[methods in molecular biology] vaccine adjuvants volume 626 || micro/nanoparticle adjuvants:...

Chapter 7

Micro/Nanoparticle Adjuvants: Preparation and Formulationwith Antigens

Padma Malyala and Manmohan Singh

Abstract

Recombinant proteins are increasingly being used as a novel approach for antigens in vaccines. Thesegenetically engineered antigens are poorly immunogenic and require a delivery system and adjuvant toelicit their effect at targeted site of action. A delivery system transports the antigen to site of action andan adjuvant activates the cells via interaction with cell receptors and enhances the potency of the antigen.Micro/nanoparticles made from biodegradable and biocompatible polyesters, polylactide-co-glycolides(PLG), have been extensively used as an adjuvant and delivery system. This chapter discusses the appli-cations of PLG micro/nanoparticles as delivery systems and adjuvant for antigens. PLG microparticlesare prepared by a solvent evaporation method while nanoparticles are prepared by solvent displacementmethod. Synthesis of PLG nanoparticles is simpler in comparison to microparticles and unlike micropar-ticles, it also enables particles to be sterile filtered. In a direct comparison using mouse animal model,our group found that microparticles and nanoparticles exhibited similar immunogenic responses. Mate-rials and methods for synthesis and characterization of micro/nanoparticles with adsorbed antigens arediscussed in detail.

Key words: PLG, microparticles, nanoparticles, antigen, adjuvant, adjuvants, delivery vehicle.

1. Introduction

Recombinant proteins are increasingly being used as a novelapproach for antigens in vaccines. While these genetically engi-neered antigens are safer when compared to traditional vaccines,they are poorly immunogenic and require a delivery system andadjuvant to elicit their effect at targeted site of action. A deliv-ery system transports the antigen to site of action, which are thecells responsible for induction of immune responses. An adjuvant

G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626,DOI 10.1007/978-1-60761-585-9_7, © Springer Science+Business Media, LLC 2010

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92 Malyala and Singh

activates the cells via interaction with cell receptors and enhancesthe potency of the antigen. Several particulate systems have beenevaluated as vaccine delivery systems and adjuvants (1). Par-ticulate systems prepared from biodegradable polymers such ashyaluronic acid and poly (lactide-co-glycolide) (PLG), chitosan,and starch are a few examples that have been investigated as deliv-ery systems (2).

Micro/nanoparticles made from biodegradable and biocom-patible polyesters, poly (lactide-co-glycolide) (PLG), have beenextensively used as an adjuvant and delivery system. This poly-mer has a long safety profile in humans as it has been used inresorbable sutures and controlled-release drug delivery. Theseparticulate systems function effectively as delivery systems due totheir uptake by antigen presenting cells (3). PLG particles of size5 μm and lesser are similar in size to pathogens and are takenup by phagocytic antigen presenting cells (APCs) (4–6). Particlesare then carried to lymph nodes by these macrophages, whichmature into dendritic cells (DCs), in addition to direct uptake byDCs (4–6). The particles not only promote trapping and reten-tion of antigens in local lymph nodes but also present multiplecopies of antigens to the immune system by particulate deliverysystems (7).

Several groups have reported that antigens adsorbed orencapsulated with PLG micro/nanoparticles induce potent anti-body and CTL responses in mice, rodents, and nonhuman pri-mates (1, 7–10). Our group has used PLG micro/nanoparticles asa delivery system by preparing charged micro/nanoparticles andadsorbing antigen on surface of the particles (7, 11–14). Depend-ing on the charge of antigen, cationic or anionic particles are usedfor adsorption by electrostatic attraction (14). Antigen adsorptionis influenced by several factors including electrostatic attractionand hydrophobic forces (14). Adsorption on surface of particlesinstead of encapsulation helps preserve the structure of the anti-gen and prevents degradation and denaturation of antigen duringthe synthesis of micro/nanoparticles (2). Some antigens we eval-uated with PLG particles are HIV gp140, recombinant proteinsfrom Neisseria meningitides, and HIV gag DNA (5, 15–17).

