Zwitterionic gel encapsulation promotes proteinstability, enhances pharmacokinetics, andreduces immunogenicityPeng Zhanga, Fang Suna, Caroline Tsaoa, Sijun Liub, Priyesh Jaina, Andrew Sinclaira, Hsiang-Chieh Hunga, Tao Baia,Kan Wua, and Shaoyi Jianga,b,1

aDepartment of Chemical Engineering, University of Washington, Seattle, WA 98195; and bDepartment of Bioengineering, University of Washington,Seattle, WA 98195

Edited by Joseph M. DeSimone, University of North Carolina at Chapel Hill, Chapel Hill, NC, and approved August 25, 2015 (received for review June 25, 2015)

Advances in protein therapy are hindered by the poor stability,inadequate pharmacokinetic (PK) profiles, and immunogenicityof many therapeutic proteins. Polyethylene glycol conjugation(PEGylation) is the most successful strategy to date to overcomethese shortcomings, and more than 10 PEGylated proteins havebeen brought to market. However, anti-PEG antibodies inducedby treatment raise serious concerns about the future of PEGylatedtherapeutics. Here, we demonstrate a zwitterionic polymer networkencapsulation technology that effectively enhances protein stabil-ity and PK while mitigating the immune response. Uricase modi-fied with a comprehensive zwitterionic polycarboxybetaine (PCB)network exhibited exceptional stability and a greatly prolongedcirculation half-life. More importantly, the PK behavior was un-changed, and neither anti-uricase nor anti-PCB antibodies weredetected after threeweekly injections in a rat model. This technologyis applicable to a variety of proteins and unlocks the possibilityof adopting highly immunogenic proteins for therapeutic or pro-tective applications.

protein delivery | immune response | zwitterionic polymer | hydrogel |nanomedicine

As an emerging class of therapeutics, protein drugs offer theadvantages of higher specificity and potency than small

molecules due to their structural complexity (1). However, thiscomplexity increases their vulnerability to environmental changesand immune system recognition and thus hinders their formu-lation and delivery. Additionally, the immunogenicity of manyprotein drugs not only limits their therapeutic efficacy, but alsothreatens patients’ lives with adverse effects including anaphy-laxis and infusion reactions (2). As a result, only minimally im-munogenic proteins can be developed into human therapeutics,which greatly restricts the possibilities and diversity of proteintherapies. Numerous efforts have been made to humanize non-human sourced proteins, but limited successes have been achievedcompared with the bright initial prospects.Conjugating polyethylene glycol (PEG) to proteins, known as

PEGylation, is thus far the most successful tactic used to mitigatean immune response to therapeutic proteins (3–5). When it wasfirst introduced to alter protein immunogenicity in the 1970s,PEG was thought to be nonimmunogenic and nonantigenic (6).However, several studies in the subsequent decades have foundanti-PEG antibodies after treatment with PEGylated therapies,with titers strongly related to modification density and the im-munogenicity of the anchoring protein (5, 7). The haptenic char-acter of PEG is considered the main culprit in the therapeuticefficacy loss of PEGylated products. Recent clinical studies ofPegloticase (PEGylated uricase) in refractory chronic gout pa-tients unequivocally demonstrated that anti-PEG antibodiescorrelate with a reduction in drug effectiveness—Pegloticaseloses efficacy in more than 40% of patients, and the presence ofanti-PEG antibodies doubles the risk of infusion reactions (8, 9).Similar results have also been observed for other PEGylated

proteins in clinical use, including PEG-asparaginase (10) andPeginterferon alfa (11). PEGylated nanoparticles such as lipo-somes also stimulate a strong anti-PEG response, and these an-tibodies were found to cross-react with different PEGylatedproducts (12, 13). Even more concerning, the prevalence of anti-PEG was found to be 22–25% in a sample of 350 healthy blooddonors, attributed to daily exposure of PEG-containing consumerproducts (14). Altogether, these findings raise obvious concernsregarding the current and future efficacy of PEGylated drugs. Analternative to this gold standard strategy is urgently needed tosupplement or replace PEGylation.Based on the extensive studies of PEGylation, we believe that

