LETTER pubs.acs.org/NanoLett
Triggered Cargo Release by Encapsulated Enzymatic Catalysis in Capsosomes Rona Chandrawati, Pascal D. Odermatt, Siow-Feng Chong, Andrew D. Price,† Brigitte St€adler,‡ and Frank Caruso* Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia
bS Supporting Information ABSTRACT: We report the coencapsulation of glutathione reductase and disulfide-linked polymeroligopeptide conjugates into capsosomes, polymer carrier capsules containing liposomal subcompartments. The architecture of the capsosomes enables a temperaturetriggered conversion of oxidized glutathione to its reduced sulfhydryl form by the encapsulated glutathione reductase. The reduced glutathione subsequently induces the release of the encapsulated oligopeptides from the capsosomes by reducing the disulfide linkages of the conjugates. This study highlights the potential of capsosomes to continuously generate a potent antioxidant while simultaneously releasing small molecule therapeutics. KEYWORDS: Capsosome, liposome, polymer capsule, subcompartments, glutathione reductase, peptide
B
iomimetic cellular structures hold tremendous promise for treating diseases on the cellular and subcellular levels. A bottomup approach to designing artificial cells13 requires reconstruction of the structure and function of biological cells for the purpose of replenishing missing or deficient cellular activity. A simplified celllike system can be based on a subcompartmentalized assembly,4 which is one of the features of biological cells that enable the performance of multistep biochemical reactions in a confined environment.5 Current artificial designs of subcompartmentalized assemblies incorporate lipids or (bio)polymers and include vesosomes (multiple liposomal subcompartments within a liposome),6 polymersomes within a polymersome,7,8 polymer subcompartments within a polymer capsule,9 and shell-in-shell microcapsules10,11—all based on single-component systems. We recently reported a new class of subcompartmentalized assemblies, termed capsosomes, which utilize two fundamentally different materials: polymer capsules and liposomes.12 The polymer capsules1315 are prepared via layer-by-layer (LbL) adsorption of interacting polymers onto sacrificial template particles, allowing for fine control over the capsule permeability by the choice and structure of the deposited polymer and the number of layers adsorbed. Liposomes, on the other hand, are able to encapsulate small, fragile hydrophobic and hydrophilic cargo. This nature-inspired hierarchical architecture has been shown to exhibit negligible inherent cytotoxicity16,17 and has the potential to serve as a mimic for biological cells toward enzymatic applications.4,13 We previously demonstrated the stable incorporation of liposomes composed of zwitterionic or negatively charged, unsaturated or saturated phospholipids into polymer films18,19 and showed the ability to control the spatial positioning of these liposomes within the polymer hydrogel carrier capsules to obtain capsosomes with membrane-associated1619 or “free-floating”20,21 liposomal r 2011 American Chemical Society
subcompartments. The former approach allows the assembly of multilayers of liposomal subcompartments; that is, a strata-like film consisting of alternating liposome and polymer layers. This design has potential benefits in, for example, the delivery of high dosages of therapeutic agents, coadministration of complementary therapeutics with the ability to control the ratio of encapsulated cargo, or coloading of enzymes for the performance of one-pot enzymatic cascade reactions or multiple parallel reactions in the same vessel. The latter design contains liposomal subunits that are encapsulated in the void of the carrier capsules and is suitable for the performance of multistep catalytic cascade processes that require rapid reagent mixing within a confined environment. Using membrane-associated capsosomes containing β-lactamase, we recently demonstrated a (one-step) successive temperature-triggered enzymatic catalytic reaction and confirmed that the temperature-triggered approach neither destroys the liposomal subcompartments nor causes release of the enzymes.19 However, many biological systems utilize multiple, coupled reactions, and mimicking cells in this regard still represents a significant challenge. Herein, to further our aim in addressing complexity in therapeutic cell mimicry, we demonstrate the ability of capsosomes to perform an encapsulated two-step enzymatic catalytic reaction, whereby reduction of a substrate is exploited to subsequently trigger cargo release. To achieve this, we coencapsulate two different biomolecules, enzymes and oligopeptides, into capsosomes and the action of the enzyme on a substrate allows the subsequent release of the oligopeptides at a designated temperature. In particular, we (i) assemble capsosomes containing glutathione reductase-loaded liposomes, (ii) confirm the functionality of the Received: August 22, 2011 Revised: September 27, 2011 Published: October 12, 2011 4958
