Factors Affecting Enzymatic Degradation of Microgel-Bound Peptides

2013, 14 (7), pp 2317–2325. DOI: 10.1021/bm400431f. Publication Date (Web): June 4, 2013. Copyright © 2013 American Chemical Society. *E-mail: ...
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Factors Affecting Enzymatic Degradation of Microgel-Bound Peptides Ronja Månsson,* Göran Frenning, and Martin Malmsten Department of Pharmacy, Uppsala University, P.O. Box 580, SE-751 23 Uppsala, Sweden S Supporting Information *

ABSTRACT: Proteolytic degradation and release of microgel-bound peptides was investigated for trypsin, poly(acrylic acid-co-acrylamide) microgels (70−90 μm in diameter), and oppositely charged polylysine, using a method combination of confocal microscopy and micromanipulator-assisted light microscopy. Results show that trypsin-induced release of polylysine increased with increasing trypsin concentration, decreasing microgel charge density and decreasing peptide molecular weight. While the microgel offered good protection against enzymatic degradation at high microgel charge density, it was also observed that the cationic peptide enabled trypsin to bind throughout the peptide-loaded microgels, even when it did not bind to the peptide-void ones. With the exception of highly charged microgels, proteolytic degradation throughout the peptide-loaded microgel resulted in the generation of short and non-adsorbing peptide stretches, giving rise to the concentration and peptide length dependence observed. A simple random scission model was able to qualitatively capture these experimental findings. Collectively, the results demonstrate that microgel charge density, peptide molecular weight, and enzyme concentration greatly influence degradation/release of microgel-bound peptides and need to be considered in the use of microgels, e.g., as carriers for protein and peptide drugs.

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INTRODUCTION Microgels are lightly cross-linked hydrogel particles in the 10 nm to 100 μm range, with a capacity to drastically change their volume in response to changes in the external environment, e.g., pH, ionic strength, temperature, specific metabolites, reducing conditions, or external fields.1−3 This enables microgels to bind and store substances, e.g., drugs, and to release them upon stimuli, making them potential candidates as drug delivery vehicles and functional biomaterials.1,2,4−10 Apart from opportunities offered as delivery systems for injectables, microgels are promising in other delivery routes due to fast response.11,12 Other prospectives for microgels, with or without drug loading, include biomaterials (e.g., as surface coatings to reduce chronic inflammation and to improve biocompatibility), regenerative medicine, and depot formulations.11,13,14 Of particular interest to microgels as delivery systems are biomacromolecular drugs, notably proteins and peptides, which have become progressively more important in drug development during the past decades, e.g., as a result of the decoding of the human proteome. Microgels potentially offer a range of advantages as delivery systems for such drugs, including controlled drug release rate, protection from enzymatic and chemical degradation, preservation of protein secondary and tertiary structure, and avoidance of aggregation, all translating into maintained biological activity, as well as decreased toxicity, immunogenicity, and other biological side effects.11,15,16 As has been recently summarized,2,11,17−20 some of these functional advantages with microgels as delivery systems for proteins, peptides, and DNA/siRNA have indeed been directly demonstrated in literature. Furthermore, a series of studies during the past few years have elucidated significant parameters © XXXX American Chemical Society

affecting peptide binding, distribution, and release from microgels,2 including effects of electrostatic and hydrophobic interactions,21−24 peptide length,25−27 cyclization,28 aggregation,29,30 and secondary structure.31 Apart from operational observations,32 however, little is known about factors determining enzymatic degradation of microgel-bound peptides. In the work presented here, microgel charge density, peptide length, and enzyme concentration are therefore investigated regarding their effect on peptide degradation and release and how this can be coupled to peptide and enzyme distribution within the microgels. In doing so, poly(acrylic acid-coacrylamide) microgels of various charge densities are employed, along with polylysine of varying length, and trypsin, a serine protease that cleaves peptides at positively charged lysine and arginine residues,33 and which is overexpressed, e.g., in inflammatory and cancerous tissues.34 Since it was previously found that polylysine could be prevented from accessing the entire microgel at low pH due to network collapse,25,27 pH variations were kept to a minimum to avoid such complicating factors affecting material distribution, as well as pH-dependent enzyme activity. Furthermore, peptide and enzyme distribution was monitored through confocal microscopy, while network swelling was monitored through micromanipulator-assisted light microscopy. The experimental results obtained were compared to predictions from a random scission model for enzymatic degradation of microgel-bound peptides. Together, this enables a comprehensive Received: March 27, 2013 Revised: May 31, 2013

