The preparation and characterization of polymeric antigen delivery systems for oral administration

LAdvanced Drug Delivery Reviews 34 (1998) 285–304

The preparation and characterization of polymeric antigendelivery systems for oral administration

*Manmohan Singh , Derek O’Hagan

Adjuvant Research Division, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608, USA

Received 20 October 1997; received in revised form 24 November 1997; accepted 2 July 1998

Abstract

Although polymeric delivery systems are well established for the oral administration of conventional drugs, they have notyet been commercially developed for vaccine delivery. The problems inherent with the oral route of delivery, including lowpH, gastric enzymes, rapid transit and poor absorption of large molecules, has made the goal of oral delivery of antigens verychallenging. Nevertheless, several polymeric delivery systems for the oral administration of vaccines are currently beingevaluated, including microencapsulation in poly(lactide-co-glycolides), alginates, polyanhydrides, starch, polymethacrylates,polyamino acids and enteric coating polymers. These approaches are designed to protect the antigen in the gut, to target theantigen to the gut-associated lymphoid tissue, or to increase the residence time of the antigen in the gut through bioadhesion.Each of these approaches is discussed in relation to antigen encapsulation and integrity, process reproducibility, ease ofpreparation and encapsulation efficiency. Potential problems associated with the scale-up of these approaches are also brieflyaddressed. Of particular relevance are the prospects for the application of these formulation processes for commercialdevelopment. 1998 Elsevier Science B.V. All rights reserved.

Keywords: Oral administration; Vaccine delivery; Antigen encapsulation; Reproducibility; Ease of preparation; Encapsula-tion efficiency

Contents

1. Introduction ............................................................................................................................................................................ 2862. Preparation methods for oral polymeric antigen delivery systems................................................................................................ 287

2.1. Solvent evaporation /extraction method for polylactide-co-glycolide (PLG) microparticles .................................................... 2872.1.1. Polymer and solvent selection ................................................................................................................................. 2872.1.2. Emulsion preparation.............................................................................................................................................. 2882.1.3. Emulsion stabilizer................................................................................................................................................. 2902.1.4. Solvent removal ..................................................................................................................................................... 2902.1.5. Microparticle characterization ................................................................................................................................. 2912.1.6. Alternative process................................................................................................................................................. 294

2.2. Preparation of alginate microcapsules ................................................................................................................................ 2952.3. Preparation of grafted starch microcapsules........................................................................................................................ 2962.4. Preparation of polyamino acid microspheres ...................................................................................................................... 2962.5. Enteric coated microcapsules prepared by the coacervation phase separation method ............................................................ 297

*Corresponding author. Tel.: 1 1-510-923-7877; fax: 1 1-510-923-2586; e-mail: manmohan [email protected]]

0169-409X/98/$ – see front matter 1998 Elsevier Science B.V. All rights reserved.PI I : S0169-409X( 98 )00044-1

286 M. Singh, D. O’Hagan / Advanced Drug Delivery Reviews 34 (1998) 285 –304

2.6. Enteric coated microcapsules prepared by spray drying....................................................................................................... 2972.7. Coating of non-pareil seeds with acrylic resins ................................................................................................................... 2982.8. Preparation of granules for oral delivery ............................................................................................................................ 2982.9. Preparation of poly(methacrylic acid) hydrogels ................................................................................................................. 2992.10. Polyanhydride co-polymers for oral antigen delivery......................................................................................................... 299

3. Conclusions ............................................................................................................................................................................ 299References .................................................................................................................................................................................. 301