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Scheme 1. Schematic Illustration of the Assembly of Capsosomes Coencapsulating Enzyme-Loaded Liposomes and PolymerPeptide Conjugates into the Cavity of the Polymer Carrier Capsules. a
a
A silica particle is coated with a PVPc precursor layer (a), followed by the deposition of zwitterionic, saturated glutathione reductase-loaded liposomes (Ls,zwGR) (b) and a PVPc capping layer (c). Sacrificial layers, PMA and PVP, are then adsorbed onto the coated particle (d), followed by the adsorption of PMAKP9 polymerpeptide conjugates (e). The assembly is continued with the LbL deposition of three bilayers of PVP and PMASH to form the shell of the carrier (f). The multilayer film is stabilized by cross-linking of the thiols within the polymer layers and core dissolution (g) results in a capsosome with “free-floating” glutathionereductase liposomal subunits and PMAKP9 conjugates at physiological pH.
encapsulated enzyme to reduce glutathione disulfide, (iii) coencapsulate disulfide-linked polymerpeptide conjugates and glutathione reductase-loaded liposomes within the capsosomes, and (iv) demonstrate the controlled release of the oligopeptides from the capsosomes by action of the enzyme. Glutathione (GSH) is an important cellular antioxidant that prevents damage to cell components caused by free radicals. This predominant sulfhydryl reducing agent exists at a concentration of approximately 5 mM in cells due to the activity of glutathione reductase (GR), an enzyme that actively reduces glutathione disulfide (GSSG) to its sulfhydryl form (GSH).22 The presence of this enzyme is essential for maintaining intracellular levels of GSH and to mitigate oxidation to avoid dysfunction of biologically active molecules. The ratio of GSH to GSSG within cells is often used as a measure of cellular cytotoxicity. Several studies have shown that a decrease in glutathione reductase activity is age-related and a low GSH/GSSG ratio results in an increased level of oxidative stress and suboptimal immune responses.22,23
Supplementation of glutathione reductase to replenish the depleted enzymatic activity would allow for the continual reduction of glutathione disulfide. In this study, we encapsulated glutathione reductase into liposomes, which were embedded into polymer carrier capsules to form capsosomes. We used 3 μm diameter capsosomes with “free-floating” liposomal subunits20 to perform the temperaturetriggered enzymatic conversion of GSSG to GSH (Scheme 1). The tailor-made cholesterol containing block copolymer, poly(N-vinylpyrrolidone)-block-(cholesteryl acrylate) (PVPc),20 was adsorbed onto silica particles, followed by the deposition of zwitterionic, saturated glutathione reductase-loaded cholesterolcontaining liposomes (Ls,zwGR) composed of 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC) and cholesterol (DPPC: chol = 4:1, at 5 mM lipid concentration) in HEPES buffer (10 mM HEPES (4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid), 150 mM NaCl, pH 7.4). This lipid composition was chosen to trigger the enzymatic reaction solely at elevated temperature. 4959
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Scheme 2. (a) Temperature-Triggered Catalysis of Encapsulated Glutathione Reductase in the “Free-Floating” Liposomal Subcompartments of Capsosomes, Which Reduces Glutathione Disulfide (GSSG) to Its Sulfhydryl Form (GSH)a and (b) Release of Encapsulated Oligopeptides Triggered by the Catalytic Activity of Glutathione Reductase in Capsosomesb
a The production of GSH is measured using Ellman’s reagent as an indicator. b Reduction of GSSG to GSH by the activity of encapsulated glutathione reductase in the liposomal subcompartments (1) facilitates release of the encapsulated peptide due to the cleavage of disulfide bonds linking the polymer carrier (PMA) and the peptide (KP9) (2).