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uptake in gels and enables switching of solution composition (e.g., peptide and enzyme concentration) during the run of the experiment. Furthermore, the setup provides laminar flow with a controllable (flow-dependent) unstirred layer thickness, enabling theoretical analysis of diffusion.30 Captured gel particles were photographed using Viewfinder, Studio 3.0.1 (Pixera, San Jose, USA), and the gel particle diameter was measured using Olympus DP-soft (Olympus, Tokyo, Japan). The volume ratio is expressed as V/V0, were V is the volume of a gel particle after peptide/enzyme exposure for a certain time, and V0 is the volume of a gel particle in 150 mM borate buffer, pH 7.4. The peptideinduced volume response of single poly(acrylic-co-acrylamide) microgels (70−90 μm in diameter when fully swollen) upon peptide binding (5 μM) was studied in borate buffer at pH 7.4 and 150 mM ionic strength by peptide flushing until the gel was fully loaded. To investigate enzymatic degradation of peptide incorporated in microgel, microgels were flushed with trypsin solution of 50, 100, 200, 500, or 1000 U/mL in borate buffer after peptide binding, until the microgels were fully swollen again. The results are presented as the average and standard deviation of reswelling ratios obtained from at least triplicate experiments at 20 °C. Confocal Laser Scanning Microscopy. Peptide and Enzyme Labeling. Polylysine and trypsin were labeled with Alexa Fluor-488 or -633 dye according to a standard protocol recommended by the supplier. In brief, ∼5−10 μg of dye per milligram of peptide/enzyme was reacted for 1 h at room temperature in basic conditions (150 mM carbonate or borate buffer, pH 8.3). Unreacted dye was removed by size-exclusion chromatography using PD-10 columns (GE Health Care, Uppsala, Sweden). The concentration of Alexa-488 or -633 was determined by absorbance measurements at 495 or 632 nm, respectively, with a Helios γ 4.60 spectrophotometer (Thermospectronic, Cambridge, U.K.), while the concentration of peptide or enzyme was measured spectrophotometrically in duplicate experiments after complexation with bisinchoninic acid.35 Absorbance measurements were performed on a Saphire plate reader (Tecan, Männedorf, Switzerland) at 562 nm. The labeling density (Alexa/pLys or Alexa/trypsin molar ratio) thus measured was 0.12 for Alexa488-pLys 10 kDa (195 μM pLys in carbonate buffer), 1.91 for Alexa488-pLys 200 kDa (10 μM pLys in carbonate buffer), 0.15 for Alexa488-trypsin (93 μM trypsin in borate buffer), and 0.034 for Alexa633pLys 10 kDa (159 μM pLys in borate buffer). Labeled and unlabeled polylysine was previously found to result in similar microgel deswelling.26 In addition, comparable findings were found in the present investigation with structurally different Alexa 488 and 633, as was a correlation between results on the effects of molecular weight, enzyme concentration, and microgel charge density for confocal microscopy (using labeled compounds) and micromanipulator-assisted light microscopy (using unlabeled compounds). This indicates that the effect of labeling is not important (at these labeling densities) for the presently investigated systems. Peptide Distribution and Degradation. Five microliters of microgel solution (0.1 w/w%) was equilibrated for at least 24 h with 100−300 μL of fluorescently labeled peptide solution. The distribution and intensity of the labeled peptides within the microgel particles was monitored with a Confocal Leica DM IRE2 laser scanning microscope (CLSM; Leica Microsystems, Wetzlar, Germany) equipped with an Ar laser, using a 63 × 1.2 water objective and LeicaTCSSLsoftware (Leica Microsystems, Wetzlar, Germany). (Note that in contrast to more heavily cross-linked polymer beads, which complicate confocal microscopy due to optical effects,36 these weakly cross-linked microgels do not suffer from such complications.) To investigate the extent of enzymatic degradationinduced peptide detachment in single gel particles, 20 μL of peptidemicrogel solution was vortexed with 750 μL of enzyme solution of 100 or 1000 U/mL for 10 s at 1400 rpm. The solution was then transferred into a confocal microscopy cuvette, and a gel particle was chosen for analysis. Scanning in xz-mode could thus be initialized 2−4 min after mixing, and the middle section of the gel particle scanned every 15 s. To evaluate the average fluorescence intensity in the microgels, ROI (region of interest) analysis was performed. The intensity ratio is expressed as I/I0, where I is the intensity of a gel particle after enzyme exposure for a certain time, and I0 is the intensity of a gel particle at the first measurement (4 min) after mixing with enzyme solution. In analogy, the volume ratio is expressed as A/A0, where A is the projected area of a gel particle after enzyme exposure for a certain time, and A0 is the area of a gel particle at 4 min after mixing with

approach for elucidating mechanisms affecting enzymatic degradation of microgel-bound peptides.