two issues are responsible for its immune response: the immuno-genicity/antigenicity of PEG itself and limited PEG coverage ofprotein surfaces. Systematic studies have shown that PEG immu-nogenicity strongly correlates with the hydrophobic characteristicsof this amphiphilic polymer (15–17); we thus hypothesize that atruly hydrophilic polymer should do a better job. Zwitterionicmaterials have emerged as an important class of extremely hy-drophilic biomaterials, demonstrating particularly high hydrationand ultra-low protein adsorption (18–20). Carboxybetaine is uniqueamong these zwitterionic materials due to its structural similarityto glycine betaine, a naturally occurring compound in the humanbody (18). Surfaces modified with polycarboxybetaine (PCB) haveexhibited protein adsorption below 0.3 ng/cm2 from 100% bloodplasma or serum (21). In a more recent study, zwitterionic hy-drogels comprising carboxybetaine monomer and cross-linkerwere shown to resist the foreign body reaction when implanted inmice for 3 months, due to the ultra-hydrophilic and biocompatiblenature of the gel (22). Moreover, PCB-coated gold nanoparticles

Significance

A protein modification technology has been developed inthis study to overcome current problems faced by proteintherapy. After being individually encapsulated in a super-hydrophilic zwitterionic gel, a therapeutic protein showed ex-ceptional stability and long in vivo circulation half-life. Moreimportantly, no immune response against either the protein orthe polymer was observed following repeated injections. Thistechnology will benefit patients by reducing administrationfrequency and mitigating adverse reactions and could allowmore immunogenic proteins to enter into human therapeuticor protective applications.

Author contributions: P.Z., F.S., and S.J. designed research; P.Z., F.S., C.T., S.L., P.J., H.-C.H.,T.B., and K.W. performed experiments; P.Z. and F.S. analyzed data; and P.Z., S.L., A.S., andS.J. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: sjiang@uw.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1512465112/-/DCSupplemental.

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were shown not to induce a measurable antibody response in ratsafter repeated i.v. injections (23). These findings motivated us totest polymer-protein constructs using PCB as a minimally- or non-immunogenic alternative to PEGylation. In terms of methodology,PEGylation processes conjugate multiple linear polymers toa protein surface to camouflage surface epitopes. Due to thelimited number of conjugation sites available, or as a tradeoff topreserve protein activity, conjugated polymers may be insuffi-cient to conceal all “danger signals.” The anchoring protein mayin turn amplify the immunogenicity of conjugated polymers,causing the inverse correlation reported between the graft den-sity and immune response (7). To overcome this limitation, weadopted bifunctional CB monomers to cross-link conjugatedpolymers into a “mesh” network, designed to possibly maskprotein surfaces completely and maximize surface hydration. Weevaluated the in vitro and in vivo performance of this zwitterionicpolymer network encapsulation (ZPNE) technology by choosinghighly immunogenic fungal-derived uricase as a model therapy andcompared the stability, pharmacokinetics (PK), and immunoge-nicity to its PEGylated counterpart.

Results and DiscussionZwitterionic Encapsulation of Uricase. In our hypothesis, there aretwo crucial aspects to the design of protein protection strategies:the adoption of a superhydrophilic biomaterial and the com-prehensive coverage of protein surface epitopes. A cross-linkedcarboxybetaine-based gel coating meets these criteria. However,incorporating a conventional cross-linker (e.g., ethylene glycoldimethylacrylate) may introduce unwanted hydrophobicity andcompromise the hydrophilicity of the cross-linked gel coating. Toavoid this detrimental possibility, we designed a bifunctional CBcross-linker carboxybetaine acrylamide cross-linker (CBAAX)(Fig. 1A) to maximize surface hydration. This carboxybetainecross-linker has proven to be a key for PCB hydrogels to becapable of resisting the foreign body reaction (22) and restrain

