Characterizing the antitumor response in mice treated with ...2 Characterizing the antitumor...

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Acta Biomaterialia xxx (2012) xxx–xxx

ACTBIO 2468 No. of Pages 7, Model 5G

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Contents lists available at SciVerse ScienceDirect

Acta Biomaterialia

journal homepage: www.elsevier .com/locate /actabiomat

Characterizing the antitumor response in mice treated with antigen-loadedpolyanhydride microparticles

Vijaya B. Joshi a, Sean M. Geary a, Brenda R. Carrillo-Conde b, Balaji Narasimhan b, Aliasger K. Salem a,⇑a Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USAb Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, USA

a r t i c l e i n f o

232425262728293031323334

Article history:Received 27 July 2012Received in revised form 19 October 2012Accepted 1 November 2012Available online xxxx

Keywords:PolyanhydrideAntitumor immune responseMicroparticlesBiodegradable polymerAntigen

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1742-7061/$ - see front matter � 2012 Acta Materialhttp://dx.doi.org/10.1016/j.actbio.2012.11.001

⇑ Corresponding author. Tel.: +1 319 335 8810.E-mail address: [email protected] (A.K. Sa

Please cite this article in press as: Joshi VB et al.Acta Biomater (2012), http://dx.doi.org/10.1016

a b s t r a c t

Delivery of vaccine antigens with an appropriate adjuvant can trigger potential immune responsesagainst cancer leading to reduced tumor growth and improved survival. In this study, various formula-tions of a bioerodible amphiphilic polyanhydride copolymer based on 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane (CPH) with inherent adjuvant propertieswere evaluated for antigen-loading properties, immunogenicity and antitumor activity. Mice were vacci-nated with 50:50 CPTEG:CPH microparticles encapsulating a model tumor antigen, ovalbumin (OVA), incombination with the Toll-like receptor-9 agonist, CpGoligonucleotide 1826 (CpG ODN). Mice treatedwith OVA-encapsulated CPTEG:CPH particles elicited the highest CD8+ T cell responses on days 14 and20 when compared to other treatment groups. This treatment group also displayed the most delayedtumor progression and the most extended survival times. Particles encapsulating OVA and CpG ODN gen-erated the highest anti-OVA IgG1 antibody responses in mice serum but these mice did not show signif-icant tumor protection. These results suggest that antigen-loaded CPTEG:CPH microparticles canstimulate antigen-specific cellular responses and could therefore potentially be used to promote antitu-mor responses in cancer patients.

� 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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1. Introduction

Cancer is responsible for one in every four deaths in the USA andis still not effectively managed therapeutically [1]. The currentparadigm of chemotherapy and surgery requires improvements soas to enhance the overall survival rate of cancer patients and to limitthe toxic side effects of the current chemotherapeutic approaches.Therapeutic cancer vaccines have received substantial impetusthrough a succession of findings over the past two decades that in-clude: (i) the discovery of tumor-associated antigens (TAAs) thatpotentially flag the presence of tumor cells to the host’s immune sys-tem [2]; (ii) the finding that dendritic cells (DCs) orchestrates thecourse of immune responses [3]; and (iii) the observation thatpathogen-associated molecular patterns derived from microbesare strong inducers of DC maturation and resultant cellular immuneresponses [4]. Most recently, US Food and Drug Administrationapproval of the first cancer vaccine, Sipuleucel-T (Provenge™), hasgiven the field of cancer immunotherapy a further boost [5].

The delivery of a cancer vaccine through the use of nano- andmicroparticle-based vectors is showing promise in both clinicaland preclinical settings [6]. Ideally, such vectors should possess a

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lem).