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EXPERIMENTAL SECTION

Materials. For microgel synthesis, N,N′-methylenebisacrylamide (BIS), N,N,N′,N′-tetramethyl-ethylenediamine (TEMED), ammonium persulfate, acrylic acid (AAc), acrylamide (AAm), and acrylamidopropyltrimethylammoniumchloride (APTAC) were obtained from SigmaAldrich (Steinheim, Germany), and sorbitan monostearate (Span 60) was from Carl ROTH (Karlsruhe, Germany). Poly-L-lysine of different molecular weights (pLys 10 kDa, pLys 30 kDa, and pLys 200 kDa) was obtained from Sigma-Aldrich (Schnelldorf, Germany) and used without further purification. Trypsin from bovine pancreas Type I (Product Number T8003, Lot Number 120M7005 V, 12238 BAEE units/mg protein), horseradish peroxidase (HRP) Type VI (Product Number P8375, Lot Number SLBC6822 V, 253 units/mg protein), and Ampliflu Red were from Sigma-Aldrich (Schnelldorf, Germany). Fluorescent tags Alexa Fluor-488 and -633 carboxylic acid succinimidyl ester, mixed isomers, were from Invitrogen (Eugene, USA), while the bisinchoninic acid (BCA) assay kit was from Pierce (Rockford, USA). All other chemicals were of analytical grade. Purified Milli-Q water was used throughout. For pH control, borate, carbonate, or acetate buffers were used, with sodium chloride added to obtain appropriate ionic strength. Microgel Preparation. Poly(acrylic acid-co-acrylamide) microgel particles were synthesized by inverse suspension polymerization as described previously,22 but with a BIS stock solution of 80 mM. In brief, 0.09 g of Span 60 was dissolved in 50 mL of cyclohexane, and the resulting continuous phase was preheated to 45 °C and stirred at 1100 rpm under nitrogen atmosphere. The charge content in the microgels was controlled by varying the amounts of acrylic acid/acrylamide in the monomer solutions (25/75, 50/50, 75/25, and 100/0 mol %).22 Acrylic acid was partly neutralized (60%) by dropwise addition of NaOH (2 M), followed by addition of acrylamide and BIS (1.4 mol %, final concentration of 26 mM in monomer solution), and finally diluted with water to 20 mL. Subsequently, 2.75 mL of the monomer solution was mixed with 30 μL of TEMED (accelerator) and 100 μL of 0.18 M ammonium persulfate solution (initiator) and added to the preheated continuous phase. The polymerization was allowed to proceed at 45 °C for 15 min, whereafter temperature was raised to 65 °C under nitrogen atmosphere to prevent quenching by oxygen. The reaction was stopped after 30−45 min by addition of 40 mL of methanol. Gel particles were left to sediment overnight and then washed repeatedly with methanol and dried in a vacuum oven (Lab-line, Melrose Park, USA). The microgels prepared in this way have previously been characterized in regards to swelling/deswelling as a function of pH, ionic strength, and ionic group content and in regards to charged group titration.22 Quaternary ammonium salt microgels from cationic APTAC were also synthesized with the emulsion polymerization method described above. As described previously,24 a solution of 0.001 g of BIS, 2.5 g of purified water, 5 g of APTAC (75 w/w % in water), and 375 μL of TEMED was prepared. One milliliter of this solution was mixed with 90 μL of ammonium persulfate (0.18 M) and added to a preheated (70 °C) and stirred (1200 rpm) cyclohexane solution (0.09 g of Span 60 in 40 mL of cyclohexane). Gelation occurred almost immediately, and the reaction was stopped after 30 min. Gel particles were repeatedly washed with acetone/water on a P4 vacuum filter. Peptide-Induced Microgel Deswelling/Swelling. Changes in microgel volume upon peptide binding and release were monitored by micromanipulator-assisted light microscopy, as described previously,31 using an Olympus Bx-51 light microscope (Olympus, Tokyo, Japan) equipped with an ONM-1 manipulator (Narishige, Tokyo, Japan) and a DP 50 digital camera (Olympus, Tokyo, Japan). Micropipets (10−20 μm in diameter) were prepared with a PC-10 puller and a MF-9 forger (both Narishige, Tokyo, Japan). Gel particles were captured by micropipet suction using an IM-5A injector (Narishige, Tokyo, Japan), placed inside a 2 mm diameter flow pipet, and flushed with peptide solution using a Peristaltic pump P-1 (Pharmacia, Uppsala, Sweden) at a flow rate of 1.9 mL/min. This experimental setup ensures that the solute composition surrounding the microgel remains constant and unaffected by solution B

