at 18
17
a concentration of 50 mg/ml and NHS was dissolved in ethanol at a concentration of 16 mg/ml. 19 20
The EDC and NHS solutions were added to the polymer solution at a ratio of 6:3:1 21 23
2
polymer:EDC:NHS by weight. The solution was mixed by vortex, then allowed to react for ~3 25
24
min with shaking. The pH was raised to ~8 by the addition of 1 N sodium hydroxide (NaOH), 26 28
27
and then a volume of 100 mg/ml peptide in ethanol solution was added to achieve a 2:1 weight 30
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ratio of polymer:peptide. The mixture was allowed to react overnight with shaking then purified 32
31
by three wash cycles with water and centrifugation at 10,000 x g for 5 minutes. Following the 3 35
34
washes, the polymer was frozen in liquid nitrogen and lyophilized for at least 24 hours. 36 38
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After lyophilization, the polymer was milled into a fine power by crushing with mortar and 39 40
pestle. The powder was sifted to the size ranges of 30-75 µm and less than 30 µm by 41 43
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ultraprecision ASTM sieves (Precision Eforming, Cortland, NY). 4 46
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Characterization 47 48 50
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Potentiometric Titration 51 53
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To determine the MAA content of the linear polymer, a 3.5 mg/ml solution of polymer in 54 5
deionized water was titrated to pH 11.5 using 0.2 N NaOH (standardized with potassium 56 57 58 59 60 ACS Paragon Plus Environment
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hydrogen phthalate) at 25°C with constant stirring. pH was measured with a Mettler-Toledo 5
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SevenEasy™ (Columbus, OH) pH probe and was recorded when the pH reached a steady value 6 8
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(±0.01 pH units in three consecutive measurements over 5 minutes). The equivalence point was 10
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used in conjunction with a charge balance to determine the amount of MAA present in each 1 12
formulation. 13 14 16
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Fluorescamine Assay 17 19
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The fluorescamine solution was prepared fresh before each test by dissolving 3 mg of 20 21
fluorescamine in 10 ml filtered acetone. Supernatant from the EDC-NHS reactions was mixed in 2 24
23
a range of dilutions with phosphate buffered saline (PBS) and the fluorescamine solution with 26
25
agitation. After reacting at room temperature with shaking for 15 min, 200 µl of each sample 27 29
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was transferred in triplicate to a black 96-well plate and the fluorescence at 360 ex/460 em was 31
30
measured using a Bio-Tek Synergy™ HT multi-mode plate reader(Winooski, VT), 32 3
sensitivity=85. 34 35 37
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Fourier Transform Infrared Spectroscopy 38 40
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Fourier transform infrared spectroscopy (FTIR) spectra were obtained using a Thermo Mattson 41 42
Infinity Gold spectrometer (Thermo Fisher Scientific Inc., Waltham, MA). The incubation buffer 43 45
4
of degraded hydrogel samples was exchanged with water using 30,000 MWCO centrifugal filters 47
46
(Millipore, Billerica, MA) over 5 washes. Samples were lyophilized and then pressed in KBr 48 49
(Sigma-Aldrich) disks. For each sample, 512 scans were performed with a resolution of 4 cm-1 50 52
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and gain of 1.0, and background spectra of a KBr blank disk was subtracted from the sample 54
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spectra. 5 56 57 58 59 60 ACS Paragon Plus Environment
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Scanning Electron Microscopy 4 6
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Scanning electron microscopy (SEM) samples were prepared by dusting carbon tape-covered 7 9
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aluminum stubs with lyophilized, crushed microgels. The samples were coated with 8-10 nm of 1
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Pt/Pd coating using a Cressington 208 Benchtop sputter coater (Watford, England). Scanning 12 13
electron microscopy images were obtained using an FEI Quanta 650 FEG scanning electron 14 16
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microscope (Hillsboro, OR) and a Zeiss Supra 40V scanning electron microscope (Jena, 18
17
Germany). 19 20 2
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Degradation 23 25
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Microgels were degraded at various trypsin concentrations in 1X phosphate buffered saline 27
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solution (pH 7.4), simulated gastric fluid, simulated intestinal fluid, rat gastric fluid or rat 28 30
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intestinal fluid. Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared 32
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according to USP 29 37. Briefly, the SGF was prepared by dissolving 2 g of sodium chloride and 3 34
3.2 g of purified pepsin from porcine stomach mucosa in ~800 ml deionized water. 7 ml of HCl 35 37
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was added, followed by enough water to make up to 1 L and the pH adjusted to 1.2. SIF was 39
38
prepared by dissolving 6.8 g monobasic potassium phosphate in 250 ml deionized water, then 77 40 41
ml of 0.2 N NaOH was added while stirring. 500 ml additional water was added then 10 g 42 4