mesenchymal stem cell differentiation (24). To realize ZPNE inthis work, each protein was encapsulated in a hydrogel nano-particle through surface acrylation and in situ free radical poly-merization reactions. Basic characterization data are shown inFig. 1 and Table S1. Both PEGylated uricase and ZPNE-modi-fied uricase (denoted as uricase-PCBX) shared a similar hydro-dynamic size of around 30 nm, much larger than the ∼10-nmdiameter of the native uricase tetramer. Zeta potential mea-surements of uricase-PCBX showed a neutral surface charge(−2.2 mV), indicating more comprehensive surface coveragethan that of uricase-PEG (−8.4 mV). Atomic force microscopy(AFM) results showed that uricase-PCBX nanoparticles have amean diameter of 28.6 ± 6.1 nm, consistent with those results fromdynamic light scattering (DLS) measurements. Gel permeationchromatography (GPC) results revealed that five PEG moleculeswere conjugated to each uricase subunit. It should be noted thatdue to the difficult “graft-to” conjugation of a large polymer to aprotein, this was the maximum conjugation number obtainedafter conjugation conditions were optimized. In contrast, it wasmuch easier to achieve higher degrees of surface modificationwith the ZPNE technique. To maintain a high residual activity(>90%), we limited the surface conjugation number to around12. The whole modification process is simple and applicable to awide variety of proteins. The core-shell structure of these zwit-terionic nanocapsules exploits a highly hydrated polymer shell torender the protein core “stealth” to its surroundings and con-ceivably to immune system recognition. Compared with con-ventional protein-polymer conjugates, this core-shell structureprovides better protection for its inner cargo.

In Vitro Protein Stability. Preparation of therapeutic proteins isoften prone to instability during storage and use, and enhancedstructural stability is a desirable attribute of modification tech-nologies. Thermal stress tests at elevated temperatures are afacile approach to evaluate differences in protein stabilities in a

Fig. 1. Zwitterionic protein encapsulation and characterization. (A) Molecular structure of CBAA monomer and CBAAX cross-linker and the process of ZPNEmodification of a protein surface. (B) Size distributions and (C) zeta potentials of modified uricase vs. native uricase. (D) AFM image of formed zwitterionicnanocapsules. (E) Thermal stability study of modified uricase vs. native uricase, as retained activity after incubation at 65 °C.

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relatively short time (25–27). A protein conjugate or nano-capsule stable at higher temperatures is likely to be stable atambient temperatures for a longer period. To test the stability ofmodified proteins to environmental stressors, their enzymaticactivities were assayed after incubation at 65 °C for differenttime periods and presented as the percentage of preheating ac-tivity retained. As shown in Fig. 1E, uricase-PCBX has excep-tional thermal stability and retained 100% activity after 2 h ofincubation. In contrast, uricase-PEG only exhibited a modeststabilizing effect.The present result, that uricase-PCBX remains stable at 65 °C,

suggests that it would not require low-temperature storage orformulation with stabilizing excipients as is the case for the na-tive protein. This high stability is important when consideringsituations in which low-temperature storage or transportation isunavailable, and may help reduce the high cost of therapeuticprotein production and use. In a previous study, PCB was shownto impart a superior protein-stabilizing effect and increase thesubstrate affinity to protein binding sites when similar proteinconjugation structures were tested (26). The CB monomer unit isa derivative of glycine betaine, a natural molecule found in manyliving systems as a stabilizing agent. Encapsulating a protein in aPCB gel network is analogous to placing it into a highly con-centrated protein stabilizing solution, equipping it with a pro-tective microenvironment that accompanies the protein bothin vitro and in vivo. Furthermore, the nanocapsule structure hasbeen reported to stabilize enzymes through multiple covalentattachments between the enzyme core and polymer shell, whicheffectively hinder conformational changes in adverse environ-ments (27, 28). Combining these two distinct mechanisms, it isunsurprising that PCB gel encapsulation affords superior stabil-ity to protein cargos.