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Sn(t ) = S0 e−keff t

enzyme solution. A minimum of three separate gel particles was studied at each condition, and one representative gel particle was chosen for timeresolved diagrams. Activity Measurements. Twenty microliters of microgel solution (0.1 % w/w) was equilibrated for at least 24 h with 200 μL of polylysine 10 kDa (200 μM in carbonate buffer). Thereafter, 200 μL of HRP (1000 U/mL in carbonate buffer) was added and left to equilibrate for at least another 12 h. To compare peptide activity before and after trypsin exposure, 40 μL of pLys-HRP-microgel solution was vortexed with 750 μL of either buffer solution or trypsin (1000 U/mL) for 10 s and thereafter left on a shaking board for two hours. For activity investigations, 2,5 μL of the substrate Ampliflu Red (10 mM in carbonate buffer), and 25 μL of freshly prepared H2O2 (20 mM in water) was added to the above solution and vortexed for 10 s. The mixture was then transferred into a confocal microscopy cuvette, and gel particles chosen for analysis. The conversion of Ampliflu Red to fluorescent resorufin by HRP was monitored using 543 nm laser excitation and 580−680 nm emission collection. Five minutes after mixing with Ampliflu Red and H2O2, the microgel was scanned, and ROI analysis was performed to evaluate the average fluorescence intensity in the microgel. A minimum of three separate gel particles was studied at each condition, and one representative gel particle was chosen for diagrams. Analysis of Peptide Degradation. The analysis assumessequential degradation, for which the enzyme randomly combines with and cleaves peptides at any peptide bond.37 Starting with a peptide containing n residues, the degradation reaction can be schematically represented as

E + Sn ⇌ Cni → E + Si + Sn − i

for i = 1, 2, ..., n − 1

Next, one can introduce functions of time, Pi(t), such that

Si(t ) = S0Pi(t ) e−keff t

E + S2 ⇌ C21 → E + S1 + S1

Pn(t ) = 1

t

n j=i+1

Sj j−1

⎛

n

∫0 ⎜⎜ ∑

⎝ j=i+1

pj (t ′) ⎞ ⎟dt ′ j − 1 ⎟⎠

for i = 2, 3, ..., n − 1 (13)

where t′ is a dummy variable. Hence Pi(t) (i = 2, 3, ..., n) are all polynomials in time. The monomer concentration can be obtained from eq 6 by direct integration. This can be cumbersome when n is large; however, the following shortcut can be used instead. It is clear that the total number of monomers per volume unit, denoted by N, cannot change with time, and since this quantity equals nS0 initially, one obtains n

N=

∑ iSi = nS0

(14)

i=1

(1)

That eq 14 indeed follows from the rate equations can be seen by expanding the time derivative of N with the aid of eqs 4−6, to obtain dN/dt = 0, and using the initial conditions 8 and 9 to determine the constant value as nS0. Hence

(2)

n

S1(t ) = nS0 −

∑ iSi(t ) = S0[n − P1(t ) e−keff t ] i=2

(15)

where n

P1(t ) =

∑ iPi(t )

(16)

i=2

Having obtained all peptide concentrations, the average peptide length is calculated as n

L(t ) =

∑i = 1 iSi(t ) n

∑i = 1 Si(t )

=

N n ∑i = 1 Si(t )

(17)

Finally, the fraction of monomers present in fragments not larger than m can be expressed as

(4)

∑

(12)

Pi(t ) = 2keff

Thus, the enzyme E first combines with an n-mer substrate Sn at peptide bond i to reversibly form a complex Cin. The complex is subsequently irreversibly degraded to fragments of length i and n − i, denoted by Si and Sn−i, respectively. Each fragment is analogously degraded into smaller fragments until only monomers remain. For simplicity, the reaction constants are assumed to be the same for all reactions, a reasonable assumption for homopolypeptides. Specifically, k1 represents the rate of complex formation, k−1 the rate of the reverse process, and k2 the rate of irreversible peptide degradation. When the enzyme concentration is considered as constant and the pseudo-steady-state approximation is used, the rate equations take the form37

dSi + keff Si = 2keff dt

(11)

whereas

(3)

dSn + keff Sn = 0 dt

for i = 2, 3, ..., n

Clearly,

E + Si ⇌ Cij → E + Sj + Si − j for i = 3, 4, ..., n − 1 and j = 1, 2, ..., i − 1

(10)

m

for i = 2, 3, ..., n − 1

Fm(t ) =

(5)

∑i = 1 iSi(t ) n

∑i = 1 iSi(t )

m

=

∑i = 1 iSi(t ) N

(18)

n

Si dS1 = 2keff ∑ dt i 1 − i=2

(6)

where E, Sn, etc. represent concentrations rather than chemical entities, t is time, and keff =

k1k 2E k −1 + k 2

(7)

is an effective rate constant. Assuming that only n-mers exist initially (i.e., ideal monodispersity), the initial conditions are

Sn(0) = S0 Si(0) = 0

(8)

for i = 1, 2, ..., n − 1

(9) Figure 1. Trypsin-induced microgel reswelling kinetics at different enzyme concentrations, for pLys 10 kDa preadsorbed to 25% AAc microgels in 150 mM borate buffer, pH 7.4.

