Polymer nanocarriers protecting active enzyme cargo against proteolysis

Polymer nanocarriers protecting active enzyme cargo against proteolysis

Journal of Controlled Release 102 (2005) 427 – 439 www.elsevier.com/locate/jconrel Polymer nanocarriers protecting active enzyme cargo against proteo...

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Journal of Controlled Release 102 (2005) 427 – 439 www.elsevier.com/locate/jconrel

Polymer nanocarriers protecting active enzyme cargo against proteolysis Thomas D. Dziublaa,*, Adnan Karima, Vladimir R. Muzykantova,b a

Institute for Environmental Medicine, University of Pennsylvania School of Medicine, 1 John Morgan/6068, 3620 Hamilton Walk, Philadelphia, PA 19104, United States b Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, United States Received 29 July 2004; accepted 15 October 2004 Available online 19 November 2004

Abstract Polymeric nanocarriers (PNCs), proposed as an attractive vehicle for vascular drug delivery, remain an orphan technology for enzyme therapies due to poor loading and inactivation of protein cargoes. To unite enzyme delivery by PNC with a clinically relevant goal of containment of vascular oxidative stress, a novel freeze–thaw encapsulation strategy was designed and provides ~20% efficiency loading of an active large antioxidant enzyme, catalase, into PNC (200–300 nm) composed of biodegradable block copolymers poly(ethylene glycol)-b-poly(lactic-glycolic acid). Catalase’s substrate, H2O2, was freely diffusible in the PNC polymer. Furthermore, PNC-loaded catalase stably retained 25–30% of H2O2-degrading activity for at least 18 h in a proteolytic environment, while free catalase lost activity within 1 h. Delivery and protection of catalase from lysosomal degradation afforded by PNC nanotechnology may advance effectiveness and duration of treatment of diverse disease conditions associated with vascular oxidative stress. D 2004 Elsevier B.V. All rights reserved. Keywords: Antioxidant delivery; Protein loading; Catalase; Nanoparticle; Biodegradable

1. Introduction Containment of vascular oxidative stress induced by reactive oxygen species including H2O2 produced by leukocytes and vascular cells themselves is an important, yet elusive goal. Small antioxidants, scavengers,

* Corresponding author. Tel.: +1 215 898 2449; fax: +1 215 898 0868. E-mail address: [email protected] (T.D. Dziubla). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.10.017

and antioxidant inducers have some utility for alleviating subtle chronic oxidant stress, but have little value for protection against severe acute insults. In theory, antioxidant enzymes (e.g., catalase reducing H2O2 into water) could afford more potent protection. However, effects of antioxidant enzymes are suboptimal due to unfavorable pharmacokinetics and inadequate delivery to endothelial cells lining vascular lumen [1]. In order to improve delivery to endothelium representing both a source and a critically important, vulnerable target of oxidants [2–4], diverse means

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have been designed [5,6]. For example, targeting of catalase conjugated with antibodies against endothelial cell adhesion molecules (CAMs) ICAM-1 and PECAM-1 boosts vascular antioxidant defense and

alleviates perfused [5], and addition

oxidative stress in cell cultures [7,8], organs [9], lung transplantation in rats lung injury in mice [10] (Fig. 1A). In to enhanced delivery of therapeutics,

Fig. 1. The concept of vascular delivery of catalase by PNC and features of synthesized block-copolymer. (A) Catalase conjugates with antibodies against endothelial cell adhesion molecules (CAM) bind to endothelial cells, gets internalized via CAM-mediated endocytosis, and remains active intracellularly for a few hours, thus protecting cells against injurious effects of H2O2, either produced by endothelial cells (e.g., in mitochondria, M) or diffusing into endothelium after being released by activated white blood cells (WBC). In addition to detoxification of H2O2, blocking CAM by conjugates inhibits WBC adhesion. (B) Once immunoconjugates are trafficked into the lysosomes, proteases degrade catalase and H2O2 escapes enzymatic interception (left). In contrast, catalase loaded into permeable, biodegradable polymer, nanoparticles (PNC with diameter below 500 nm and thus permissive of CAM-endocytosis) will remain protected against lysosomal proteolysis and decompose H2O2 even after degradation of proteins on PNC surface. (C) IR spectra of the synthesized polymers verified the existence of the diblock PEG-PLA and PEG-PLGA polymers. The peak at 2900 cm1 is the CH stretch predominantly found in the PEG backbone and the bond at 1750 cm1 is indicative of the ester group repeat unit in both PLA and PLGA. Pure PEG, PLGA, and PLA spectra are also presented. The inset represents a typical diffusional analysis plot of H2O2 in PLGA. The diffusivity, which was found to be 3.3107 cm2 s1, was obtained by plotting the normalized cumulative flux (defined as Vl(2A)1 ln(12Ct Co1) from Eq. (1)) vs. time and obtaining a linear regression.