PK Study. Since the 1970s, PEGylation has been the gold standardmethod to alter PK properties of both proteins and a varietyof nanoparticle platforms for drug delivery (3, 29). Surface at-tachment of PEG significantly prolongs in vivo circulation timesby increasing hydrodynamic size to avoid rapid renal clearance

(for small particles) and reducing interactions with both bloodcomponents (opsonization) and immune cells. The PK profile ofZPNE-modified uricase was compared with this gold standard inhealthy Sprague–Dawley rats through repeated i.v. injections.Fig. 2 shows the circulation profiles of native and modified uri-case after each injection. All concentration-time curves fit a two-compartment model. PK parameters are listed in Table 1. On theinitial injection, uricase-PEG exhibited an elimination half-life(t1/2β) of 38.2 h, threefold higher than the 13.9 h t1/2β of nativeuricase. The area under curve (AUC∞) of PEGylated uricase isfive times that of the native enzyme, roughly in agreement withvalues reported in the literature (30). In comparison, uricase-PCBXshowed exceptionally prolonged circulation behavior, with a t1/2βof 134.9 h, a 3.6-fold improvement over uricase-PEG, and 10-foldhigher than the native enzyme. Moreover, the AUC∞ of uricase-PCBX was 4.2 times that of uricase-PEG, indicating its significantlyhigher systemic availability.Uricase is a large tetrameric protein with a molecular weight

of ∼130 kDa. The rapid clearance of native uricase is consideredto be associated with an irreversible uptake process, likely by thereticuloendothelial system (RES), rather than renal filtration(31). Conjugating a total of 20 large PEG molecules (each witha molecular weight of 10 kDa) to uricase can effectively covera large portion of its surface, minimizing nonspecific uptakeclearance. This phenomenon is closely related to material hy-dration and nonfouling character. As a well-known class of ultra-low fouling materials, zwitterionic polymers have indeed beenused to extend the circulation lifetime of nanoparticles (32–34).Apart from material properties, recent research has shown sur-face coverage density to be another important factor affectingparticle uptake by immune cells and in vivo circulation behavior(35). A high surface grafting density of PEG molecules, ex-ceeding the minimum for a brush conformation, is required fornanoparticles to evade immune cell uptake and clearance. How-ever, in the case of protein modification, there is a certain limitto conjugation density due to the restricted number of availablefunctional groups on the surface of a protein. A cross-linkedhydrogel “mesh,” which does not require a high number of

Fig. 2. Blood circulation profiles of modified uricase vs. native uricase. Circulation curves after the (A) first i.v. administration, (B) second administration, and(C) third administration.

Table 1. PK parameters after repeated injections

Parameters

Native uricase (different injections) Uricase-PEG (different injections) Uricase-PCBX (different injections)

1 2 3 1 2 3 1 2 3

t1/2α (h) 1.7 0.4 0.5 3.7 2.1 1.9 2.9 3.7 4.0t1/2β (h) 13.9 12.5 13.2 38.2 30.6 24.8 134.9 121.3 145.5AUC∞ (μg/mL × h) 296 85 90 1,394 590 237 5,796 5,658 6,126CL (mL/h) 3.38 14.17 16.68 0.72 2.03 6.34 0.17 0.21 0.24MRT (h) 11.6 11.2 9.7 48.1 33.6 11.5 190 170 203