These liposomes were capped with PVPc and subsequently a poly(methacrylic acid) (PMA) sacrificial layer was adsorbed in sodium acetate buffer (20 mM NaOAc, pH 4.0). The membrane of the polymer carrier capsules was then assembled by sequential deposition of three bilayers of PVP and thiol-modified PMA (PMASH) via hydrogen bonding interaction.2426 The thiol groups within the polymer layers were cross-linked with nonreducible 1,8-bis(maleimido)diethylene glycol ((BM)PEG2),9 and capsosomes were obtained by dissolving the silica core using a 2 M hydrofluoric acid (HF)/8 M ammonium fluoride (NH4F) solution followed by several washing steps in phosphate buffered saline (PBS) (10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM NaCl, pH 7.4). The activity of glutathione reductase encapsulated in Ls,zw was examined by the reduction of GSSG to GSH, as measured by Ellman’s assay (Scheme 2a). Equal numbers of capsosomes containing Ls,zwGR were incubated with GSSG, β-nicotinamide adenine dinucleotide 20 phosphate (NADPH), and Ellman’s reagent at 24 or 37 °C in PBS
(1 mol of NADPH is required to reduce 1 mol of GSSG to produce 2 mol of GSH22). In the presence of GSH, the disulfide bond of Ellman’s reagent is cleaved, releasing the 2-nitro-5-mercaptobenzoic acid (TNB) chromophore with an absorbance maximum at 412 nm. Figure 1 shows the GSH production over time via the temperature-triggered enzymatic assay in capsosomes using glutathione reductase encapsulated within the “free-floating” liposomal subcompartments. The enzymatic conversion of GSSG to GSH was only observed for capsosomes incubated at 37 °C (red, closed symbol) but not at 24 °C (red, open symbol), confirming the temperature dependency of the capsosome-confined reaction. Below the phase transition temperature (Tm) of the liposomes, the reagents freely permeate the polymer component (PMASH layers) but not the liposomes. Near the phase transition temperature, substrate molecules pass through transient defects produced by disturbances to the packing order of the lipids.27 The production of GSH for capsosomes incubated at 37 °C was observed at 5 h, and the absorbance readings continued to 4960
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Figure 1. Temperature-triggered reduction of GSSG to GSH, catalyzed by glutathione reductase encapsulated within the “free-floating” liposomal subcompartments of capsosomes at T = 24 or 37 °C. An increase in absorbance at 412 nm is due to the reduction of Ellman’s reagent by GSH, which is only observed at T = 37 °C (red, closed symbol). Negative control samples were run in parallel without the addition of GSSG (blue).
increase until ∼30 h. Negative control samples (i.e., without the addition of GSSG) did not show any measurable increase in absorbance. A similar increase in absorbance readings observed for the coreshell particles and capsosomes substantiates that core removal does not cause a loss of glutathione reductase activity in the capsosomes (Figure S1, Supporting Information). In addition, there was no production of GSH observed when glutathione reductase was encapsulated into the cavity of the polymer carrier capsules without the use of liposomal subcompartments (Figure S2, Supporting Information). This demonstrates the benefit and necessity of liposomal encapsulation in protecting fragile biomolecules and maintaining their functional activity. A further advantage of this approach is the ability to trigger the enzymatic assay. We next demonstrate controlled release of the peptides from capsosomes by the action of glutathione reductase by coencapsulating both polymerpeptide conjugates (PMAKP9) and glutathione reductase-loaded liposomes (Ls,zwGR) in the cavity of the polymer carrier capsules. KP9 oligopeptide is a wild-type antigen used in vaccination28 and has been successfully encapsulated into disulfide-stabilized PMA capsules via their conjugation to a carrier polymer.29 In our previous work, release of these therapeutic agents from the carrier capsules was triggered by the intracellular reductive environment and their immunostimulatory activity was confirmed by stimulation of specific T cells.29,30 In the current study, Alexa Fluor 488-labeled PMA-KP9 (PMAKP9488) conjugates, which are stabilized by a disulfide linkage, were synthesized according to a previously published protocol.29 The encapsulation of PMAKP9488 conjugates into capsosomes was achieved by their adsorption onto polymer/liposome-coated particles prior to the assembly of the membrane of the carrier capsules (Scheme 1). Figure 2a compares the encapsulation efficiency of PMAKP9488 conjugates in 3 μm diameter PMA capsules (i) to capsosomes (ii). A similar increase in fluorescence intensity of PMAKP9488 was measured using flow cytometry for both assemblies, indicating a negligible effect of the presence of liposomes on the adsorption of the conjugates.