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targeting CAM inhibits leukocytes adhesion to endothelium, thus attenuating their proinflammatory functions [11–13]. Delivered catalase enters endothelial cells via a novel internalization mechanism, CAM-mediated endocytosis [14], which provides a pathway for intracellular drug delivery when nanoparticles are 100–500 nm in size [15]. This enhances detoxification of injurious diffusible intracellular oxidants and minimizes catalase shedding from cell surface [14]. From previous experiments using a model polystyrene nanoparticle system with surface-absorbed catalase, it was found that the subsequent intracellular trafficking lead to a lysosomal destination and degradation of catalase within 3 h after delivery, restricting the duration of antioxidant protection [16]. During the mid- to late-1970s, it was discovered that the immobilization of enzymes inside polymeric supports works to protect enzyme from thermal and proteolytic degradation (please see Torchilin [17] and Klibanov [18] for reviews). As such, one method to lengthen the therapeutic window is to design biodegradable polymer nanocarriers, which protects encapsulated catalase from lysosomal proteolysis, thus prolonging duration of desired effects (Fig. 1B). Application of nanocarriers for drug delivery is an expanding area of research [19,20] that provided the design of biomaterials with controlled rates of drug release [21,22] and delivery of small therapeutic molecules by nanocarriers [23–25]. It is tempting to expand the utility of nanocarriers for delivery of therapeutic enzymes. However, loading of such large fragile cargoes into nanoparticles without loss of activity has been elusive. Catalase delivery seems an ideal testing model for evaluating some key aspects of enzyme delivery by nanocarriers. Firstly, catalase substrate H2O2, an injurious reactive oxygen species, permeates membranes like water and internalized catalase enclosed in intracellular vesicles decomposes H2O2 diffusing from the medium [8,16]. Secondly, catalase possesses a wide functional pH range (4.0–9.0) [26] and is therefore capable of maintaining its activity in the acidic environment of the lysosomes and polymeric nanocarrier (PNC) core [27]. In theory, as long as polymer shell restricts proteolysis of catalase loaded inside polymeric water-permeable PNC, it will

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reduce H2O2 even in lysosomes (Fig. 1B). Thus, the duration of antioxidant protection will then be controlled by degradation rate of the PNC inside the lysosomes. This study for the first time describes loading and protection of an active large enzyme (catalase) into PNC composed of diblock PEG-PLGA copolymers, formulated within the size limits allowing endocytosis (200–500 nm diameter). At least 25% of loaded catalase was fully protected from external proteases by loading into these PNC.

2. Materials and methods 2.1. Reagents All reagents were used as received unless stated otherwise. Methoxypoly(ethylene glycol) 5000 MW (mPEG) was purchased from Polysciences (Warrington, PA). Poly(lactic-co-glycolic acid) (50:50) in the free acid (38,000 MW) and esterified (33,000) forms was purchased from Alkermes (Cincinnati, OH). Bovine liver catalase (242,000 MW) and horseradish peroxidase were obtained from Calbiochem (EMD Biosciences, San Diego, CA). 10-acetyl-3,7-dihydroxyphenoxazine (amplex red) was purchased from Molecular Probes (Eugene, OR). All other reagents and solvents were obtained from Sigma-Aldrich (St. Louis, MO). 2.2. Synthesis of diblock copolymers Diblock copolymers were prepared by two different schemes. Scheme One: PLGA (50:50) polymer containing a carboxylate end group and PEG (10,000 MW) was freeze dried overnight to remove bound water. The polymers were mixed in a 4:1 molar ratio (PEG/PLGA) in anhydrous dichloromethane (DCM) to a final polymer concentration of 10 wt.%. Next, 2,2-dicyclocarbodiimide (DCC) and N,N’-dimethylaminopyridine (DMAP) were added to the PLGA at the molar ratio of, 4:1:2. Conjugation was carried out under a N2 atmosphere at room temperature for 18 h. The resulting precipitate, dicyclohexylurea, was filtered out, and the polymer was precipitated twice in anhydrous ether. The filtrate was then dried and dissolved in cold acetone. The insoluble fraction was