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conjugation sites, can effectively overcome this problem. Athin hydrogel layer can ideally mask the entire protein surface,making it “stealth” to surroundings and slowing down RESclearance. The results of this study confirm our hypothesis thatgreatly extended circulation profiles of protein therapeutics canbe realized by PCB nanocapsule encapsulation. Patients couldbenefit greatly from this advance by enjoying a reduced adminis-tration frequency compared with PEGylated products.Uricase is administered chronically in clinical practice to treat

refractory chronic gout, so repeated injections were performedto evaluate the long-term performance of ZPNE-modified andPEGylated proteins. The second and third injections were givenat days 7 and 14 after the initial injection. It should be noted thatsevere infusion reactions were observed 3–5 min after the secondand third injections of both native uricase and uricase-PEG, withsymptoms including hypopiesia and dyspnea. In contrast, noadverse effects were observed following the uricase-PCBX in-jections. Although the calculated t1/2β of native uricase appearscomparable after each injection, the sampling frequency waslikely inadequate for its rapid elimination; its accelerated elim-ination after repeated injections is more clearly reflected in thesignificantly reduced AUC∞. Following the second and thirdinjections of uricase-PEG, an obvious accelerated blood clear-ance (ABC) phenomenon was noted, with t1/2β decreasing to 30.6and 24.8 h, and AUC∞ values dropping 2.8-fold and 5.9-fold,respectively. This accelerated elimination process is most likelydue to the induction of anti-PEG antibodies, which will be dis-cussed in the following section. Unlike PEGylated uricase, thePK profiles of uricase-PCBX in this study did not show anysignificant change following three injections, indicating thaturicase-PCBX is likely to maintain its superior PK performancein repeated applications.

Detection of Antibodies. Serum collected at day 35 after the firstinjection was tested for both anti-uricase and anti-polymer an-tibodies using direct ELISA. As shown in Fig. 3 A and B, IgG wasthe major isotype of anti-uricase observed after repeated in-jections. The measured antibody titers are listed in Table S2.

Due to the strong immunogenicity of the fungal enzyme, thenative uricase group developed extremely high IgG antibody titersalong with relatively high titers of IgM. In the group treated withPEGylated uricase, the titers of anti-protein antibodies were sig-nificantly lower than the native enzyme but still clearly elevated.In contrast, neither anti-uricase IgG nor IgM were detected inthe rat group treated with uricase-PCBX. Our results here areconsistent with clinical findings, showing that PEGylation cannotcompletely prevent the generation of anti-protein antibodies, atleast for highly immunogenic uricase (9). This finding supports ourhypothesis that comprehensive surface coverage of a protein with astealth polymer network can effectively render the protein “in-visible” to the immune system and thus mitigate antibody induction.The severe infusion reactions and accelerated drug elimination

seen in the native uricase group is apparently related to strongproduction of anti-uricase antibodies. However, for PEGylatedprotein therapeutics, clinical trial evidence suggests anti-PEGantibodies rather than anti-protein antibodies are primarily re-sponsible for infusion reactions and efficacy loss over chronicinjections (8, 9). Accordingly, strategies to suppress the pro-duction of anti-polymer antibodies remain a critical challenge inthe development of polymer-protein conjugates. Uricase-PEGand uricase-PCBX groups were tested for anti-PEG and anti-PCB anti-polymer antibodies, respectively, and the results areshown in Fig. 3 C and D. Notably, at day 35, the major anti-PEGresponse comes from IgM (titers 1:6,400) rather than IgG (titers1:800), suggesting differences in adaptive immunity pathwaysfor the protein and polymer components in the conjugates.Compared with the strong response to PEGylated uricase, noIgG or IgM were detected against the polymeric component ofuricase-PCBX.As repeated injections are commonly needed for therapeutic

proteins, the antibody response issue should not be overlookedand merits careful evaluation. It should be noted that in theearlier work establishing PEGylation as a method to alter proteinimmunogenicity (6), neither anti-BSA nor anti-PEG antibodieswere detected after injecting BSA-PEG conjugates into a rabbitmodel. Three reasons may account for their observations. First,

Fig. 3. Detection of antibodies by direct ELISAs. (A) Anti-uricase IgM, (B) anti-uricase IgG, (C) anti-polymer IgM, and (D) anti-polymer IgG. Serum from eachrat is plotted separately.