Microparticles are prepared by double emulsification usingsolvent evaporation method (17). Briefly, primary emulsion ofwater in oil is prepared by homogenization with a probe, which isthen added to a water phase and emulsified by homogenization,resulting in water-in-oil-in-water emulsion. The solvent is thenallowed to evaporate resulting in a suspension of PLG micropar-ticles in water. In comparison to microparticles, synthesis of PLGnanoparticles is simpler and prepared by solvent displacementmethod (8). Nanoparticles are prepared under magnetic stirringby addition of oil phase to water. Organic solvent is allowed toevaporate, yielding PLG nanoparticles. This process also enables

Micro/Nanoparticle Adjuvants: Preparation and Formulation with Antigens 93

particles to be sterile filtered through a 0.2 μm filter. Size of PLGnanoparticles prepared by our group is typically around 100 nmwhile microparticles are around 1 μm.

A direct comparison of PLG microparticles and nanoparticleswas made by several groups using systemic and mucosal adminis-trations, and with different antigens (8). Conclusions from thesestudies were varied; some groups showed microparticles were bet-ter than nanoparticles while some showed the latter is better (7).Our group conducted three studies with two protein antigensand found that microparticles and nanoparticles exhibited simi-lar responses (7).

Formulation process for PLG micro/nanoparticles as a deliv-ery system for antigens is discussed in the sections below. Antigensdescribed in the methods are Escherichia coli-derived recombi-nant meningococcal vaccine candidate MB1 for Neisseria menin-gitides serotype B (IRIS, Novartis Corporation, S.r.l., Siena, Italy)for protein antigens (7) and HIV-1 pCMVkm p55 gag plas-mid (made at Chiron) for DNA plasmid antigens (5). In thedescription for encapsulated PLG microparticles, CpG is used asan example for encapsulated adjuvant (17). Instruments speci-fied are those that have been used by our group and can bereplaced with other suitable instruments. A detailed descriptionof the materials and methods for synthesis and characterization ofmicro/nanoparticles along with critical notes is given.

2. Materials

2.1. Synthesis ofAnionic Blank PLGMicroparticles

1. RG503, poly (D,L-lactide-co-glycolide) 50:50 copolymercomposition (intrinsic viscosity 0.4 from manufacturer’sspecifications) (Boehringer Ingelheim)

2. Dioctylsulfosuccinate (Sigma Chemical, St. Louis, MO)3. PBS buffer4. Methylene chloride (Sigma Chemical, St. Louis, MO)5. Water6. Ultra-Turrax T25 homogenizer (IKA-Labortechnik,

Germany)7. ES-15 (Omni International, Jarrenton, VA)8. Ice

2.2. Synthesis ofCationic Blank PLGMicroparticles

1. RG504, poly (D,L-lactide-co-glycolide) 50:50 copolymercomposition (molecular weight of 48,000 Da from manu-facturer’s specifications) (Boehringer Ingelheim)

2. Methylene chloride (Sigma Chemical, St. Louis, MO)


3. TE buffer: 10 mM Tris, Na+, Cl– counterions (pH 8.0),1 mM EDTA

4. Ultra-Turrax T25 homogenizer (IKA-Labortechnik, Ger-many)

5. Water6. Cetyl trimethyl ammonium bromide (CTAB) (Sigma Chem-

ical, St. Louis, MO)7. ES-15 (Omni International, Jarrenton, VA)8. Ice

2.3. Synthesis ofEncapsulated PLGMicroparticles

1. CpG oligonucleotide 1826, 5′-TCC ATG ACG TTC CTGACG TT-3′, synthesized with phosphorothioate backbones(Oligos Etc., Wilsonville, OR)

2. Low molecular weight chitosan (Fluka—Sigma-AldrichChemie GmbH Industriestrasse 25 CH-9471 Buchs SGSwitzerland)

3. TE buffer: 10 mM Tris, Na+, Cl– counterions (pH 8.0),1 mM EDTA


5. Methylene chloride (Sigma Chemical, St. Louis, MO)6. Ultra-Turrax T25 homogenizer (IKA-Labortechnik,

Germany)7. Water8. Dioctylsulfosuccinate (Sigma Chemical St. Louis, MO)9. ES-15 homogenizer (Omni International, Jarrenton, VA)

2.4. Synthesis ofBlank PLGNanoparticles


2. Acetone3. Water4. Acrodisc 0.2 μm Supor membrane syringe filters (Pall, East

Hills, NY)