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filtered out, and once more precipitated in cold ethanol. Scheme Two: dl-lactide was recrystalized twice in anhydrous ether, and then mixed with mPEG in weight ratios predetermining their molecular weight. The bulk material was raised to 140 8C for 2 h under a reduced nitrogen atmosphere. After this time, the temperature was reduced to 120 8C, 1 wt.% stannous 2-ethyl-hexanoate was added, and the polymerization proceeded for 6 h. The resulting polymer was dissolved in DCM, and precipitated twice in cold diethyl ether. The final product was then serially dried in a rotovap (Safety Vap 205, Buchi, Switzerland) and a freeze dryer (RCT 60, Jouan, Winchester, VA) to remove any residual solvent. The chemistry of the polymer was verified by Fourier transform infrared spectroscopy (FTIR, Nicolet Magna IR560, Madison, WI), gel permeation chromatography (GPC) using 2 serial pLGel Mixed C columns 3007.5 mm (Polymer Laboratories, Amherst, MA) with an Acuflow Series III pump and a Differential Refractometer (Knauer, Berlin, Germany) calibrated using polystyrene standards was used to evaluated polymer MW and the polydispersity index (PDI). Relative PEG content was determined by the chemical assay described next. 2.3. PEG and PLA content determination A 50 Al aliquot of the concentrated nanoparticle prep was saponified by adding 200 Al of 5 M NaOH and reacting overnight at 80 8C. This solution was then neutralized with 200 Al of 5 M HCl. PEG concentration was determined by a colorimetric assay based off of the PEG–Barium Iodide complex. Absorbance of the color product was measured at 550 nm using a microplate reader (Model 2550-UV, Bio-Rad Labs, Hercules, CA) [28]. To measure PLA concentration, an enzymatic assay for l-lactic acid was used. 5 Al of sample was added to 45 Al of 50 mM PBS in a microplate well. To this well, 50 Al of assay buffer was added. The assay buffer consisted of 2 U ml1 horseradish peroxidase, 20 mU lactate oxidase and 1 Ag ml1 of Amplex Red. After 10 min of incubation at room temperature, the product concentration, resorufin, was determined by UV absorbance at 550 nm. Concentrations were

measured in triplicate for each individual particle preparation. 2.4. Determination of H2O2 diffusivity in PLGA The diffusivity of H2O2 through PLGA was determined by using a two chamber diffusion apparatus. Polymer films of esterified PLGA (34,000 MW) were prepared via solvent casting procedure. The donor cell contained a 5 mM H2O2 solution in PBS (50 mM, 7.4 pH), and the receptor cell contained pure PBS buffer. At specific time intervals (15 and 30 min), the receptor cell contents were removed and replaced with fresh buffer. The concentration of the H2O2 in the receptor cell was determined by UV absorbance at 242 nm (Cary 50 UV-Vis, Varian, Palo Alto, CA). Diffusivity studies were performed in triplicate for two independently cast polymer films. 2.5. Nanoparticle synthesis A schematic representation of the double emulsion synthesis procedure used is outlined in Fig. 2A. The primary emulsion was formed by homogenizing at 15 krpm for 1 min (80 8C, dry ice/acetone bath) a 100 Al aqueous drug solution (1 mg ml1 catalase in PBS) in a 1 ml organic polymer solution (25 mg ml1 in DCM), using a 7-mm blade homogenizer (Kinemetica Polytron 3100 equipped with a PTDA3007/2 generator, Brinkmann Instruments, Westbury, NY). This primary solution was immediately pipetted into a secondary aqueous phase (5 ml) containing 2 wt.% poly(vinyl alcohol) (PVA, 10,000 MW, 80% hydrolyzed) and was homogenized at 15 krpm for 1 min. This second homogenization was added to 10 ml more of the same surfactant solution, and stirred overnight under mild agitation to remove residual solvent. To purify the resultant nanoparticles, a serial centrifugation scheme was used. The solution was first centrifuged at 1000g for 15 min to remove the large microparticle/macroaggregate fraction. The supernatant was then centrifuged at 22,000g for 30 min. Nanoparticles were rinsed twice more to remove residual surfactant and unloaded protein. All preparations were performed in triplicate. Particle sizes were determined by dynamic light scattering (DLS, 90PLUS Particle Sizer, Brookhaven Instruments, Holtsville, NY).