Figure 2. Fluorescence intensity of PMA capsules encapsulating PMAKP9488 (a, i) or capsosomes encapsulating Ls,zwNBD and PMA KP9488 (a, ii), as measured by flow cytometry. A similar increase in fluorescence intensity of PMAKP9488 indicates a negligible effect of the presence of liposomes on the encapsulation of the polymerpeptide conjugates. CLSM images of capsosomes encapsulating Ls,zwGR and PMAKP9488, dispersed at pH 4 (b, i) or pH 7.4 (b, ii). At physiological pH (pH 7.4), electrostatic repulsion between the conjugates and the negatively charged hydrogel membrane results in changes in the spatial position of the encapsulated polymerpeptide conjugates, which are now contained inside the cavity of the carrier capsules. Scale bars are 5 μm.
Confocal laser scanning microscopy (CLSM) images confirmed the encapsulation of PMAKP9488 in the capsosomes (Figure 2b). In NaOAc buffer (pH 4), the presence of the fluorescently labeled conjugates is seen as a homogeneous green corona on the membrane of the capsosomes (Figure 2b,i). When these capsosomes were dispersed in PBS (pH 7.4), PVP and the sacrificial PMA were released from the multilayer film, resulting in single-component cross-linked PMA carrier capsule walls.20 Deprotonation of the PMAKP9488 and the PMASH causes electrostatic repulsion between the conjugates and the negatively charged hydrogel membrane, changing the spatial position of the encapsulated PMA KP9488 conjugates, which are now contained inside the cavity of the carrier capsules together with the Ls,zwGR (Figure 2b,ii). By correlation with a calibration curve of KP9488 (Supporting Information, Figure S3), the number of KP9 oligopeptides encapsulated in the capsosomes was quantified as 5 105 copies per 3 μm diameter capsosome. Release of encapsulated KP9 oligopeptides from PMA capsules has previously been demonstrated by incubating the loaded carrier capsules in 5 mM GSH.29 In this study, the GSH generated from the encapsulated enzyme glutathione reductase cleaves the disulfide linkage between the polymer and the peptides (Scheme 2b). Equal numbers of capsosomes encapsulating Ls, 4961
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Figure 3. (a) Changes in normalized fluorescence intensity of capsosomes encapsulating Ls,zwGR and PMAKP9488 upon incubation with GSSG and NADPH, as monitored by flow cytometry as a function of time. A decrease in fluorescence intensity indicates release of the encapsulated KP9488 due to cleavage of the disulfide linkage between the polymer carrier and the peptide by GSH produced by glutathione reductase. (b) Flow cytometry dot plot of capsosomes encapsulating Ls,zwGR and PMAKP9488 incubated with GSSG and NADPH at 37 °C, before (i) and after (ii) release of the peptide (24 h). A decrease in fluorescence is indicative of peptide release, while similar light scattering indicates a minimal change in the shape and the composition of the capsosomes. Corresponding DIC and fluorescence microscopy images of these capsosomes incubated at 37 °C, before (c, i and c, ii) and after (d, i and d, ii) the glutathione reductase-induced cleavage and subsequent release of KP9488 (24 h) are shown. Scale bars are 5 μm. zw