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BSA shares a large degree of similarity with rabbit serum albu-min (RSA) (36), which makes it less immunogenic as a modelprotein. In a later study, the generation of antibodies againstPEGylated proteins was shown to be strongly related to theprotein immunogenicity (7). This result explains why PEGylatednonhuman enzymes, e.g., uricase and asparaginase, have beenreported to present the most severe issues in clinical trialsresulting from anti-PEG antibodies (8–10). Second, a high de-gree of modification can be easily attained with BSA. The re-lationship between modification degree and immunogenicity ofconjugates was also clearly demonstrated in the later study (7).Serum albumin is a class of protein with extraordinary ligandbinding capacity (36), but this is not the case for most therapeuticproteins. Finally, immunological assays of the time, e.g., immu-nodiffusion and complement fixation tests, were not capable ofdetecting low-titer antibodies.Because there is no standard method to evaluate anti-polymer

antibodies, and especially as no positive control is available forthe detection of anti-PCB antibodies, we validated our resultsusing a surface plasmonic resonance (SPR) sensor. SPR is anultra-sensitive technique used to study specific/nonspecificprotein adsorption on surfaces with nanogram-level detectionlimits (21, 37). Compared with ELISA, SPR analysis is morestraightforward, as the procedures are simpler, and each step canbe carefully controlled and monitored. To detect anti-PEG oranti-PCB antibodies, we modified the surface of gold chips withpoly(ethylene glycol) methyl ether methacrylate (PEGMA) orPCB polymer brush layers. Both PEGMA and PCB are non-fouling materials that resist nonspecific protein adsorption fromdiluted serum. When serum samples flow over the modified chipsurface, only the polymer-specific antibodies can bind to thesurface and generate an SPR signal through specific adsorption(Fig. 4A). The results are shown in Fig. 4. Sera obtained pre-injections were used as a negative control. When sera from uri-case-PEG–treated rats were flowed over a PEGMA modifiedchip, adsorption curves showed typical specific binding kineticswith adsorbed mass increasing linearly with time. Protein bindingreached 40–50 ng/cm2 in 15 min under experimental conditions.No nonspecific binding was detected on the same chip from

either control serum or uricase-PCBX serum, indicating all ad-sorption came from anti-PEG antibodies. In contrast, none ofthe sera showed detectable adsorption on a PCB-coated chipsurface, suggesting that there is no antibody in the sera that canrecognize and bind to PCB polymers.

Biodistribution of Modified Uricase. To examine the biodistributionof modified uricase samples following chronic use, we gatheredblood and tissue samples from a treated rat 72 h after the thirdinjection for further analysis. The third doses of uricase, uricase-PEG, and uricase-PCBX were labeled with a fluorescent probefor this test, whereas unlabeled formulations were given for theinitial and second doses to avoid interference. As shown in Fig. 5,the data are presented as recovered fluorescence per gram oftissue. Uricase-PCBX sustained a high serum concentration,whereas all three samples accumulated primarily in the liver.Native uricase was observed to have less organ accumulationcompared with protected enzymes three days after injection.This phenomenon can be explained by their different levels ofprotease stability. Native, unprotected uricase was readily de-graded by proteases after liver accumulation, but protecteduricase was partially shielded from participation in this meta-bolic process. As uricase nanocapsules were prepared usingamide-containing cross-linkers, they are expected to degradethrough slow hydrolysis. One potential advantage of this per-sistence is that these nanocapsule constructs may retain theirability to catalyze the oxidation of uric acid even after distri-bution into organs.

ConclusionsIn summary, we demonstrate here that a polycarboxybetainepolymer network mesh coating on a protein effectively improvesprotein stability, extends PK, and mitigates immune responsewith superior efficacy to PEGylation. PCB-encapsulated uricasedid not suffer the accelerated blood clearance encountered byPEGylated uricase during repeated injections in a rat model, andneither anti-uricase nor anti-PCB antibodies were detected in theserum. With its superior ability to mitigate protein immunogenicity,this reported technique makes it possible to adopt highly immu-nogenic proteins for human therapeutic or protective applications.