1. Introduction be successfully modified to allow the development oflive oral vaccines. Some organisms are difficult or

Oral administration continues to be the most impossible to grow in culture and many cannot beattractive route for the delivery of all medicinal easily manipulated using existing techniques inagents. The ease of delivery, lack of pain associated molecular biology. Therefore, there is considerableand cost effectiveness has made oral dosage forms need for the development of new vaccine delivery(ODF) the most commonly used products for drug systems which can be applied to a range of differentadministration. The ODFs currently available on the pathogens, and can be administered orally. Principal-market constitute almost 60% of all pharmaceutical ly for reasons of safety, it would be highly desirableproducts. However, although the oral route is exten- if the delivery approach was based on a non-livingsively exploited for drug delivery, it is yet to become delivery system, rather than on an attenuated organ-a prominent route for the administration of vaccines. ism.Nevertheless, because of the attractiveness of the One approach to the development of new deliveryroute of delivery and the associated increase in systems for oral vaccines involves the use of poly-compliance, all vaccine products would be signifi- mer encapsulation of antigens. A number of differentcantly improved if they could be administered by the polymers have been evaluated for the developmentoral route. Several groups have reported interesting of oral vaccines, including naturally occurring poly-findings in relation to the development of oral mers (e.g. starch, alginates and gelatin) and syntheticvaccine delivery systems in the last few years [1– polymers (e.g. polylactide-co-glycolides (PLG),16]. Oral administration of vaccines might result in polyanhydrides and phthalates). Of primary concernimprovements in efficacy, since oral immunization are considerations of toxicity, irritancy and al-would stimulate mucosal immunity at the sites at lergenicity, and the need for a biodegradable or awhich many pathogens initially infect the host. The soluble coating. The advantages of using naturalinduction of mucosal immunity might prove to be polymers include their low cost, biocompatibility andparticularly advantageous in the elderly, since unlike aqueous solubility. However, the natural polymerssystemic immunity, mucosal immunity does not may also be limited in their use due to the presenceappear to be subject to age-associated dysfunction. In of extraneous contaminants, variability from lot toaddition, mucosal immunity appears to develop lot and low hydrophobicity. In contrast, syntheticearlier in life than systemic immunity. Therefore, polymers are more reproducible and can be preparedoral administration of vaccines might be exploitable with desired degradation rates, molecular weightsin very young subjects. and co-polymer compositions. Nevertheless, synthet-

To date, there are only a limited number of ic polymers may be disadvantageous due to theirlicensed oral vaccines available, including polio limited solubility, they are often soluble only invaccine and a live typhoid vaccine Ty21a (Vivotif). organic solvents and consequently may not releaseHowever, several additional oral vaccines are in late biologically active antigen. Both natural and syn-stages of development, including improved vaccines thetic polymers are available in bulk quantities fromagainst V. cholerae and Salmonella typhi, and a commercial suppliers.rotavirus vaccine. These vaccines were mainly de- Several approaches involving polymeric coatingsveloped as live attenuated organisms, and provide or encapsulation are currently being evaluated for theprotection only against the pathogen which was development of oral delivery systems for vaccines.originally attenuated. However, not all pathogens can These various approaches include, PLG microparti-

M. Singh, D. O’Hagan / Advanced Drug Delivery Reviews 34 (1998) 285 –304 287

cles [1–7], enteric coating polymers [17–21], algi- 2.1. Solvent evaporation /extraction method fornate microcapsules [22–25], tablets [21], granules polylactide-co-glycolide (PLG) microparticles[21], starch microcapsules [26], gelatin capsules[27], polymethacrylates [28] and polyamino acid Solvent evaporation is the most widely usedmicrospheres [29,30]. The objectives with all of method for the preparation of microparticles withthese approaches is to improve upon one or more of entrapped antigens [34–59]. The method is based onthe following parameters, to reduce gastric and the formation of a multiple emulsion (w/o /w) fromintestinal degradation of the antigen, to enhance which the oil phase (organic solvent) is evaporated toantigen absorption, to extend residence time in the yield free flowing microparticles. Fig. 1 illustratesgut, to promote uptake of the antigen by the gut- various preparation approaches that could be used toassociated lymphoid tissue (GALT) and to reduce prepare microparticles for oral vaccine development.the dose of antigen needed to induce a significantimmune response. In general, microparticle formula- 2.1.1. Polymer and solvent selectiontions made from synthetic or natural polymers, The most commonly used polymers for solventwhich are prepared with a mean size of 10 mm or evaporation are PLG, which are biodegradable poly-less, are mainly designed to target antigens to the mers with a long history of safe use in humans asGALT (or Peyer’s patches). Although small particles surgical sutures and as drug delivery systems [60–may also protect incorporated antigens against degra- 69]. PLG polymers are available in various co-dation in the gut and may promote interaction with polymer ratios and molecular weights and are com-the gut wall through bioadhesive mechanisms. In mercially available in bulk quantities. The polymerscontrast, formulations prepared with particles of a also have high reproducibility from lot to lot forlarger size ( . 10 mm) are generally designed simply monomer ratios and molecular weights. In most ofto protect antigens against gastric acidity and en- the work described in the literature, PLG microparti-zymes and to deliver the antigen into the gut. The cles have been prepared from low to mediumuptake of microparticles made from various poly- molecular weight polymers with lactide /glycolidemers across the gut has been extensively evaluated in monomer ratios of about 50/50 [6,44,47,48]. Thisthe literature [8,31,33,38]. Preliminary findings in type of polymer has been used because of itssmall animal models suggest that oral delivery of relatively rapid degradation rate (biodegradable inantigens in microparticles may eventually be pos- vivo within 2–3 months) in comparison to alternativesible. For example, Jones et al. [7] showed the PLG polymers [67–69].induction of protective immunity against Bordetella During microparticle preparation by solventpertussis in mice, following oral administration of evaporation, one important parameter is the choice ofmicroencapsulated fimbriae. organic solvent to form the primary emulsion. This