2.5. Adsorption ofProtein Antigens

1. Escherichia coli-derived recombinant meningococcal vaccinecandidate MB1 for Neisseria meningitides serotype B (IRIS,Novartis Corporation, S.r.l., Siena, Italy)

2. Recombinant glycoprotein from HIV (Novartis Corpora-tion)

3. Concentrated histidine buffer: 100 mM L-histidine, Na+

counterion, pH 5.0


4. Concentrated (10x) PBS (Novartis Corporation)5. Lab rocker (aliquot mixer, Miles Laboratories)

2.6. Adsorption ofDNA PlasmidAntigens

1. HIV-1 pCMVkm p55 gag plasmid (made at Novartis Cor-poration)

2.7. Lyophilization ofPLG/AntigenFormulations

1. Sucrose2. Mannitol3. Polyvinyl alcohol (PVA) (MW = 15,000) (MP Biomedicals,

Irvine, CA)4. FreeZone 4.5 L benchtop freeze dry system (LABCONCO,

Kansas City, MO)

2.8. Characterizationof Formulations

1. Horiba LA-930 (Irvine, CA)2. Malvern zeta analyzer 3000 HSA (Malvern Instruments,

UK)3. Wescor osmometer4. Endotoxin materials as specified by USP5. SDS-PAGE and agarose gel electrophoresis6. HPLC

3. Methods

3.1. Synthesis ofAnionic PLGMicroparticles

Microparticles are prepared by the solvent evaporation method(Fig. 7.1).

1. Homogenize 30 mL of 6% w/v PLG polymer solution inmethylene chloride with 6 mL of PBS using a clean 10-mmprobe in the Ultra-Turrax T25 homogenizer (see Notes 1and 2). The water to oil ratio in the primary emulsion is 1:5and homogenization is at 24,000 rpm for 2 min.

2. Add the water-in-oil emulsion thus formed to 150 mL of dis-tilled water containing dioctylsulfosuccinate surfactant (0.5%w/w PLG) and homogenize the mixture at 7,600 rpm witha 20-mm probe in the ES-15 homogenizer for 20 min in anice bath. The oil to water ratio in the secondary emulsionprocess is 1:5.

3. This procedure results in the formation of water-in-oil-in-water emulsion. Stir at 1,000 rpm for 12 h at roomtemperature. Allow the methylene chloride to evaporate(see Note 3).

4. Cap the bottle and store at 4◦C with stirring.


PLG-DCMPBS Buffer(Aqueous Phase) (Organic Phase)

Homogenisation (w/o emulsion)

0.05 % DSS solution

w/o/w emulsion Solvent Evaporation

PLG/ DSS Microparticles

Fig. 7.1. Preparation of blank PLG microparticles.

3.2. Synthesis ofCationic Blank PLGMicroparticles

Microparticles are prepared by solvent evaporation method.1. Homogenize 75 mL of 6% w/v PLG polymer solution

in methylene chloride with 7.5 mL TE buffer using aclean 10-mm probe in the Ultra-Turrax T25 homogenizer(see Notes 1 and 2). The water to oil ratio in the primaryemulsion is 1:10 and homogenization is at 24,000 rpm for3 min.

2. Add the water-in-oil emulsion thus formed to 300 mL of dis-tilled water containing CTAB surfactant (1% w/w PLG) andhomogenize the mixture at 7,600 rpm with a 20-mm probein the ES-15 homogenizer for 20 min in an ice bath. The oilto water ratio in the secondary emulsion process is 1:4.



3.3. Synthesis ofEncapsulated PLGMicroparticles

Encapsulated microparticles (Fig. 7.2) are prepared by solventevaporation method.

1. Add 0.5% CpG (w/w PLG) to chitosan in a ratio of 1.4:1and add to 6 mL of TE buffer (see Note 2). Homogenize30 mL of 6% w/v PLG polymer solution in methylenechloride with 6 mL of TE buffer containing CpG–chitosancomplex using a clean 10-mm probe in the Ultra-TurraxT25 homogenizer (see Note 1). The water to oil ratioin the primary emulsion is 1:5 and homogenization is at24,000 rpm for 2 min.