Materials and MethodsProtein Encapsulation and Polymer Conjugation. For ZPNE, uricase was firstmodified to introduce acryloyl group onto its surface. The reaction wasperformed by dissolving 20 mg uricase into 10 mL 50 mM Hepes buffer(pH 8.5), followed by adding 75 μL N-acryloxysuccinimide (NAS) dimethyl

Fig. 4. Detection of antibodies by an SPR sensor. (A) Scheme showing thedetection setup. The gold chip surface was modified with either PEGMA orPCB polymer brushes. The polymer brush serves dual functions, providing anonfouling background while serving as the investigated antigen. (B) Sum-mary of the SPR detection results. (C) Sensor signal-time curves on PEGMA-coated chips, each curve representing one serum sample. No uricase-PCBXserum showed any adsorption onto a PEGMA surface, though only onetypical curve is shown. (D) Sensor signal-time curves on PCB-coated chips.

Fig. 5. Biodistribution of modified uricase vs. native uricase after the thirdinjection. Each value is averaged from three rats. SDs are shown as error bars.

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sulfoxide (DMSO) solution (20 mg/mL) dropwise. The reaction was stirred at4 °C for 2 h. The polymer encapsulation was done via in situ radical poly-merization by adding 400 mg CBAA monomer, 80 mg CBAAX, 10 mL Hepesbuffer, 15 mg ammonium persulfate (APS), and 60 μL tetramethylethyl-enediamine (TEMED) into the former reaction solution. After stirring foranother 2 h, the reaction mixture was concentrated and washed extensivelyby PBS 7.4 using 300-kDa molecular weight cutoff centrifugal filters.

Uricase-PEG conjugates were prepared in 50 mM Hepes buffer (pH 8.5),with uricase concentration at 1 mg/mL and mPEG-NHS concentration at10 mg/mL The reaction was stirred at 4 °C overnight. Then the conjugateswere concentrated and washed extensively by PBS, pH 7.4, using a 300-kDamolecular weight cutoff centrifugal filter. The protein residue activity wasmeasured by a commercially available uricase activity kit (Life Technologies).

PK and Biodistribution Study. The PK of native and modified uricase is studiedusing Spraque–Dawley rats (male, body weight 74–100 g) as the animalmodel. Each sample has three duplicates to generate statistical significance.All animal experiments adhered to federal guidelines and were approved bythe University of Washington Institutional Animal Care and Use Committee.For PK studies, each protein sample was administered into the rat via tailvein injection at the dose of 25 U/kg body weight. Blood samples werecollected from the tail vein at 5 min, 6 h, 24 h, 48 h, and 72 h after the in-jection. The blood samples were put in heparinized vials and centrifuged,

and the enzyme content in plasma was estimated by enzyme activity. The i.v.injections and bleeding procedure were repeated three times with 1 wk astime interval between each injection. Five weeks after the first injection,5 mL blood was drawn by using cardiac punch, and serum was prepared forantibody detections. All PK parameters were calculated using PKSolver fol-lowing the instructions.

For the biodistribution study, all rats were given unlabeled samples for thefirst and second dose, followed by FITC-labeled samples for the third dose. At72 h after injection, all rats were killed, and the heart, liver, spleen, lung,kidney, and blood were collected for further analysis. The collected organtissues were homogenized using a tissue ruptor, followed by centrifugationat 3,200 × g for 30 min at room temperature. The fluorescence of particles inthe tissues was measured using a microplate reader and compared with astandard curve generated using FITC-labeled samples added to untreatedhomogenized tissues.

Statistics. The Student t test was chosen to compare two small sets of quan-titative data when data in each sample set were related, with P < 0.05 beingconsidered as statistically significant.

ACKNOWLEDGMENTS. This work was supported by the Defense ThreatReduction Agency (HDTRA1-13-1-0044) and National Institutes of Health(1R21EB020781-01).

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