The purpose of this review is to discuss the decision is based both on the desired microparticlevarious methods used for the preparation of oral characteristics and also on the toxicity of the solventspolymeric antigen delivery systems which have been [50,58,84]. Ethyl acetate (EA) [55] is an attractivedescribed in the literature. The problems associated choice in relation to toxicity (oral LD50 in rats, 5.62with these methods will be addressed, including ease g/kg) and compares favourably with dichlorome-of manufacture, yield, reproducibility, scale-up and thane (DCM) (oral LD50 in rats, 1.6 g /kg) [50].commercial development possibilities. Moreover, EA is already used orally in humans to

enhance food flavour (apple flavour) and this is animportant consideration. However, PLG polymershave much lower solubility in EA than in DCM. In

2. Preparation methods for oral polymeric addition, DCM has greater volatility at ambientantigen delivery systems conditions (boiling point of DCM is 408C, whereas

EA has a boiling point of 778C) and this is importantSome of the methods that have been described for in the solvent evaporation process. Another issue is

the development of polymeric oral antigen delivery the solubility of the solvents in water, which governssystems are listed in Table 1. the rate of solvent removal from the multiple emul-


Table 1An outline of the various approaches evaluated for the development of oral polymeric vaccine delivery systems

Polymer Preparation method Commercial Desired objective Size range Antigens evaluatedavailability in vivoof polymer

PLG Solvent evaporation / Yes Uptake into Peyer’s patches 1–10 mm Pertussis [7]extraction etc. Ricin toxoid [9]

S. typhi [10]Influenza [12]M. hyopneumoniae [18]Rotavirus vaccine [21]

Alginates Ionotropic gelation Yes Uptake into Peyer’s patches 15 mm Rotavirus vaccine [21]Ovalbumin [22]

Starch Emulsion coacervation Yes Uptake into Peyer’s patches 10 mm Human serum albumin [26]

Proteinoid Thermal condensation No Uptake into Peyer’s patches , 5 mm Influenza [29]Ovalbumin [30]

CAP Coacervation phase separation Yes Protection (against gastric 300 mm M. hyopneumoniae [18]acidity and enzymes) Rabies antigen [19]

Vibrio vaccine [20]Cholera vaccine [79]Influenzae vaccine [80]Typhoid vaccine [81]BCG vaccine [82]Hepatitis B vaccine [83]

CAP Spray drying Yes Protection 30 mm Rotavirus vaccine [21]Eudragits Coating on non-pareil seeds Yes Protection 300 mm Vibrio vaccine [20]Starch Granulation Yes Protection 1 mm Rotavirus vaccine [21]Methacrylates Hydrogels Yes Protection 5 mm Pasteurella haemolytica [28]Polyanhydrides Phase-inversion Yes Protection /bioadhesion 5 mm DNA [74]

sion and the subsequent hardening of the microparti- of 20–100 mm. For oral antigen delivery with PLGcles. The aqueous solubility of DCM is about 2% microparticles, the desired size range is usually lessv /v whereas for EA it is about 10%. In some than 5 mm. Therefore polymer concentrations in thesituations, the removal of EA has been too rapid to 2–5% range have mostly been used.allow the reproducible formation of good quality The polymer solution in the organic solvent, whenmicroparticles [50]. As a consequence, most reports emulsified with the aqueous antigen solution, forms ain the literature, including many from our group, water-in-oil (w/o) emulsion, which is then added to ahave chosen dichloromethane over alternative sol- much larger aqueous phase. The addition of emul-vents, including EA [6,7,35,42–49,53,56]. sion stabilizers to the internal emulsion, including

gelatin, polyvinyl alcohol, cetyltrimethyl ammonium2.1.2. Emulsion preparation bromide (CTAB), methyl cellulose and sodium

The polymer concentration in the primary emul- dodecyl sulphate have been reported to enhancesion mainly controls the size of the resultant mi- emulsion stability and to help to retain the majoritycroparticles. A lower polymer concentration (be- of the antigen within the microparticles [59,70,71].tween 2–5%) is selected if the desired size of the These additives are thought to act either by stabiliz-microparticles is between 200 nm and 5 mm. Higher ing the primary emulsion or by enhancing viscositypolymer concentrations, ranging from 10–16%, may to prevent partitioning of the protein to the externalbe used to prepare larger microparticles in the range aqueous phase. The stabilizers also enhance the