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2.6. Analysis of enzymatic activity The activity of catalase was determined by using a standard catalase assay, as previously described [29]. Briefly, 950 or 900 Al of a 5 mM H2O2 solution in PBS (7.4 pH) was added to a quartz cuvette at ambient conditions. A catalase-loaded particle solution was added to bring the total volume to 1 ml. The concentration of H2O2 was monitored verses time by measuring the absorbance at 242 nm (1 Unit=23.0.[DAbs ml1]) The activity was measured twice at two different concentrations (50 and 100 Al) for each individual particle preparation. 2.7. Loading analysis Loading analysis was indirectly calculated by measuring the 125I-catalase content in solution pre-

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and post-centrifugations (n=3). Protein content was determined by radiotracing using a Wizard 1470 gamma counter (Wallac Oy, Turku, Finland). Catalase was radiolabeled with Na125I (Perkin Elmer, Boston, MA) using the Iodogen (Pierce Biotech., Rockford, IL) method, and unbound iodine was removed from protein using gel permeation chromatography (Biospin 6 Columns, Bio-Rad Labs, Hercules, CA). Conditions were set based upon manufacturer’s recommendations. 2.8. Determination of protection of enzyme To evaluate the ability of nanoparticles to protect the activity of loaded enzyme, an in vitro proteolytic assay was used. In these studies, particles were incubated at 37 8C in a PBS solution containing 0.2 wt.% pronase, a robust proteolytic enzyme. Aliquots

Fig. 2. Formulation of PLGA-PEG nanoparticles. (A) A scheme of the double-emulsion synthesis procedure. For all experiments, the conditions presented in diagram were used unless when otherwise noted. (B) Sonication (20 W) in DCM ( ) or in 50:50 DCM/acetone (z) rapidly inactivated catalase, yet it retained activity after homogenization (20 kRPM) in DCM at 4 (o) and 80 8C (5). (C) The loading efficiency was determined by tracing the amount of radiolabeled catalase contained inside either the microparticles (i.e., pellet obtained after centrifuging for 15 min at 1000g) or nanoparticles fractions (2nd pellet obtained by centrifuging for 30 min at 22,000g). Loading efficiency into the PNC was greatly enhanced (~eightfold) when a freeze thaw cycle (grey bar) was included in the primary emulsion step. Unless specified otherwise, the data in this and other figures is presented as MFS.E.M. (n=3).

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were taken at specific interval of incubation and measured for either enzymatic activity or protein loading content.

3. Results 3.1. Synthesis of PEG-PLGA polymers and H2O2 diffusion through PLGA polymer In order to achieve targeted vascular antioxidant therapy, the PNC developed herein can be designed to contain functional groups for the surface attachment of affinity moieties such as antibodies to endothelial adhesion molecules [7–11]. The PEG moiety seems the most useful in this context [30–32]. In addition, external PEG chains define stealth properties of PNC [33,34]. From this standpoint, composition of the diblock copolymer and inclusion of PEG is of specific importance. A new conjugation scheme was used to synthesize copolymer containing 38 kD PLGA and 10 kD PEG with unblocked terminal hydroxyl group. PEG and lactic acid assays showed that the polymer contained 11 wt.% PEG (50% conjugation yield). GPC confirmed that the MW was 50,000 with a PDI of 2.03. FTIR analysis verified presence of both the carbon hydrogen stretch of PEG saturated backbone at 2850 cm1 and the ester peak of the PLGA at 1790 cm1 in the copolymer (Fig. 1C). Since it is theorized that catalase will reside inside a shell of polymer (Fig. 1B), it is imperative that catalase’s substrate, H2O2, readily permeates across this barrier. One way to determine this is to measure the diffusivity of hydrogen peroxide in PLGA. Since diffusivity is a bulk property, it is insensitive to geometry. The easiest way to obtain this property is by using a classical two-chamber polymer-film diffusion study. From these experiments, H2O2 can easily diffuses through PLGA polymer (film thickness varied from 80 to 200 Am). Under the experimental conditions, steady state and a constant driving force can be assumed. The following equation, derived from Fick’s first law of diffusion, was used to calculate the film permeability [35].   2Ct 2A Dk ln 1  t ¼  V l Co