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pancreatin was mixed into the solution. The pH was adjusted to 6.8 using 0.2 N NaOH or HCl 46
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then the solution was made up to 1 L with water. 47 48 50
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Gastrointestinal fluids were harvested from Sprague Dawley juvenile male rats (250-300 g) 52
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according to a protocol published by Yamagata et al. with some modifications 38. Briefly, after 54
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sacrificing the rat the stomach was excised and ligated at both ends. A needle was inserted to 5 57
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inject 5 ml of pH 1.2 HCl-NaCl buffer (same as SGF minus pepsin) and the gastric contents were 58 59 60 ACS Paragon Plus Environment
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collected in a 50 ml centrifuge tube. Similarly, a ~20 cm section of the upper small intestine was 5
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cannulated and flushed twice with 10 ml cold PBS (1X, pH 7.4). The fluid was collected as 6 8
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intestinal fluid in a 50 ml centrifuge tube. Both the harvested fluids were centrifuged at 3,200 x 10
9
g, 4°C, for 15 min to separate solids from the fluids. The supernatants were retained as rat gastric 1 12
fluid and rat intestinal fluid, respectively. Fluids were stored at -20°C until use. 13 14 16
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Degradation was measured by relative turbidity of the solutions over time, as reported by 18
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Klinger and Landfester 39. Microgels were suspended in trypsin solutions of varying 19 20
concentration, PBS, SGF, SIF, or rat gastrointestinal fluids at various concentrations. 100 µl of 21 23
2
each sample solution, including pure solutions of PBS, SGF, SIF, or rat gastrointestinal fluids as 25
24
a control, was added to a 96-well plate in triplicate. The absorbance was then measured at 500 26 28
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nm in 5 minute intervals over 90 minutes using a Bio-Tek Synergy™ HT multi-mode plate 30
29
reader (Winooski, VT). The temperature was controlled at 37°C and the plate underwent shaking 32
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for 3 seconds before each measurement. 3 34 36
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Activity of the trypsin following incubation with particles and deactivation methods including 38
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addition of serum-containing cell culture media or 5 minutes incubation at 60°C, 70°C, or 80°C, 40
39
was evaluated using a trypsin activity assay adapted from the protocol by Yanes et al. 40. Briefly, 41 43
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degradation supernatant was combined with 1 mg/ml BAEE in PBS at a 1:9 sample:BAEE ratio 45
4
by volume. Immediately after addition of the BAEE, absorbance at 253 nm was measured at the 46 47
minimum interval (typically 40-50 seconds) for 5 minutes using a Bio-Tek Synergy™ HT multi48 50
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mode plate reader (Winooski, VT). 51 53
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In vitro Cytotoxicity Study 54 5 56 57 58 59 60 ACS Paragon Plus Environment
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L929 and RAW 264.7 cell lines were obtained from American Type Culture Collection 5
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(ATCC, Rockwell, MD). All cell lines were cultured in Dulbecco’s modified Eagle medium 6 8
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(DMEM) (Mediatech, Herndon, VA) supplemented with 10% heat-inactivated HyClone™ Fetal 10
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Bovine Serum, USDA Tested (Fisher Scientific), 1% 200 mM L-glutamine solution (Mediatech), 1 12
100 U/ml penicillin, and 100 µg/ml streptomycin (Mediatech). Cytotoxicity studies were 13 15
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performed using DMEM without phenol red supplemented with 2% heat-inactivated HyClone™ 17
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Fetal Bovine Serum, USDA Tested (Fisher Scientific), 1% non-essential amino acids 18 19
(Mediatech), 100 U/ml penicillin, and 100 µg/ml streptomycin (Mediatech). Cells were 20 2
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incubated at 37°C in a 5% CO2 environment. 23 25
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Cells were seeded at a density of 10,000 cells/well in a 96-well plate and allowed to incubate 26 28
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for 24 hours prior to the experiment. Microgels were degraded in 1.25 or 0.625 mg/ml trypsin in 30
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PBS at concentrations ranging from 1.3-6 mg/ml. Degradation took place at 37°C with shaking 32
31
for at least 4 hours. Trypsin was deactivated by addition of 2X volume DMEM without phenol 3 35
34
red containing 2% fetal bovine serum. Cells were incubated with degraded microgels for 8 hours 37
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at 37°C and 5% CO2. The cytotoxic effect of the microgels was evaluated using a CellTiter 96® 38 39
Aqueous One Solution Cell Proliferation MTS Assay (Promega, Madison, WI). MTS assay was 40 42
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added to the wells and incubated for 90 minutes at the same conditions before absorbance 4
43
measurements were made at 490 nm using a Bio-Tek Synergy™ HT multi-mode plate reader 45 46