Characterizing the antitumor re/j.actbio.2012.11.001

number of favorable traits that include: biocompatibility; beingcapable of efficient co-delivery of immunogen (e.g. TAAs) and bacte-rial adjuvants to DCs; possessing adjuvant properties; and beingcapable of being stably stored and inexpensively manufactured[7–9]. In this study we investigated the potential of amphiphilicpolyanhydride microparticles based on 1,8-bis(p-carboxyphen-oxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy)hexane (CPH) to be used as a cancer vaccine delivery vehicle. Weused 50:50 CPTEG:CPH (Fig. 1) microparticles, which have previ-ously shown adjuvant properties in generating robust antigen-specific humoral responses and preferential uptake and activationof DCs and macrophages [10–14]. In addition, CPTEG:CPH copoly-mers have been shown to be non-toxic, stabilizing to antigens andbiodegradable with CPTEG:CPH particles, providing a burst releaseof 10–30% of encapsulated antigen followed by zero-order releasefor 10–30 days [11,15–17]. Thus, these polyanhydride vectors pos-sess many of the traits desirable for a cancer vaccine vehicle. Whencombined with a potent antigen delivery vehicle, the presence of abacterial or viral adjuvant has shown to increase antigenicity. Manyunmethylated CpG motifs of bacterial DNA act as immunestimulants which can bias immune responses to a Th1 type. Thesynthetic CpG-B oligonucleotide 1826 (CpG ODN) induces DC matu-ration and B cell activation through interaction with the Toll-likereceptor 9, which leads to enhanced activation of cytotoxic T cellresponses [18].

ll rights reserved.

sponse in mice treated with antigen-loaded polyanhydride microparticles.

http://dx.doi.org/10.1016/j.actbio.2012.11.001

mailto:[email protected]


http://www.sciencedirect.com/science/journal/17427061

http://www.elsevier.com/locate/actabiomat


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Fig. 1. Chemical structure of CPTEG:CPH polymer.

2 V.B. JoshiQ1 et al. / Acta Biomaterialia xxx (2012) xxx–xxx


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In this study, we report on the successful fabrication of CPTEG:CPHmicroparticles encapsulating a model TAA, ovalbumin (OVA),either alone (CPTEG:CPH–OVA), or co-encapsulated with CpG ODN(CPTEG:CPH–OVA/CpG). We also further demonstrate theimmunogenicity and anticancer potential of these microparticles ina prophylactic mouse tumor model.

2. Materials and methods

2.1. Polymer synthesis

Synthesis of CPTEG:CPH copolymer was carried out by meltpolycondensation, as described previously [19]. In brief, CPTEGand CPH monomers were mixed in a round-bottom flask at a50:50 M ratio to provide a total of 2 g of monomers. Next, 100 mlof acetic anhydride was added to the monomer mixture and re-acted for 30 min at 125 �C. The acetic anhydride was then removedin the rotary evaporator, and the resulting viscous liquid was poly-merized in an oil bath at 140 �C under a vacuum (<0.03 torr) for90 min. The resulting polymer was dissolved in methylene chlorideand isolated by precipitation into cold hexane in a 1:15 ratio. Thepurity of the polymer and the number average molecular weightswere verified and estimated using 1H nuclear magnetic resonance(1H NMR) spectra obtained from a Varian VXR-300 MHz NMR spec-trometer (Varian Inc., Palo Alto, CA). In addition, gel permeationchromatography (GPC; Waters HPLC 277 System, Milford, MAusing Varian Inc. GPC columns) was performed to determine thepolymer’s molecular weight.

2.2. Fabrication and characterization of microparticles loaded withCpG ODN and OVA

Microparticles were prepared using a double emulsion solventevaporation method derived from Intra and Salem [20]. Briefly, puri-fied endotoxin-free chicken egg white OVA (Sigma, St. Louis, MO)and endotoxin-free CpG ODN (Integrated DNA Technologies, Coral-ville, IA) were dissolved in 100 ll of 1% poly(vinyl alcohol) (PVA,Mowiol�; Sigma, Allentown, PA) solution. This solution was soni-cated for 30 s on power setting #10, (Sonic Dismembrator Model100, Fisher Scientific, Pittsburgh, PA) in 1.5 ml of dichloromethane(DCM) containing 200 mg of 50:50 CPTEG:CPH copolymer. This pri-mary emulsion was then sonicated in 8 ml of 1% PVA solution to gen-erate a secondary emulsion, which was then added to 22 ml of 1%PVA solution. The secondary emulsions were stirred in a fume hoodfor 2 h to allow evaporation of DCM. Microparticles were centri-fuged at 2880 g for 5 min. The pellets obtained were washed twicewith distilled water, followed by freeze drying using FreeZone 4.5(Labconco Corporation, Kansas City, MO). Particles were stored in

Please cite this article in press as: Joshi VB et al. Characterizing the antitumor reActa Biomater (2012), http://dx.doi.org/10.1016/j.actbio.2012.11.001

sealed containers at �20 �C. Size distribution and zeta potentialwere measured using a Zetasizer Nano ZS (Malvern, Southborough,MA).