ð1Þ

where A is the area of the diffusion plane, V is the volume of the receptor cell, D the diffusivity, k the partition coefficient, l the thickness of the polymer film, C o the concentration of the donor cell and C t the calculated cumulative concentration of the receptor cell at time, t. A typical analysis plot of experimental data is shown in inset (Fig. 1C). Averaging data of two PLGA films with 3 replicates each, diffusivity of H2O2 through PLGA was found to be 3.3F0.37107 cm2 s1. Since the H2O2 diffusivity across the lipid bilayer of cells is ~2.0108 cm2 s1 [36], as long as the diffusion path length is less than 10 times the lipid bilayer thickness, diffusion limitations can be ignored. 3.2. Mechanical homogenization with a freeze-thaw cycle results in a significant degree of active catalase loading Recent studies on the synthesis of PNC consisting of PEG-PLGA and other biodegradable polymers provide examples of PNC loading by small soluble drugs resistant to harsh conditions of PNC formulation [23–25]. However, loading of large enzymatically active proteins into PNC is a significant and yet unsolved problem. For example, a probe sonication double emulsion followed by rapid solvent evaporation employed for encapsulation of l-aspariginase and interferon-alpha impaired the activity of both proteins [37,38]. Loading of protein C (a 60-kD monomer serine protease that cleaves coagulation factors) into mPEG-PLGA PNC using a similar probe sonication approach using an acetone/DCM mixture caused protein C inactivation, yet a fraction of released protein C could be recovered [39]. Likewise, in the presence of DCM, catalase activity was reduced by 80% after 10 s of sonication, while the acetone/DCM cosolvent mixture actually exacerbated inactivation, with 60% activity loss even without sonication (Fig. 2B). This data implies low endurance of large multimeric enzymes to loading, which likely depends on complex cargo quaternary structure (catalase is a 240-kD tetramer containing central coordinated heme redox group). Since this sonicationinduced deactivation was not seen in a pure aqueous system, it is believed that an interfacial-mediated unfolding of catalase is the likely mechanism of deactivation. To load catalase into PEG-PLGA matrix during PNC formation without loss of enzymatic

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activity, a double emulsion–solvent evaporation method [23,39], which can utilize either ultrasound or mechanical homogenization for emulsification steps (Fig. 2A), was used. When the latter approach was employed, catalase retained ~90% activity after 1 min 15 krpm homogenization in acetone–DCM at 4 8C and ~60% when a 80 8C freeze–thaw cycle was included. Since mechanical emulsification produces particles with a wide size-distribution, serial centrifugations and filtration through micron filter were employed to isolate the PNC fraction (particles b700 nm diameter) from larger microparticles. Theoretically, double emulsion PNC possesses both an inner and external aqueous phase, resulting in an enhanced energy penalty that makes the loading of aqueous drug inside PNC pockets unlikely. Since entropy works against loading, conceivably the PNC loading is not determined by equilibrium partition of the drug into the PN, but rather by the kinetic effects of polymer gelation that reduces the inner aqueous domain release into the outer aqueous compartment than. Indeed, standard loading conditions resulted in poor loading (2% of 125I-catalase, Fig. 2C). However, when polymer precipitation/gelation was induced by a freeze–thaw cycle in the primary emulsion step, catalase loading into PNC fraction was enhanced to 15% (Fig. 2C).

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size determined by the surfactant load of the system. Plotting of PNC size as a function of rate2/time, a scaling factor for energy input, illustrates this effect (Fig. 3). The average PNC size decreased with an increase in homogenization rate and time (Fig. 3A). PNC decreased in size from 350 to 250 nm as the homogenization rate increased from 5 to 20 krpm (o). When the PEG content in the PEG-PLGA was decreased from 11 to 5 wt.%, this size dependence became even more evident, varying from 700 to 350 nm (.). In order to analyze significant number of samples and estimate reproducibility, DLS measurements were used to determine PNC size in most

3.3. PNC size is determined by energy input, surfactant concentration, and polymer composition/ concentration PNC formation can be considered a balance of kinetic and thermodynamic effects. As particle size decreases, the interface between the oil and water phase increase. In order to initially overcome the energy barrier, energy (e.g., mechanical homogenization) is added to the system. However, once energy input is ceased, the oil phase starts to coalesce into increasingly larger sizes unless enough surfactant is present to stabilize the system, or the polymer solidifies prior to coalescence. This hardening of the polymer phase is controlled predominately by the solvent evaporation rate (a kinetic parameter) but is also determined by the polymer MW and intrinsic features of a solvent. In agreement with this theory, size of the PNC decreases with increasing energy input in the secondary emulsion down to a minimum

Fig. 3. Effects of mechanical energy input on PNC size. Panels show average PNC sizes determined by DLS for PNC synthesized using PEG-PLGA with 11% PEG content (o, right axis) and 5% PEG content ( , left axis). PVA concentration was 4 wt.%. (A) PNC size decreases as homogenization rate and time (inset) of the secondary emulsion increases. (B) By plotting rate2/time (an energy scaling factor) vs. particle size, the global dependence of PNC size on mechanical emulsification is apparent. Increased mechanical energy input decreases particle size down to the size allowable by the surfactant capacity of the system. TEM (inset) verified the particle sizes (stained by uranyl acetate) measured by DLS. White bar=500nm.