The solution of eqs 4−6, subject to the initial conditions 8 and 9, can be conveniently determined recursively as follows. First, the solution of eq 4 is immediately obtained as C

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RESULTS AND DISCUSSION Effect of Enzyme Concentration. As discussed before,27 incorporation of cationic polylysine into anionic polyacrylate (-containing) microgels results in osmotic deswelling of the microgel network. On peptide release, triggered, e.g., by electrostatic screening at high ionic strength, the collapsed microgel network expands to its dimensions prior to peptide incorporation.22 Similarly, peptide desorption caused by enzymatic degradation can be expected to result in microgel reswelling, at least as long as the enzyme in itself does not cause network collapse. Indeed, as shown in Figure 1, this is the case, as the rate of microgel reswelling increases with enzyme concentration. As most clearly seen for the lower enzyme concentrations, there is an induction time for microgel reswelling that also scales with enzyme concentration. At sufficiently long degradation times, however, complete microgel reswelling is observed at all enzyme concentrations. As shown in Figure 2a, 10 kDa polylysine is evenly distributed throughout the microgel, as is trypsin added subsequently to peptide loading. Thus, the enzyme has access to its peptide substrate throughout the microgel network, and consequently degradation occurs gradually with time throughout the microgel. While degradation-induced desorption occurs relatively similarly in the different microgel regions, a slightly faster intensity decrease is observed in the outermost regions of the microgel, an effect most likely due to a shorter diffusion distance. Furthermore, as can be seen in Figure 2b, the gel reswelling is correlated with a decrease in polylysine concentration, and at sufficiently long degradation time, complete microgel reswelling corresponds to complete polylysine desorption. Thus, as the polylysine concentration in the microgel decreases, osmotic pressure drives water uptake and causes microgel reswelling. A striking result in Figure 2b is that as the peptide intensity in the gel decreases, so does the enzyme intensity, and both peptide and enzyme intensity is down to zero when the gel is fully swollen. In principle, this decrease could be caused by trypsin autolysis, which has previously been demonstrated to occur at hydrophilic surfaces.38 However, since freshly prepared trypsin does not diffuse into poly(acrylic acid-co-acrylamide) microgels in the absence of polylysine at basic pH, this mechanism is less likely. In contrast, trypsin binds readily to positively charged APTAC microgels at the same conditions, whereas at pH 3, the situation is reversed (Supporting Information, Figure S1). A similar behavior has previously been reported by Gai and Wu, who found trypsin uptake in poly(acrylic acid/methylene-bisacryl-amide) microspheres at pH

Figure 2. CLSM images (a) and corresponding intensity (I/I0) plots (b), displaying the distribution of Alexa633-pLys 10 kDa (red) and Alexa488trypsin (green) in pLys-loaded 25% AAc microgels, subsequently exposed to trypsin at 1000 U/mL in 150 mM borate buffer, pH 8.3. In panel b, also degradation-induced microgel reswelling, expressed as projected area (A/A0), is shown. Before exposure to trypsin, microgels were equilibrated with labeled peptide solution for at least 24 h.

Figure 3. Trypsin-induced microgel reswelling for different microgel charge densities. The trypsin concentration was 1000 U/mL, added to microgels with preadsorbed pLys 10 kDa, in 150 mM borate buffer, pH 7.4.

Figure 4. CLSM intensity plots displaying the intensity (I/I0) over time for Alexa488-pLys 10 kDa in 25% (a) and 50% (b) AAc microgels, exposed to trypsin at 100 or 1000 U/mL in 150 mM carbonate buffer, pH 8.3. Before exposure to trypsin, microgels were equilibrated with labeled peptide solution for at least 24 h. D