Fig. 1. An outline of the various approaches to the preparation of PLG microparticles with entrapped vaccines.

stability of the primary emulsion and help to prevent tageous because this equipment is available in a widedroplet coalescence. However, in many cases, the range of sizes, appropriate for a 5-ml lab scale batchpresence of protein (antigen) is sufficient to stabilize to a 500-l commercial scale batch [59]. Therefore,the primary emulsion and the addition of stabilizers the Silverson homogenizer seems an ideal emulsifiermay not be necessary [47–53]. The requirement for for pre-clinical formulation preparations when usedan additional emulsion stabilizer in the primary w/o at rev. /min of 8000–12 000 [56–58]. However, labemulsion needs to be determined for each individual scale batches have often been prepared with alter-antigen. Our experience with a number of antigens native homogenizers which provide higher shearhas indicated that additional stabilizers are not rates to the emulsion mix, for example the Ultratur-required to prepare a stable primary emulsion with rax from IKA works, Wilmington, USA. Thismany protein antigens. homogenizer can run at up to 25 000 rev. /min and

The primary emulsion is formed using either a can work with small volumes. The choice ofhomogenizer or a sonicator. Various equipment has homogenizer is usually based on the followingbeen used to obtain small droplet sizes in the primary parameters, the maximum rev. /min achievable withemulsion [41–53]. The use of a Silverson homogen- a small volume probe, a stainless steel construction,iser (Silverson Machines, Chesam, Bucks) is advan- autoclavable parts and an accurate tachometer.


2.1.3. Emulsion stabilizer gradual evaporation of the organic solvent from theOnce the primary w/o emulsion is formed, it is multiple emulsion by stirring under ambient con-

then added to a larger aqueous phase containing a ditions or under nitrogen flushing. In this situation,stabilizer. The choice and concentration of the the low boiling point of methylene chloride isstabilizer in the aqueous phase are based on the advantageous and results in uniform solvent removaldesired microparticle characteristics, including the within a few hours, depending upon stirring speedssize range and porosity, the type of solvent used (EA and surface area. Solvent evaporation promotesor DCM) and the amount of protein being entrapped. relatively slow gelling of the microparticle boundarySome of the stabilizers that have been evaluated and results in the preparation of smooth and uniforminclude, polyvinyl alcohol (PVA) [56–58], polyvinyl microparticles, with no pores on the surface. Duringpyrrolidone (PVP) [44,45] and methyl cellulose [58]. solvent extraction, the multiple emulsion (w/o /w) isPVA is the most commonly used stabilizer mainly poured into an excess of aqueous phase to extract alldue to its low toxicity (oral LD50 in rats, 20 g/kg vs. the organic solvent out of the microparticles and to8 g/kg for PVP), good aqueous solubility and its rapidly harden the polymer droplets to form mi-availability in a range of molecular weights [56–59]. croparticles. The total volume of the extraction phaseStabilizer concentrations used for microparticle prep- is governed by the solubility of the solvent in wateraration have ranged from 0.1% w/w to 10% w/w. ( | 2% for DCM and | 10% for EA) and the volumeHowever, a lower stabilizer concentration results in needed to extract the total amount of solvent froman irregular size distribution of the microparticles, the multiple emulsion. Usually 4–6 times the volumelow loading levels and low entrapment efficiency. of the multiple emulsion (w/o /w) are required forOptimum microparticle formulations are usually complete extraction. The solvent extraction processobtained with a 5–10% stabilizer concentration is relatively quick and is complete within 30–60[56,59]. Following microparticle preparation, PVA is min. However, due to the rapid removal of thebound into the structure of the microparticle and solvent, pores and cracks are sometimes visible oncannot be washed away [88]. Table 2 shows the the surface of the microparticles prepared by thisresidual levels of PVA in microparticles prepared method. In general, the porosity of the microparticleswith varying PVA concentrations. The residual is also higher by this method and could lead to aamount of PVA in the particles did not change with higher burst release of entrapped antigens [55,71].increasing PVA concentration from 1 to 10%. Moreover, much larger volumes must be handled

with the solvent extraction process and this can2.1.4. Solvent removal rapidly become prohibitive for larger batches of

After addition of the primary w/o emulsion to the microparticles.aqueous stabilizer solution, a w/o /w emulsion is Once the microparticles have been formed, theyformed. This multiple emulsion is then subjected to are recovered mainly by high speed centrifugation,solvent removal to form discrete microparticles. This followed by washing with distilled water. The mi-step is normally carried out by one of two alternative croparticles are then either air-dried under vacuum ormethods, solvent evaporation [56–59] or solvent under nitrogen. The microparticles may also beextraction [49,55]. Solvent evaporation involves a freeze dried to yield a dry product [56–59]. Freeze