-- --- - -

Proteinn

Protein

+++

++ +

++

+

--- - -

Proteinn

Proteinn

+++

+ ++

+IP

ProteinPLG Microparticlep

1µm

---

-

-++++ +

+++

Protein-

-

---

---

n ++++ +

++

+IP

IP IP

IP

IP

IPp-

-- -- ---Protei

n

Protein

++

+ + + ++

+ -

-- --

--

--Protei Proteinin

++

++ + + +

++

+IPIP

IPIP

Protein

PLG / Protein / IP PLG/Protein + IP

n in+ +

PLG /IP/ Protein

The IP gets incorporated into the PLG microparticles

PLG/Protein + IP

The IP is added before or after the l hili ti

plyophilization process

Fig. 7.2. Anionic PLG microparticles with protein and immunopotentiator (IP).

2. Add the water-in-oil emulsion thus formed to 150 mL ofdistilled water containing surfactant (0.05% w/w PLG).Homogenize the mixture at 7,600 rpm with a 20-mm probein the ES-15 homogenizer for 8 min in an ice bath. The oilto water ratio in the secondary emulsion process is 1:5.



3.4. Synthesis ofBlank PLGNanoparticles

PLG nanoparticles were prepared by the solvent displacementmethod (Fig. 7.3).

1. Add dropwise the organic phase comprising PLG dissolvedin acetone to 50 mL of pure, distilled water with magneticstirring. The concentration of polymer solution is chosenbased on PLG content required.

2. Allow the acetone to evaporate overnight (see Note 3).3. There was no surfactant present in the aqueous phase.4. Sterilize particles by filtration.

3.5. Adsorption ofProtein Antigens

1. Incubate a suspension containing 100 mg of blankanionic PLG microparticles or encapsulated microparticlesor nanoparticles with protein antigen (1% w/w PLG).

2. Add concentrated buffer solution to affect a final concentra-tion of 10 mM histidine buffer pH 5 for MB1 protein, and1× PBS for gp120, in a 10 mL total volume.

3. Allow the suspension to mix overnight on a lab rocker at4◦C (see Note 4).

4. Remove about 1 mL of suspension for the determination ofadsorption efficiency.


Nanoparticles Microparticles

dropwiseaddition

Water

PLG+

Acetone

500 rpm

allowsolvent toevaporate

nanoparticlesuspension

O/W emulsion

homogenize @2,000 rpm for 2

min

aqueous phase: PBS

oil phase: PLG in dichloromethane

solvent evaporationsolvent displacement

Water+

DSS

W/O/W emulsion

homogenize @12,000 rpm for 8 min

add O/W toexternal water

phase

+

on ice bathallow solventto evaporate

microparticlesuspension

dioctyl sodiumsulfosuccinate (DSS)at 0.05 % wt/ wt PLG

Fig. 7.3. Particle preparation methods: a comparison.

5. Aliquot volumes containing the required dose of proteininto small glass vials.

3.6. Adsorption ofDNA PlasmidAntigens

1. Take a suspension containing 100 mg of cationic blank PLGmicroparticles in a beaker.

2. Add DNA plasmid antigen at a typical load of 4% w/w PLGin a dropwise fashion to the PLG microparticles under con-stant stirring (see Note 5).

3. Allow the suspension to stir for 30 min after complete addi-tion of the antigen.

4. Remove about 1 mL of suspension for the determination ofadsorption efficiency.

5. Aliquot volumes containing required dose of antigen intosmall glass vials.

3.7. Lyophilization ofPLG/AntigenFormulations

1. To the aliquots from Sections 3.5 and 3.6, Step 5, add man-nitol and sucrose to affect a final concentration of 4.5 and1.5%, respectively, upon reconstitution.

2. Cap the vials with a stretch plastic film. Prepare the vials forlyophilization by providing vents for evaporation on the film.

3. Freeze the vials at –20◦C.4. Lyophilize the vials at −50◦C and 90×10−3 mbar.5. Cap the vials after lyophilization and store at 4◦C.

3.8. Characterizationof Formulations (seeNotes 6 and 7)3.8.1. Sizing

1. Take 1 mL of PLG microparticles and add dropwise in a par-ticle size analyzer until the particles are in the required rangefor transmission and obscuration of the instrument. Measure


size of the particles following directions of the instrument.Record the size distribution of the microparticles.