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4 and pH-triggered release at pH 8.39 Given that the isoelectric point of trypsin is about 10.8,40 this pH and microgel charge behavior is unexpected. As shown in Supplementary Figure S2, charge distribution is relatively even over the protein surface, and hence one would not expect electrostatic interactions to favor trypsin binding to acrylic acid based microgels at low, but not high, pH or to cationic APTAC microgels but not to anionic acrylic acid based ones. Instead, non-electrostatic contributions, such as van der Waals and hydrophobic interactions, are likely to play an important role, considering the relatively low net charge of trypsin at these conditions (+7)41 and propensity of trypsin to favor electrostatically and non-electrostatically driven adsorption relatively equally.42 Thus, at low pH, poly(acrylic acid)-containing microgels are deswollen, resulting in increased protein-microgel van der Waals interactions. For APTAC, on the other hand, hydrophobic interactions are likely to contribute to the trypsin-microgel interaction. In analogy, the presence of polylysine in poly(acrylic acid)-containing microgels causes dramatic microgel deswelling, resulting in promoted van der Waals and hydrophobic contributions to trypsin binding. The presence of polylysine is not expected to completely eliminate trypsin autolysis. However, as discussed above, trypsin is strongly attracted to polylysine, indicated by trypsin binding to polylysine-loaded microgel, but not to the same microgel in the absence of polylysine. In contrast, there is no self-association of trypsin in solution. Thus, by preferential localization at the polylysine, proteolysis of the latter is facilitated and autolysis is suppressed. In line with this, qualitatively similar results were obtained in borate buffer (suppressing trypsin autolysis) and carbonate buffer (not doing so), arguing against pronounced trypsin autolysis. Effect of Microgel Charge Density. Enzyme-induced microgel reswelling rate decreases with increasing microgel charge density (Figure 3), and for the two highest charge densities, 75% and 100%, no degradation-induced peptide desorption is observed. In parallel, Figure 4 shows a faster decrease in microgel-bound polylysine for the lower charge density microgel, while Figure 5 shows a correspondingly faster decrease in trypsin Figure 6. (a) CLSM images and corresponding intensity profiles displaying the distribution of Alexa488-pLys 10 kDa in microgels containing 25% (left) and 50% AAc (right) after equilibration with labeled peptide solution for at least 24 h in 150 mM carbonate buffer, pH 8.3. (b) Effect of microgel charge density on the mean fluorescent intensity from at least 15 separate gels.

significantly contribute to the overall degradation processes.) Together, these results show that the overall degradation process is suppressed at higher microgel charge density. Since electrostatic interactions increase with charge contrast, i.e., at higher microgel charge density, peptide binding affinity increases. This is expected to result in a decreased peptide desorption rate, as previously demonstrated for electrolyteinduced peptide desorption at high ionic strength.22 This means that a lower critical peptide length (i.e., more extensive degradation) is required for peptide desorption to occur. In line with this, facilitated peptide desorption at high ionic strength was found to result in an accelerated complete microgel reswelling (Supporting Information, Figure S4), the latter in agreement with previous findings on effects of microgel charge density on peptide binding, desorption, and electrolyte elutability.22 In Figure 6, representative images and line profiles are shown for 25% and 50% microgels, fully loaded with peptide without

Figure 5. CLSM intensity plot displaying the intensity (I/I0) over time for Alexa488-trypsin in 25% or 50% AAc microgels loaded with 10 kDa pLys, subsequently exposed to trypsin at 1000 U/mL in 150 mM borate buffer, pH 8.3. Before exposure to labeled trypsin, microgels were equilibrated with peptide solution for at least 24 h.

concentration after degradation-induced polylysine desorption. (Supplementary Figure S3 demonstrates that in pure buffer, i.e., in the absence of enzyme, there are some differences in self-leakage. However, since microgel reswelling was essentially unaffected by this minor spontaneous desorption, this is not expected to E

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With increasing peptide length, both the reswelling induction time and the overall reswelling time increase. At sufficiently long degradation times, however, the microgel is completely reswollen for both molecular weights. At this stage, there is no peptide (Figure 8 and Supporting Information, Figure S6) nor trypsin (Figures 2 and 10) remaining in the microgel. However, the peptide distribution within the microgels is different depending on peptide molecular weight, as demonstrated in Figure 9. Thus, while 10 kDa polylysine distributes readily

Figure 7. Trypsin-induced microgel reswelling for different pLys molecular weights. The trypsin concentration was 1000 U/mL, added to 25% AAc microgels with preadsorbed pLys of 10, 30, or 200 kDa molecular weight in 150 mM borate buffer, pH 7.4.

exposure to enzyme. Results show that more polylysine is bound to the higher charged microgel, in agreement with previous studies on the effect of charge contrast on peptide binding to oppositely charged microgels.22,24,25 Therefore, yet another contribution to the slower degradation-induced reswelling kinetics observed for the higher charged microgel (Figure 4) could be that a longer time is required to degrade the larger amount of polylysine bound. Furthermore, as seen in Figure 3, the lower charged gel is less contracted when fully loaded with peptide compared to the higher charged gels. Therefore, access for trypsin to the entire peptide-loaded microgel network is higher for the less charged microgel. The importance of the latter effect is demonstrated in Figure S5 (Supporting Information), where the partially loaded and less deswelled microgel displays much faster enzyme-induced reswelling than the fully loaded and more compact one. Finally, polylysine is expected to bind in a flatter conformation at more highly charged microgel network chains, in analogy to polyelectrolyte adsorption at solid surfaces,42 which may result in a reduced specific activity of trypsin due to difficulties of accessing the flat polylysine chains for scission. Together, these factors provide the higher charge density microgels with a protective effect against proteolytic degradation of incorporated polylysine. Effect of Peptide Molecular Weight. The molecular weight dependence of enzyme-induced microgel reswelling is shown in Figure 7, while Figure 8 shows the polylysine concentration within the microgels during the degradation process.