To estimate the loading of OVA and CpG ODN, 20 mg of micro-particles from each batch was incubated with 0.2 N NaOH forapproximately 12 h at room temperature or until microparticleshad fully degraded. This solution was then neutralized using 1 NHCl and loading was calculated using Eq. (1). The percentageencapsulation efficiency (EE) of the fabrication process was calcu-lated as described in Eq. (2).

loading ¼ ðconcentration� volumeÞ=weight of particles ðmgÞð1Þ

EE ¼ ðweight of particles ðmgÞ � loading� 100Þ=initial weightof drug ðlgÞ ð2Þ

Here, loading = lg of OVA or CpG ODN encapsulated per mg ofparticles, concentration = calculated concentration of OVA or CpGfrom the standard curve (lg ml�1), and volume = volume of OVAor CpG ODN solution (ml).

The surface morphology and shape of microparticles were exam-ined using scanning electron microscopy (SEM). Briefly, a suspen-sion of particles was plated onto a silicon wafer mounted on anscanning electron microscope stub. This was then coated withgold–palladium by an argon beam K550 sputter coater (EmitechLtd., Kent, England). Images were captured using a Hitachi S-4800scanning electron microscope (Hitachi High-Technologies, Ontario,Canada) at 5 kV accelerating voltage.

2.3. Prophylactic murine tumor model

Eight- to twelve week-old male wild-type C57BL/6 mice (JacksonLaboratory, Bar Harbor, Maine; n = 4 per group) were treated withintraperitoneal injections of the following six groups of treatments:(i) OVA and CpG ODN encapsulated in 50:50 CPTEG:CPH micropar-ticles (CPTEG:CPH–OVA/CpG); (ii) OVA encapsulated in 50:50CPTEG:CPH microparticles (CPTEG:CPH–OVA); (iii) OVA encapsu-lated in 50:50 CPTEG:CPH microparticles with soluble CpG ODN;(iv) blank 50:50 CPTEG:CPH microparticles at an equivalent doseto the OVA particles; (v) soluble OVA and CpG ODN; and (vi) naive(untreated). For mice treated with soluble CpG, the particles or solu-tion of OVA was admixed with CpG solution immediately prior toinjections. Each mouse was primed on day 0 and similarly boostedon day 7 with the indicated treatments. Doses of 100 lg of OVAand 50 lg of CpG ODN per mouse were consistently used. On day21 OVA-specific CD8+ CD3+ T lymphocyte levels were determinedfrom peripheral blood harvested by submandibular bleeds (see



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Section 2.4). On day 28 OVA-specific IgG2a and IgG1 antibody titerswere measured in serum harvested by submandibular bleeds (seeSection 2.5). On day 35, mice were subcutaneously challenged with2 � 106 OVA-expressing E.G7 cells (American Type Culture Collec-tion, Manassas, VA) and tumor volumes were monitored over timeusing Eq. (3) [21]. All animal experiments were carried out in accor-dance with current institutional guidelines for the care and use ofexperimental animals.

tumor volume ¼ p=6� ðdiameter1 ðmmÞ� diameter2 ðmmÞ � height ðmmÞÞ ð3Þ

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Table 1Loading efficiency and loading mass of OVA- and CpG-encapsulated CPTEG:CPHmicroparticles.

Group Loadingefficiency (%)

Loading mass(lg per mg of polymer)

OVA-loaded CPTEG:CPH 78.6 OVA:11.8OVA- and CpG-loaded CPTEG:CPH OVA: 12.0 OVA:1.8

CpG: 21.0 CpG: 0.2

Table 2Particle size and zeta potential of CPTEG:CPH microparticles as measured by zetasizerNano ZS.