GR and PMAKP9488 were incubated with GSSG and NADPH in PBS at 24 or 37 °C and the release of KP9488 was monitored over time by flow cytometry (Figure 3a). Approximately a 50% decrease of the fluorescence intensity was observed within 24 h at 37 °C, followed by a plateau through to 72 h. In contrast, control samples showed a much smaller decrease in fluorescence (∼1020%) in the same time frame. These data are in agreement with the temperature-triggered activity of the glutathione reductase in capsosomes (Figure 1); that is, the conversion of GSSG to GSH only occurred at 37 °C, thus the induced release of the oligopeptides only occurred at this temperature. Figure 3b shows the corresponding flow cytometry dot plot of these capsosomes before (i) and after (ii) incubation at 37 °C in the glutathione reductase assay for 24 h. A decrease of fluorescence in the population was observed, while there was no change in the light scattering measurement. The latter confirmed that the presence of GSH did not compromise the structural integrity of the carrier capsules because the membrane of the capsosomes in this study is stabilized via a noncleavable crosslinker, (BM)PEG2. Corresponding differential interference contrast (DIC) and fluorescence microscopy images of these capsosomes before and after peptide release are shown in panels c and d of Figure 3, respectively. DIC images confirmed that the capsosomes remained intact after the enzyme-mediated peptide release (Figure 3c,i and 3d,i). Fluorescence microscopy images showed that upon release of the cargo, some of the peptides
remained associated with the membrane of the capsosomes (Figure 3d,ii). This is likely due to nonspecific adsorption and possible covalent interactions between the cysteine groups of the KP9488 and maleimide ends of partially cross-linked (BM)PEG2. Capsosomes were also incubated with 5 mM GSH at 37 °C to serve as a positive control. As expected, the flow cytometry dot plot of these capsosomes, with the corresponding DIC and fluorescence microscopy images, resemble those of capsosomes subjected to the glutathione reductase assay (Figure S4, Supporting Information). Taken together, the results demonstrate that capsosomes encapsulating glutathione reductase may be used to reduce glutathione disulfide, which subsequently mediates the release of a small peptide. In summary, we have demonstrated the assembly of capsosomes coencapsulating glutathione reductase-loaded liposomal subcompartments and PMAKP9 conjugates, both of which are confined to the interior void of the carrier capsules. Ellman’s test confirmed the functionality of the encapsulated enzyme, which exhibited a temperature-triggered enzymatic conversion that reduced oxidized glutathione to its sulfhydryl form. The reduced glutathione subsequently cleaved the disulfide bonds that link the small peptide (KP9) to polymer carrier (PMA), enabling its release from the capsosomes. These initial results demonstrate the potential of capsosomes to continuously generate a potent antioxidant at human body temperature while simultaneously releasing small molecule therapeutics. In our future work, we aim 4962
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’ ASSOCIATED CONTENT
bS
Supporting Information. Absorbance readings of Ellman’s assay using coreshell particles or glutathione reductase-loaded PMA hydrogel capsules, fluorescence calibration curve for KP9488, flow cytometry dot plot of capsosomes after the enzymatic assay at 8 h or after 24 h of incubation with 5 mM GSH at 37 °C (with the corresponding DIC and fluorescence microscopy images), experimental details, and instrument specifications. This material is available free of charge via Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Present Addresses †
Department of CINT Science, Sandia National Laboratories, Albuquerque, NM 87185. ‡ Interdisciplinary Nanoscience Center, Aarhus University, Aarhus 8000, Denmark.
’ ACKNOWLEDGMENT This work was supported by the Australian Research Council under the Federation Fellowship (F.C.) and Discovery Project (F.C.) schemes. We thank Dr. Almar Postma (CSIRO Molecular and Health Technologies) for the synthesis of PVPc.
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