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experiments. Electron microscopy confirmed size of PNC determined by DLS (Fig. 3B, inset). To assess the role of surfactant stabilization in PNC synthesis, the effect of PVA surfactant was tested. As PVA concentration increased (from 0.1 to 4 wt.%), the size of the PNC increased for both second homogenization rates tested, 15 and 20 krpm (Fig. 4A). At each PVA concentration (except 4 wt.%), the PNC formed at 20 krpm was smaller than the 15 krpm counterpart. Increased surfactant concentration also increased the PNC yield (5 to 25 wt.%; Fig. 4B, inset). The interfacial area (IA) of the oil to water phase was defined by the following equation IA ¼ Cparticles SAparticle ¼

3Cmass qr

ð2Þ

where C particles is the number concentration of PNC, SAparticle is the surface area of the PNC, C mass is the

mass concentration of the particles, q is the polymer density (assumed to be 1.2 g cm3), and r is the mean particle radius. This analysis shows an overall increase in oil/water surface area per volume of emulsion (Fig. 4B). Results of chemical assays of the conjugated diblock PEG-PLGA confirmed the conjugation efficiency to be 50%. This result represents a relatively under appreciated aspect of PNC synthesis, i.e., role of diblock PEG-PLGA to monoblock PLGA feed ratio. The majority of PNC in this study was synthesized using either PEG-PLGA in its pure form or mixed with bulk PLGA. It was found that the diblock PEG-PLGA to monoblock PLGA feed ratio represented a significant factor that controls size of the resultant polymer particles (Figs. 3 and 4). Increases in PEG-PLGA diblock content decreased the subsequent PNC size, presumably due to the surfactant

Fig. 4. Effect of surfactant and polymer concentration on nanoparticles size. (A) For the 5 wt.% PEG content PEG-PLGA polymer, when the PVA concentration increased, the mean particle size also increased for both 2nd homogenization rates, 15 (o) and 20 krpm ( ). For each given surfactant concentration, the lower homogenization rate possessed a larger particle size, in agreement with the trend noticed in Fig. 2. (B) Merging data on PNC size with PNC yield (inset) shows that the net result from increasing PVA content is an increase in oil/water interfacial area. (C) When the concentration of PEG-PLGA (5 wt.% PEG) polymer increased ( ), the PNC size decreased down to a minimum level and the total mass of PNC increased. When the PEG-PLGA concentration was kept constant (10 mg ml1) and pure PLGA was added to increase the net polymer concentration (o), the PNC size increased with increasing concentration. (D) The increase in polymer concentration also resulted in a net increase in the oil/water contact interfacial area.

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qualities afforded by this amphiphile. This hypothesis is further substantiated by the fact that increasing polymer concentration with a constant wt.% of PEG resulted in a decrease in PNC size (from 400 to 250 nm) and an increase in PNC yield (from 1 to 5 mg). In contrast, when the polymer concentration was increased with a constant 10 mg ml1 PEG-PLGA concentration, particle size actually increased from 400 to 700 nm and the interfacial area per volume remained relatively constant (Fig. 4C and D, o). The measured PEG content of the PNC matched that of the feed conditions, confirming that (A) PEG-PLGA did not preferentially self-associated into micelle structures and (B) that PNC polymer composition can be predetermined by simply altering the copolymer mixtures. 3.4. Effect of energy input on catalase loading and activity in PNC The activity of loaded catalase was determined by the direct monitoring A 242 nm absorbance of H2O2 that is relatively stable in water in minute time scale (Fig. 5A, inset, curve a). The addition of unloaded PNC elevated a net absorbance due to light scattering, but produced no measurable subsequent decrease in H2O2 (curve c). In a sharp contrast, catalase-loaded PNC produced the same increase in absorbance that was immediately followed by a decrease in H2O2 absorbance (curve d), compatible by the kinetics with effect of a similar amount of free catalase (curve b). Therefore, catalase loaded into PNC retains its

Fig. 5. Loading of catalase into nanoparticles. (A) Effect of primary homogenization time on catalase activity of nanoparticles. (Inset) A representative printout of kinetic data obtained in catalase activity assays. H2O2 optical density was stable in the absence (curve a) and rapidly declined in the presence (curve b) of catalase. There was a stable increase in the measured absorbance due to scattering induced by the unloaded PNC (curve c). Catalase-loaded PNC caused H2O2 decay (curve d). (B) As second homogenization rate increases beyond optimal level (15 krpm), particle size and catalase loading decreases. (Note: surfactant=2 wt.% sodium cholate). Freeze–thaw cycle in the primary emulsion ( ) preserves activity of PNC-loaded catalase vs. loading at 4 8C (o). (Inset) PNC-associated catalase activity decreased with prolongation of the secondary homogenization (1 vs. 5 min). (C) 125I-catalase loading into PNC depends on the secondary homogenization rate, 15 krpm (black bar) 25 krpm (gray bar), but not polymer MW and PEG content.