Particle size(lm)

Polydispersityindex (PDI)

Zeta potential(mV)

Blank particles 3.08 0.25 �0.63 ± 3.81OVA-loaded CPTEG:CPH 2.02 0.54 �3.50 ± 10.10OVA- and CpG-loaded

CPTEG:CPH1.57 0.52 �5.24 ± 7.24

2.4. Tetramer staining on peripheral blood lymphocytes

The frequency of OVA-specific CD3+ CD8+ T lymphocytes wasdetermined by tetramer staining and direct immunofluorescence,as previously described [22]. The tetramer used was the H-2Kb

SIINFEKL Class I iTAg™ MHC Tetramer (Kb-OVA257) labeled withphycoerythrin (PE; Beckman Coulter, Fullerton, CA). Surface CD8and CD3 were stained with fluorescein isothiocyanate-labeled ratanti-mouse CD8 (eBioscience, San Diego, CA) and PE–Cy5-labeledhamster anti-mouse CD3 (eBioscience, San Diego, CA) antibodies,respectively. Samples were acquired using a FACScan flow cytom-eter (Becton Dickinson, NJ) and analyzed with FlowJo software(TreeStar, OR).

2.5. Estimation of anti-OVA antibodies in peripheral blood using theenzyme-linked immunosorbent assay (ELISA)

Measuring antigen-specific IgG1 and IgG2a levels can provideinformation not only with respect to the degree of humoral stimula-tion but also as to the type (Th1 or Th2) of antigen-specific immuneresponse generated through the determination of IgG2a:IgG1 ratios[23]. High IgG2a:IgG1 ratios tend to indicate potential for a Th1 typeor cellular immune response whilst a low IgG2a:IgG1 ratio is moreindicative of a Th2 type or primarily humoral response. OVA-specificIgG1 and IgG2a antibodies in peripheral blood were quantified usinga modification of a standard ELISA protocol that has been describedin detail previously [24]. Briefly, serum samples were collected viasubmandibular bleeding and serial dilutions of the serum sampleswere incubated in wells of Immulon� 2HB 96-well high bindingpolystyrene microtiter plates (Thermo, Milford, MA) that had beenpreviously coated with 5 lg ml�1 of OVA solution in phosphate-buf-fered saline (PBS). Plates were washed with PBS–Tween, followed byincubation with alkaline phosphatase conjugated goat anti-mouseIgG antibodies (Southern Biotech, Birmingham, AL). Excess antibodywas washed away before p-nitrophenylphosphate (Sigma, St. Louis,MO) was added in the dark. Absorbance was measured after 2 h at405 nm using a SpectraMax� Plus384 microplate reader (MolecularDevices LLC, Sunnyvale, CA).

2.6. Statistical analysis

Groups were compared by one-way analysis of variance (ANOVA)followed by a Tukey post-test to compare all pairs of treatments.Analysis of survival curves was performed using the Mantel–Coxlog-rank test (GraphPad Prism, La Jolla, CA). Results presented arerepresentative of two or three repeats.

3. Results

3.1. Polymer characterization

The purity of the 50:50 CPTEG:CPH copolymer was verifiedusing 1H NMR spectroscopy. The NMR spectra indicated that theactual composition of the copolymer was in agreement with the


molar feed ratio (data not shown). GPC analysis showed that the50:50 CPTEG:CPH copolymer had a weight-average molecularweight of 8500 g mol�1, with a polydispersity index of 1.7. Theseresults are consistent with previously published data [14,25].

3.2. Characterization of 50:50 CPTEG:CPH microparticles loaded withCpG ODN and OVA or OVA alone

The 50:50 CPTEG:CPH microparticles were prepared using adouble emulsion solvent evaporation process as described in Mate-rials and methods. When OVA alone was used, loading efficiencies of>78% were obtained, yielding 11.8 lg of OVA encapsulated per mg ofmicroparticles (Table 1). When CpG ODN and OVA were co-encapsu-lated, the loading efficiency of OVA was only 12%, but the amount ofOVA in the microparticles was enough to be used in subsequentin vivo studies. The mean diameter of all microparticle preparationswas between 1 and 3 lm. The zeta potential of blank microparticleswas �0.63 mV, which did not change significantly upon encapsula-tion of OVA and CpG ODN in the microparticles (Table 2). SEMrevealed that the various microparticle preparations possessed asmooth morphology (Fig. 2).