Figure 9. CLSM images and corresponding intensity profiles, displaying the distribution of Alexa488-pLys 10 kDa (left) and Alexa488-pLys 200 kDa (right) in 25% AAc microgels after equilibration with labeled peptide solution for at least 24 h in 150 mM carbonate buffer, pH 8.3.

throughout the microgel network, 200 kDa polylysine displays a higher peptide concentration in the outermost regions, bordering on the shell formation previously demonstrated for high molecular weight peptides with hydrodynamic radii larger than the microgel mesh size.24,27 When exposed to trypsin, the dense shell formed by the longer peptide partially disintegrates,

Figure 8. CLSM intensity plots, displaying the intensity (I/I0) over time for Alexa488-pLys 10 kDa (a) and Alexa488-pLys 200 kDa (b) in 25% AAc microgels exposed to trypsin at 100 or 1000 U/mL in 150 mM carbonate buffer, pH 8.3. Before trypsin exposure, microgels were equilibrated with labeled peptide solution for at least 24 h. F

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to peptide-void ones, competitive displacement, i.e., desorption by polylysine caused by adsorption of trypsin, can be excluded as origin of the effects observed. Furthermore, the kinetics of trypsin and polylysine distribution within the microgels for the 200 kDa peptide clearly demonstrates enzyme-induced generation of smaller peptide fragments. Given this, the origin of the effects observed is the formation of ever smaller peptide fragments through chain scission by trypsin, previously shown to proceed until 2−3 amino acid oligomers,43,44 until sufficiently short and non-adsorbing peptide fragments are formed. For 50% charged consensus peptides and a 100% acrylic acid microgel, the cutoff between binding and non-binding was previously found to occur at a peptide length between 6 and 12 amino acids,25 while for fully charged oligolysine and 100% acrylic acid microgel, the corresponding cutoff is between 3 and 5 amino acids.45 Employing a simple random scission model for trypsin degradation of polylysine, in which trypsin can bind to any lysine unit and degrade any bond in the peptide with equal rate constant (obviously a simplification, but reasonable for homopolypeptides), results in decay curves of the average peptide length as indicated in Figure 11a. As seen, degradation within the model proceeds monotonically until only lysine monomers remain. While this is a simplification in relation to experimental findings that trypsin degradation proceeds only until 2−3 mers remain,43,44 it is still clear from Figure 11a that degradation proceeds until only peptides shorter than the critical desorption length remains. On the basis of findings by Bysell et al.,25 the cutoff length was assumed to be 10 lysine units for the model calculations, but the precise value of this is not important, as qualitatively similar results are obtained with other cutoff lengths. As seen in Figure 11b, the fraction of lysine residues in peptide fragments shorter than the critical desorption length increases with degradation time. With increasing peptide molecular weight, longer degradation time is obviously needed until only these short fragments remain. The model therefore qualitatively catches the experimental finding of the peptide length dependence. Furthermore, as seen from eq 7, keff scales linearly with the enzyme concentration, and hence also the dependence of the latter agrees semiquantitatively with experimental findings (Figures 1 and 11). Further comparison with experimental data is precluded, as experimental determination of the entire length distribution of the microgel-bound peptide is extremely difficult, if at all possible. The effects of peptide concentration and molecular weight on the time required to reach desorbing peptide fragment lengths are, however, in good agreement with experimental findings. Since the biological activity of proteins and peptides generally relies on their structural integrity, proteolytic degradation is expected to cause a reduction, elimination, or alteration of their biological activity. Demonstrating this to be the case also for microgel-bound peptides/proteins, horseradish peroxidase (HRP) was incorporated, together with polylysine, into acrylic acid microgels. Using resorufin as substrate, confocal microscopy could be used to visualize HRP activity (as opposed to amount incorporated) for microgel-bound HRP, as well as effects of trypsin on this activity. As shown in Supplementary Figure S7, trypsin exposure indeed causes complete elimination of HRP activity for low charge density microgels, but not for high charge density ones, in line with the findings of proteolytic degradation of pLys within the microgels at comparable conditions. To the best of our knowledge, this is the first detailed investigation of enzymatic degradation in polymer microgels and

allowing peptide to redistribute and diffuse into the microgel center, resulting in additional microgel deswelling. Finally, after sufficiently long degradation time, all polylysine is released (Figure 8) resulting in complete microgel reswelling (Supporting Information, Figure S6 and Movies SM1 (10 kDa) and SM2 (200 kDa)). From Figure 10a, it can be seen that trypsin diffuses all the way into the microgel core for both 10 and 200 kDa polylysine.