(Winooski, VT). Cytotoxicity is reported as ‘relative cell proliferation’, or normalization of the 47 49
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assay absorbance values to the average assay absorbance for cells incubated in only culture 51
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media. 52 53 5
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Insulin Loading 56 57 58 59 60 ACS Paragon Plus Environment
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Microgels were loaded by equilibrium partitioning post-synthesis with recombinant human 5
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insulin. Microgels were incubated at 37°C for 4 hours in a 0.5 mg/mL insulin solution of pH ~5.5 6 8
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at a ratio of 7:1 microgel:therapeutic by weight. The microgels were collected by centrifugation 10
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at 10,000 x g for 5 minutes then collapsed by resuspension in 0.5 N HCl. Microgels were 1 12
separated from supernatant by centrifugation at 10,000 xg for 5 minutes. The loaded microgels 13 15
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were lyophilized and stored at -20°C for further studies. Protein loading was evaluated with a 17
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MicroBCA assay protein quantification assay (Pierce-Thermo, Rockford, IL). 18 19 20
Results and Discussion 21 23
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Synthesis, purification and lyophilization of the uncrosslinked P(MAA-co-NVP) yielded 25
24
white, fluffy polymer. Due to the phase transition of MAA from hydrophobic to hydrophilic 26 28
27
above pH ~5, adjusting the pH of the solution to neutral prior to freeze drying facilitated the 30
29
solubilization of the dried polymer into aqueous solution. Potentiometric titration was used to 32
31
determine that the linear polymer was approximately 45 mol% or 39 wt% MAA. 3 35
34
The number average molecular weight of linear chains, Mn, may be calculated from the 37
36
kinetics of free-radical polymerization for each polymer 41. 39
38
= 42
41
40
(1)
/
Values for the rate constants were found for both methacrylic acid and N-vinylpyrrolidone42, 45
4
43
the initiator efficiency, f, was approximated as 0.75, and the value of a, a constant related to the 46 47
method of termination was 1.33 48
41
. Actual values were input for monomer concentration [M],
50
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initiator concentration [I], and molecular weight of the repeat unit M0. The number average 52
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molecular weight of linear chains of each polymer was found to be on the order of 10,000, which 54
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in in agreement with other values reported 43, thus linear chains of the copolymer are expected to 5 57
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Scheme 1 shows the mechanism of the EDC-NHS crosslinking reaction with the linear 5
4
P(MAA-co-NVP). Upon solubilization of the polymer in the ethanol-water solution, the pH was 6 8
7
adjusted to ~5 to favor the activation of the carboxylic acid groups by EDC and increase the 10
9
stability of the active ester intermediate 44. EDC was added at a molar ratio of 1:2 to the MAA 1 12
groups on the linear chains, and NHS was added at a molar ratio of 1:1.8 to the EDC. Upon 13 15
14
addition of the EDC and NHS the solution became turbid but no precipitation was evident. Both 17
16
the EDC and NHS were dissolved in ethanol to limit instability due to hydrolysis while 18 19
maintaining polymer solubility. 20 21 2 23
O
H3C Cl H N+ CH3
24 25
H3C + H N CH3
OH
26 27
H3C O
HN
NH2
HN
HN
HN
NH2
NH2
+
O
N
28
NH
H3C O
O H2N
O NH
NH
CH3
O
NH O
NH
OH
NH O
N
29
CH3
32
31
Scheme 1. 3
HN
HN
O NH
NH
O
O
O
NH
NH O
OH
NH O
HN
HN
30
H3C
CH3
NH2
HN
O
O NH
HN
HN
HN
N
+
NH2
HN
NH2
NH2
Peptide crosslinking reaction scheme. Carboxylic acid groups on the
35
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poly(methacrylic acid-co-N-vinylpyrrolidone) linear polymer are activated by EDC, then react 37
36
with at least two of the five primary amine groups on the GRRRGK peptide to form a 38 39
crosslinked hydrogel network. 40 41 43
42
After activation of the carboxylic acid groups, the pH was raised to ~7-8 to facilitate attack on 45
4
the primary amines of the oligopeptide 47
46
44
. Various polymer:EDC:NHS:peptide weight ratios
were tested to maximize peptide incorporation. All formulations with peptide content below a 48 50
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polymer:peptide ratio of ~3:1 failed to produce hydrogels. A best performing formulation of 52
51
solvents, polymer, and EDC-NHS based on reaction efficiency and reproducibility was an 53 54
ethanol-water mixture with a polymer:EDC:NHS:peptide weight ratio of 20:10:3.3:10. 5 56 57 58 59 60 ACS Paragon Plus Environment
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The peptide was added at a molar ratio of 1:3.6 relative to the EDC; the free amine groups 5
4
were in 1.4x excess relative to the theoretical maximum of activated carboxyl groups. At this 6 8
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feed ratio, the degree of crosslinking is expected to be about 3. Upon addition of the peptide 10
9
solution the mixture was immediately turbid and precipitation of crosslinked polymer was 1 12
evident. After reacting for at least 8 hours the crosslinked polymer typically resembled an 13 15
14
amorphous hydrogel. 17
16
Following washes and lyophilization the hydrogel appeared as fluffy white chunks, as seen in 18 19
Figure S.1 (initial). The dried hydrogel was easily crushed into a powder consisting of particles 20 2
21