3.3. Immunogenicity of different CPTEG:CPH formulations

Immunocompetent C57BL/6 mice were given prime/boost vac-cinations with CPTEG:CPH microparticles encapsulating OVAplus/minus soluble or co-encapsulated CpG ODN. On day 28 afterthe priming vaccinationm serum titers of OVA-specific IgG1 andIgG2a antibodies were measured using ELISA. Results revealed thatmice receiving CPTEG:CPH–OVA/CpG generated significantly high-er OVA-specific antibody responses (both IgG1 and IgG2a) than allother formulations (Fig. 3). Using a tetramer binding assay, OVA-specific T lymphocytes in the peripheral blood from mice on days14 and 20 revealed no significant changes, with the exception ofthe group vaccinated with CPTEG:CPH–OVA, which displayed thegreatest levels of OVA-specific T lymphocytes on both days. Inaddition, the levels of OVA-specific T lymphocytes in the groupvaccinated with CPTEG:CPH–OVA were found to be significantlyincreased by day 20 (Fig. 4).

3.4. Tumor protection studies

Thirty-five days after the initial boost with the various micropar-ticle formulations, C57BL/6 mice were challenged with a lethal doseof an OVA-expressing tumor cell line (E.G7). Tumor volumes were



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Fig. 2. SEM microphotographs of different CPTEG:CPH microparticle formulations. SEM images of: (A) blank CPTEG:CPH microparticles; (B) CPTEG:CPH microparticlesencapsulating OVA; (C) CPTEG:CPH microparticles encapsulating OVA and CpG-ODN. The size bar represents 10 lm.

Fig. 3. Comparative serum titers of IgG2a and IgG1 OVA-specific antibodies aftervaccination with different CPTEG:CPH microparticle formulations. On day 28 post-primary vaccination with indicated treatments, OVA-specific IgG1 and IgG2a titerswere measured in sera using ELISA (as described in Materials and methods). Allgroups were compared using ANOVA followed by Tukey’s post-test (⁄p < 0.01). TheCPTEG:CPH/OVA CpG group showed significantly higher serum titers for IgG1 whencompared with all groups.

Fig. 4. Analysis of OVA-specific T cell frequency in PBLs of mice vaccinated withdifferent CPTEG:CPH microparticle formulations. Mice were primed (day 0) andboosted (day 7) with the indicated formulations. PBLs were stained using afluorescently tagged tetramer, designed to bind to OVA-specific CD8 + T cells, onday 14 (i) and day 20 (ii) post-primary vaccination with the indicated microparticleformulations. All groups were compared statistically using ANOVA followed byTukey’s post-test (⁄p < 0.05).



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monitored over the subsequent 28 days and revealed all formula-tions to have significantly enhanced protective effects when


comparing tumor volumes on day 14 to untreated (naive) miceand mice treated with blank microparticles (Fig. 5). Analysis ofsurvival revealed that mice treated with CPTEG:CPH–OVA orCPTEG:CPH–OVA + soluble CpG ODN had significantly (p < 0.05) im-proved survival over all other groups (Fig. 6b) on day 14. At the ter-mination (day 28 post-tumor challenge) of the study, 75% of micesurvived in the CPTEG:CPH–OVA group, whilst 50% of mice survivedin the group treated with CPTEG:CPH–OVA + soluble CpG ODN. Allbut one of the surviving mice had slow-growing tumors. Ascalculated from the log-rank testm the median survival for theCPTEG:CPH–OVA group was undefinedm whilst for theCPTEG:CPH–OVA + soluble CpG ODN group the median survivalwas 27.5 days.