Figure 10. (a) CLSM images and corresponding intensity profiles for 10 kDa (left) and 200 kDa (right) in 25% AAc microgels, displaying the distribution of Alexa488-trypsin (1000 U/mL) 2 min after addition. (b) CLSM intensity plot, displaying the intensity over time for Alexa488-trypsin in 25% AAc microgels exposed to trypsin at 1000 U/mL in 150 mM borate buffer, pH 8.3. Before exposure to labeled trypsin, microgels were equilibrated with peptide solution for at least 24 h.

Results also show that more trypsin is bound to the microgel for the longer peptide (Figure 10a). In addition, the trypsin concentration stays high, or unchanged, for a longer time for the longer peptide, indicating that the longer peptide stays intact (or at least bound) for a longer time compared to the shorter one (Figure 10b), all in line with the results discussed above. Given the observation of peptide-mediated trypsin incorporation into acrylic acid containing microgels but its non-binding G

dx.doi.org/10.1021/bm400431f | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 11. (a) Average peptide length (in monomer units) during random scission by trypsin for different molecular weights pLys. (b) Fraction of peptide fragments with a length shorter than 10 monomer units. In both figures, degradation is plotted against the composite time parameter (keff·t).

proteolytic degradation was found only at sufficiently high peptide-microgel charge contrast, indicating the latter to be an important factor in the design of microgel delivery systems for peptides and protein drugs.

related systems. There are, however, some studies of related issues, notably from a functional perspective. For example, Ding et al. investigated incorporation of trypsin into vinylene carbonate/β-hydroxyethylene acrylate microbeads and found trypsin activity to increase with increasing degree of bead swelling, in agreement with results of the present investigation.46 Furthermore, Perera et al. demonstrated that insulin-loaded poly(acrylic acid)-cysteine nanoparticles protected insulin from enzymatic degradation, suggested to be due to surface inactivation of trypsin when bound to the poly(acrylic acid) nanoparticle.47 Yamamoto and Hirata furthermore showed that cross-linked polylysine-glutaraldehyde hydrogels are degraded by trypsin, with optimal conditions for trypsin units over 500, pH 7−11, and 50−200 mM salt concentrations, the latter two effects observed also in the present investigation.48 In addition, recent studies on enzyme-responsive microgel and microcapsule delivery systems have shown these to exhibit triggered release of incorporated biologics (such as IgG, DNA, and heparin) in the presence of trypsin.49,50 However, detailed studies of factors determining enzymatic degradation of peptides incorporated in microgels or related systems have not been reported previously.

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ASSOCIATED CONTENT

S Supporting Information *

Additional light microscopy and CLSM images and plots, along with structure illustrations of trypsin are provided together with two CLSM movies of the enzymatic degradation of Alexa488 labeled polylysine 10 kDa (SM1) and 200 kDa (SM2) respectively. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. Helena Bysell and Dr. Bjö rn Walse are gratefully acknowledged for support with experimental design and with protein structure representation, respectively, and Maria Høtoft Michaelsen is gratefully acknowledge for performing early trial experiments on these systems. This work was financed by the Swedish research Council.

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CONCLUSIONS Trypsin-induced release of polylysine incorporated into acrylic acid/acrylamide microgels increases with increasing trypsin concentration, decreasing microgel charge density, and decreasing peptide molecular weight. These effects are due to trypsin-induced degradation within the microgel rather than to competitive displacement, as demonstrated by non-adsorption of trypsin in the peptide-void microgels. Further demonstration of this was given by the length-dependent kinetics. Thus, while 10 kDa polylysine distributes equally throughout the microgel for both 25% and 50% charge densities, 200 kDa polylysine displayed shell formation. When exposed to trypsin, this kinetically arrested polylysine-rich shell disintegrates, allowing the (smaller) peptide (fragments) to diffuse into the microgel core, resulting in continued gel deswelling. At longer degradation times, all peptide fragments are released, and the microgel completely reswollen. A simple random scission model was able to qualitatively capture the effects observed experimentally of trypsin concentration and polylysine molecular weight. Given the unfavorable effect of degradation for the function of peptide and protein drugs, as well as claims in literature that microgels are able to provide protection from proteolytic degradation, clarifying the substance of such statements is important. As demonstrated in the present investigation, protection against

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REFERENCES

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