2. Take about 20 μL of PLG nanoparticles and dilute up to 2mL with purified water and place in sample cell of a Malvernzeta analyzer. Measure the size of the particles following thedirections of the instrument.

3.8.2. Osmolarity 1. Calibrate the osmometer before sample analysis.2. Take 10 μL of the formulation and add to sample holder.3. Measure osmolarity as directed in the manual.4. Osmolarity range should be 270–330 mOsm/kg.

3.8.3. Endotoxin 1. Use gel clot method as directed by USP.

3.8.4. SDS-PAGE andAgarose GelElectrophoresis

1. Determine the antigen loading levels and integrity of antigenusing SDS-PAGE and agarose gel electrophoresis.

2. Centrifuge 1 mL of samples in replicates at approximately9,660×g.

3. Separate the supernatant from the pellet.4. For protein antigens, load samples of supernatant and pel-

let, extracted with SDS sample buffer, along with controlsin SDS-PAGE gels (4–20% gradient tris/glycine polyacry-lamide gels).

5. For DNA plasmids, load samples of supernatant and pelletand follow standard protocol for agarose gel electrophoresis.

6. For protein samples, stain with a Coomassie stain asinstructed in a manual for SDS-PAGE.

7. Compare with control after destaining and confirm integrityof protein as well as semiquantification of protein associatedwith supernatant and pellet.

3.8.5. HPLC 1. Determine the protein loading levels and integrity of proteinusing HPLC.

2. Centrifuge 1 mL of samples in replicates at approximately9,660×g for 20 min.

3. Separate the supernatant from the pellet.4. Load supernatant samples in HPLC along with relevant con-

trols by a method suitable to protein in question.5. Quantify amount of protein present in supernatant to deter-

mine protein adsorbed on particles.


4. Notes

1. Take adequate care to ensure probes are clean before andafter use. Failure to do so might result in a poor formationof microparticles (Sections 3.1, 3.2, and 3.3).

2. Buffers in the primary emulsion can be changed, based onformulation parameters (Sections 3.1, 3.2, and 3.3).

3. After evaporation of organic solvent, measure the volume ofsuspension. Add water, if required, to compensate for waterevaporation losses (Sections 3.1, 3.2, 3.3, and 3.4).

4. Ensure gentle mixing of particles during protein adsorptionto prevent aggregation (Section 3.5).

5. During DNA adsorption, add DNA slowly to ensure com-plete adsorption on to microparticles (Section 3.6).

6. Follow instructions set up by the instruments for analyticalcharacterization.

7. Use relevant controls for all analytical characterization.

Acknowledgments

The authors deeply acknowledge the contributions of JanetWendorf and Aravind Chakrapani who worked extensively on for-mulation and characterization of PLG nanoparticles in their postdoctoral tenure with us. Thanks are also due to the Vaccine For-mulation and Delivery group in Novartis Corporation.

References

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4. O’Hagan, S., Ulmer, J. (2004) Micropar-ticles for the delivery of DNA vaccines.Immunol Rev 199, 191–200.

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8. Diwan, M., Elamanchili, P., Cao, M.,Samuel, J. (2004) Dose sparing of CpG


oligodeoxynucleotide vaccine adjuvants bynanoparticle delivery. Curr Drug Deliv 1,405–412.

9. Xie, H., Gursel, I., Ivins, B. E., et al.(2005) CpG oligodeoxynucleotides adsorbedonto polylactide-co-glycolide microparticlesimprove the immunogenicity and protectiveactivity of the licensed anthrax vaccine. InfectImmun 73, 828–833.

10. Hunter, S. K., Andracki, M. E., Krieg, A. M.(2001) Biodegradable microspheres contain-ing group B Streptococcus vaccine: immuneresponse in mice. Am J Obstet Gynecol 185,1174–1179.

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15. Chesko, J., Kazzaz, J., Ugozzoli, M., et al.(2004) Adsorption of a novel recombinantglycoprotein from HIV (env gp120dv2) toanionic PLG microparticles retains struc-tural integrity, while encapsulation in PLGmicroparticles does not. Pharm Res 21,2148–2152.

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17. Malyala, P., Chesko, J., Ugozzoli, M., et al.(2008) The potency of the adjuvant, CpGoligos, is enhanced by encapsulation in PLGmicroparticles. J Pharm Sci 97, 1155–1164.

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