4. Discussion

To date, studies with biodegradable polyanhydride particleshave involved the encapsulation of pathogen-derived antigens,which have subsequently demonstrated protection against infec-tious pathogens such as tetanus [26] and Yersinia pestis [27]. How-ever, the potential for the amphiphilic CPTEG:CPH microparticles,specifically, to be used as cancer vaccines has yet to be explored.In this study, we investigated the cancer vaccine potential ofCPTEG:CPH microparticles encapsulating a model TAA. Develop-ment of cancer vaccines are often challenging since there is a needto invoke a cell-mediated immune response instead of, or as wellas, a humoral response. Delivery of antigen in a particulate, ratherthan soluble, form is required for the successful generation of anadaptive tumor-specific cytotoxic T lymphocyte response capableof eradicating the primary tumor and, more importantly, its meta-static lesions [28]. If a particulate cancer vaccine delivery system isto be successful, it must fulfill a number of requirements [6],including: (i) protection of the TAA; (ii) efficient delivery to den-dritic cells; and (iii) concomitant stimulation of dendritic cells,leading to subsequent stimulation of an immune response capableof eradicating the tumor [6]. Microparticles made of CPTEG:CPHhave been shown to stabilize encapsulated antigens [29,30], andtheir ability to activate dendritic cells is comparable to that of lipo-polysaccharide (LPS) [16,27,31]. It has been speculated that thestructural similarity of 50:50 CPTEG:CPH particulates to LPS [32]leads to effective immune activation, resulting in long-term anti-body production [19]. Thus, 50:50 CPTEG:CPH particles fulfill manyof the traits required for a potentially successful cancer vaccinevector.

Amphiphilic CPTEG:CPH particles have previously shown highencapsulation efficiency of different model proteins using variousparticle preparation methods [16,27]. In this study, OVA wasshown to be efficiently encapsulated in CPTEG:CPH microparticlesusing a double emulsion solvent evaporation method. However,co-encapsulation of CpG ODN and OVA into CPTEG:CPH



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Fig. 5. The prophylactic antitumor effect of vaccinating mice with different CPTEG:CPH microparticle formulations. Thirty-five days prior to subcutaneous challenge withE.G7-OVA tumor cells, mice were vaccinated with the following microparticle formulations on day 0 (prime) and day 7 (boost): (i) OVA and CpG-ODN encapsulated inCPTEG:CPH; (ii) OVA encapsulated in CPTEG:CPH; (iii) OVA encapsulated in CPTEG:CPH with soluble CpG-ODN; (iv) blank CPTEG:CPH particles; (v) soluble OVA and CpG-ODN; and (vi) naive. (A) Tumor volumes were recorded and each curve represents the tumor growth for each individual mouse. Tumor volumes from each treatment groupwere compared statistically using ANOVA followed by Tukey’s post-test (⁄⁄⁄p < 0.001; ⁄⁄p < 0.01; ⁄p < 0.05). (B) Summary of those groups that were significantly different fromthe naive and blank CPTEG:CPH groups. All other group pairings showed no significant differences.

Day 14 naïve Blank CPTEG:CPH OVA CpG Solution CPTEG:CPH/OVA CPTEG:CPH/OVA+ Soluble CpG CPTEG:CPH/OVA CpG

****eviaN *

Blank CPH-CPTEG * * * *

OVA CpG Solution * *

Median survival (days) 16 15 22 undefined 27.5 23.5

(A)

(B)

Fig. 6. Survival curve of mice bearing E.G7-OVA tumors. Mice were primed (day 0) and boosted (day 7) with ( ) OVA and CpG-ODN encapsulated in CPTEG:CPHmicroparticles; ( ) OVA encapsulated in CPTEG:CPH microparticles; ( ) OVA encapsulated in CPTEG:CPH microparticles with soluble CpG-ODN; ( ) blankCPTEG:CPH microparticles; and ( ) soluble OVA and CpG-ODN. (A) Survival of mice in each treatment group was recorded along with ( ) naive group (n = 4). (B)Survival curves were analyzed using the Mantel–Cox log-rank test and the groups with significant differences between them displayed (⁄⁄p < 0.01; ⁄p < 0.05).