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enzymatic activity and effectively decomposes H2O2 diffusing through polymer shell. Increases in the rate of homogenization in the primary emulsion (up to 20 krpm) did not alter catalase activity (not shown), but homogenization times extending beyond 1 min at 15 krpm inactivated PNC-loaded catalase (Fig. 5A). While interesting from a theoretical standpoint, this negative effect seems surmountable since the first emulsion homogenization time has limited effect on PNC size and 1 min is sufficient for the effective synthesis of PNC in the size range of interest (Fig. 2). Loading of catalase into the primary emulsion without vs. with a freeze–thaw cycle endowed PNC with marginal vs. substantial catalase activity within tested range of the secondary homogenization rate (Fig. 5B), in a good correlation with 125I-catalase loading data (Fig. 2C). Second emulsion homogenization time longer than 1 min was deteriorating for catalase activity (Fig. 5B, inset). The homogenization rate of the second emulsion displayed a bell-shape optimum of PNC-loaded catalase activity (~15 krpm) followed by reduction at 20 krpm (Fig. 5B). Supporting this result, 125I-catalase loading into PNC was N20% vs. b5% at 15 vs. 25 krpm in second emulsion (Fig. 5C). Yet, even more striking was the fact that, at the polymer concentration used (25 mg ml1), the loading efficiency was independent of polymer composition (PEG content of 5–20 wt.%) and MW (32–50 kDa; Fig. 5C). This feature is of strategic importance since polymer MW and composition can be used to control PNC properties such as degradation rate, sizing, and in vivo circulation. 3.5. Nanocarriers protect loaded catalase from proteolysis After 4 h incubation with a powerful proteolytic enzyme, pronase, free catalase possessed b1% of initial H2O2-degrading activity, while PNC-loaded catalase retained 35% of initial activity (Fig. 6, inset). Co-incubation of catalase with unloaded PNC did not protect it from pronase, indicating that only catalase encapsulated into PNC is protected against proteolysis, yet is still capable of degrading H2O2 diffusing through the polymer shell. The time course of the proteolytic loss of catalase activity was also determined (Fig. 6). About 90% of

Fig. 6. Loading into nanoparticles protects catalase from proteolysis. (Inset) Free catalase retained less than 1% of its initial activity after 4 h of protease treatment (closed bar), regardless of the presence of unloaded PNC (light bar), while PNC-loaded catalase retained 35% activity (gray bar). Nanoparticles were prepared using PEG-PLGA with a 10% (o) or 20% ( ) aqueous content in the primary emulsion, and loss of activity in a pronase solution was measured as a function of time. For free catalase (z), 80% of activity is lost after 15 min, whereas PNC/catalase preparations retain a stable (25%) fraction of activity resistant to degradation. In the 10% aqueous content preparation, rate of activity loss was greatly reduced.

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free catalase activity was lost after 1-h incubation, and fell below measurable levels at 6 h. In contrast, catalase loaded into the standard PNC formulation retained 40% of its initial activity at 6 h. The activity seemed to reach a plateau with 25% of the initial activity remaining at stable level for at least ~20 h. The loss of activity in the PNC is believed to be associated with the catalase that is either surface bound or is released into the aqueous medium. However, the stable active fraction represents catalase residing inside the protease inaccessible domains of the PNC. When the catalase was loaded into PNC formed using a 20% aqueous content in the primary emulsion (instead of the 10 wt.% employed through these studies), initial rate of loss of catalase was significantly accelerated, but, after 1 h, the activity stabilized at level of ~25% of initial activity.