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microparticles (CPTEG:CPH–OVA/CpG) was found to be much lessefficient. High concentrations of CpG ODN in the water phase led


to the precipitation of polymer–DNA aggregates from the primaryemulsion. Destabilization of the primary emulsion was curtailed by



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decreasing the CpG ODN:polymer ratio from 0.015 to 0.001. Thisalteration did not affect the microparticle size, but it did result inlow loading for CpG ODN and OVA. The possibility that thepresence of nucleic acid is responsible for the destabilization ofthe primary emulsion was confirmed by the observation thatmicroparticle aggregation also occurred when herring spermDNA was used instead of CpG ODN (data not shown).

We found that certain CPTEG:CPH-based formulations could pro-vide significant, albeit incomplete, prophylactic protection againsttumor challenge. Mice vaccinated with CPTEG:CPH–OVA affordedthe greatest protection. What was particularly unexpected was thatthese mice showed both increased survival and significantlydecreased tumor volumes (day 14) compared to mice vaccinatedwith CPTEG:CPH–OVA/CpG. It has been shown previously that ahigher proportion of IgG2a antibodies relative to IgG1 antibodies fa-vors the production of antigen-specific cytotoxic T cells [23], whichis key to eradicating cancer [33]. However, we found that ratios ofIgG2a:IgG1 antibodies were small (<0.25) for all particulate treat-ment groups, suggesting Th2-biased immune responses. The ob-served Th2 response by the CPTEG:CPH–OVA formulation was notsurprising since CPTEG:CPH particles encapsulating different anti-gens have previously been shown to generate Th2 responses. How-ever, when CpG ODN was co-encapsulated (CPTEG:CPH–OVA/CpG),or co-delivered in soluble form, the IgG2a:IgG1 ratio did not increase.In other words, CpG ODN did not push the response toward a Th1, oreven a Th0, response. This was unexpected, since CpG ODN has beenpreviously reported to promote Th1 immune responses [34,35]. Onepossible explanation for this observation is that the adjuvant prop-erties of 50:50 CPTEG:CPH particles resulted in abrogation of thenormal effect of CpG ODN. In other words, 50:50 CPTEG:CPH parti-cles have been shown previously to possess strong adjuvant proper-ties [11] and therefore, when added in relatively high dosescompared to the CpG ODN, could result in a phenotypic dominance.It has also been reported that LPS can abrogate the effects of CpGODN [36] and, since 50:50 CPTEG:CPH particles are LPS-like in theireffect on dendritic cell activation [13], it is possible that 50:50CPTEG:CPH particles have a similar dominating influence over den-dritic cells and therefore subsequent immune responses. The OVA-specific CTL responses were marginally, though significantly, in-creased in mice vaccinated with CPTEG:CPH–OVA, whilst none ofthe other treatment groups displayed significant increases relativeto the naive group. This may explain why the CPTEG:CPH–OVAgroup exhibited the greatest tumor protection and improved sur-vival (p < 0.05). This further supports the development of novelCPTEG:CPH polymeric carriers as antigen delivery systems for can-cer vaccines.

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5. Conclusions

To date, the use of biodegradable microparticles in tumorimmunotherapy has primarily involved PLGA microparticles [6].Here, microparticles composed of the bioerodible amphiphilic poly-anhydride 50:50 CPTEG:CPH provided significant protection againsttumor challenge without additional adjuvants, making it a promis-ing vaccine delivery system that requires further evaluation of themechanism of action. In addition, the fact that these polyanhydridesare tunable systems indicates there is potential for modificationsthat can lead to more cellular (or Th1-biased) immune responses.

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Acknowledgements

A.K.S. gratefully acknowledges support from the AmericanCancer Society (RSG-09-015-01-CDD) and the National CancerInstitute at the National Institutes of Health (1R21CA128414-01A2/UI Mayo Clinic Lymphoma SPORE/2P50CA097274-11). B.N.


gratefully acknowledges the Vlasta Klima Balloun Professorship.The authors declare no conflict of interests.

Appendix A. Figures with essential colour discrimination

Certain figure in this article, particularly Fig. 1 is difficult tointerpret in black and white. The full colour images can be foundin the on-line version, at http://dx.doi.org/10.1016/j.actbio.2012.11.001.

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