4. Discussion The long-term goal of this research is to improve antioxidant therapies by intracellular delivery of a

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potent antioxidant enzyme catalase into endothelial cells, utilizing targeting toward CAMs, surface determinants functionally involved and up-regulated in inflammation and oxidative stress [7,40] (Fig. 1A). The degree of internalization via CAM–endocytosis pathway is greatly enhanced when PNC is between 100 and 500 nm in size [14,15]. In addition, drug delivery particles larger than a few hundred nanometer are not optimal for intravascular administration, in part due to fast uptake by reticulo-endothelial system and danger of mechanical retention in capillaries, e.g., in the pulmonary vasculature [41,42]. Hence, it is important to produce PNC within the 100–500 nm size constraint. In order to prolong antioxidant effects, we have designed stealth PNC that possesses particles size in the range dictated by the proposed delivery scheme and would protect delivered catalase in lysosomes and other aggressive compartments. Catalase residing in lysosomes effectively decomposes H2O2 that diffuses into this compartment through numerous cellular membranes including plasmalemma and the lysosomal membrane [8,14]. Diffusion of H2O2 through PLGA polymer is slower than in water (Fig. 1C), yet is still 10 times faster than in lipid membranes [36]. PNC composed of PEG-PLGA copolymers are most likely even more permeable for H2O2, due to PEG’s hydrophilicity. Proteolysis studies confirm that PNCencapsulated catalase, inaccessible for external proteases, decomposes external H2O2 (Fig. 6). Thus, the diffusional barrier formed by PNC is not expected to compromise the effectiveness of catalase delivered into endothelial cells. Mechanical homogenization was found to be less damaging than sonication (80% activity remaining after 1 min homogenization at 20 krpm), yet increased homogenization rates and time seemed to suppress loading (Fig. 5B). However, varying surfactant level and polymer concentration provided useful alternative means in controlling the size of loaded PNC. Standard double emulsion conditions at 4 8C were not conducive to effective loading of catalase into the PNC (~2%, Fig. 1C). It was hypothesized that, by incorporating a freeze–thaw cycle during PNC synthesis, the polymer phase would precipitate around the primary emulsion, improving overall encapsulation. Indeed, loading was enhanced ~10-fold using this strategy. An observation that at higher polymer concentrations (N100 mg ml1) the freeze–thaw cycle resulted in the complete precip-

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itation of the polymer phase further confirmed this hypothesis. One question that needed to be answered was whether all loaded catalase is active and available for hydrogen peroxide degradation. If there is a loss of accessibility or a significantly enhanced deactivation of enzyme in the freeze–thaw loading mechanism, it would be expected that the loading ratio by radiotracing would be significantly higher than the loading ratio as determined by activity measurements. However, the radiotraced loading ratio of the freeze–thaw to non-freeze–thaw samples was 8 (Fig. 2C). Yet, from the activity studies (Fig. 5B), the activity ratio of the freeze–thaw (1.7 Units mg1 polymer) to nonfreeze–thaw (0.13 Units mg1 polymer) samples was 13. This enhancement in the activity loading ratio as compared to the radiotraced loading ratio demonstrates that not only is the majority of loaded catalase accessible for hydrogen peroxide degradation, but that there may even exist a protective mechanism for enzyme activity. Considering the decrease in catalase activity seen in the freeze–thaw method without polymer present (Fig. 2B), it is speculated that the presence of both the amphiphilic polymer and freeze– thaw cycle contributes to this observation. Further studies are required to elucidate the extent to which this protective effect may exist. It is tempting to speculate upon theoretical considerations of PNC formation, sizing and loading as a potential basis for a more general framework for the formulation of drug delivery vehicles. Conceivably, many enzymes using small substrates diffusing through polymer shell, such as sugars (e.g., glucose oxidase, glucose dehydrogenase), amino acids (e.g., nitric oxide synthase), and glutathione (e.g., glutathione reductase) may be amenable to loading into protecting PNC. Specifically, features of catalaseloaded PNC described in this paper warrant testing of this delivery vehicle in cell culture and animal studies, as a new strategy for a prolonged protection against vascular oxidative stress.

5. Conclusion This study combines protein immobilization/ encapsulation technology and nanocarrier synthesis to create polymer nanoparticles capable of protecting

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a large enzyme cargo from external proteolysis and exerting its activity towards its substrate, H2O2 diffusing through the polymer shell (Fig. 6). Not only did PNC effectively slow the rate of catalase activity loss but that a large fraction (25%) of the loaded enzyme was also stably protected from proteolysis. Therefore, nanotechnology-based intracellular delivery of PNC-loaded catalase can be tested as a novel approach to achieve a prolonged protection against oxidative stress.

Acknowledgements The authors would like to thank Anthony Lowman and his laboratory in the Department of Chemical Engineering at Drexel University for the generous use of their FTIR and helpful comments in drafting this work. Thanks are also given to Vladimir Shuvaev for advice in determination of catalase enzymatic activity. Furthermore, thanks are given to Karen Winey and her laboratory in the Materials Science and Engineering Department at the University of Pennsylvania providing use to their GPC. Finally, Fariyal Ahmed in the laboratory of Dennis Discher in the department of chemical engineering at the University of Pennsylvania is graciously acknowledged for reviewing this manuscript. Personal assistance was provided by an NIH NRSA postdoctoral training grant. This work was funded by a grant from the National Institute of Health (NIH RO1 HL078785).

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