Table 2The residual levels of PVA in PLG microparticles with entrapped ovalbumin following preparation by solvent evaporation in the presence ofincreasing concentrations of PVA (1 to 10%) in the external phase of the water-in-oil-in-water emulsion. PVA estimated by titrimetricmethod

Batch PVA in external phase (% w/w) Volume of PVA solution Residual PVA in microparticles (% w/w)

PLG/OVA batch I 1 60 1.45PLG/OVA batch II 2 60 1.30PLG/OVA batch III 5 60 2.15PLG/OVA batch IV 10 60 2.82


drying is an attractive approach, since losses areminimal and sterile vials can be sealed during thefreeze drying. However, freeze drying may not besuitable for some antigens which are prone todegradation during this process and in this case,drying under vacuum or nitrogen may be the bestrecovery method.

2.1.5. Microparticle characterizationThe ratio of the amount of microparticles re-

covered from the formulation process versus thestarting polymer concentration is expressed as apercentage and represents the yield of the process.The typical yield of microparticles from the multipleemulsion method ranges from 50–95%, but is com-monly between 80–90% for lab scale batches [58].The ratio of antigen entrapped in the microparticlesversus the total antigen used is also expressed as a

Fig. 2. The actual loading achieved versus the theoretical loadingpercentage and represents the loading efficiency of level for five batches of PLG microparticles with entrappedthe process. The loading efficiency varies with the ovalbumin (1 to 5% w/w theoretical loading) prepared by aantigen under evaluation and typically ranges from solvent evaporation method. The reproducibility of the loading

levels is also shown (n 5 3).50–95%. The smallest quantity of antigen requiredto make a reproducible batch using PLG polymer isat least a few mg (2–5 mg). At a microparticle formulations and a proper validation needs to beloading level of 1–2%, the loading efficiency is carried out with each antigen to assure reproducibleusually higher, but can drop gradually with increas- estimations [90]. Fig. 3 shows a representative SEMing loading levels for some antigens. However, some of PLG microparticles prepared by a solvent evapo-proteins (e.g. ovalbumin) have been entrapped very ration process. Greater than 95% of the microparti-efficiently in microparticles even at high loading cles have a mean size of less than 5 mm. Fig. 4levels [58]. Fig. 2 shows microparticles prepared shows a representative in vitro release profile of awith five different theoretical loading levels of model protein (ovalbumin) from PLG microparticlesovalbumin and the actual entrapment levels prepared by the solvent evaporation method.achieved. The residual levels of solvent in the final product

Following preparation, PLG microparticles should is an important parameter to be addressed and is abe subjected to a thorough in vitro physicochemical particular concern for the regulatory agencies. Incharacterization [84]. The characterization of the general, the concern about residual levels of solventmicroparticles represents a quality control measure to in the final product are much greater for a parenteraldetermine lot to lot variability, to allow predictions formulation than for an oral product. Nevertheless,of in vivo behaviour and to screen for formulations the lowest achievable levels of residual solvents arethat meet the required specifications. Some of the desirable. The use of gas chromatography, a Car-tests carried out commonly include, determinations bopac SP-1500 column and a flame ionization detec-of size distributions by laser diffractometry, surface tor, has been used to estimate residual DCM levels inevaluation under scanning electron microscopy PLG microparticles [84]. Microparticles prepared(SEM), residual solvent and PVA determinations, with an encapsulated vaccine for oral administrationmoisture content, stability on storage, porosity mea- to humans were shown to contain less than 10 ppmsurements, bioburden, antigen loading level and of residual DCM [84]. Lupron depot, a marketedrelease rates [84,89]. Different methods are common- PLG formulation has less than 50 ppm of DCM.ly utilized to estimate the protein loading in these Removal of higher levels of DCM may be a problem


Fig. 3. A representative scanning electron micrograph of PLG microparticles for oral antigen delivery prepared using the solvent-evaporationmethod (size bar 5 5 mm). Mean size of the distribution was 550 nm.

in some cases as freeze drying does not remove theDCM completely, since DCM binds to the polymer.Vacuum drying at slightly elevated temperatures( . 208C) may aid in better removal of residualDCM.

In vivo evaluations in animals are the mostrelevant read-outs for these formulations and arenecessary to determine the integrity of antigens andtheir ability to generate neutralizing antibodies orprotective immunity. Antigens that are prone tosolvent denaturation, moisture induced-aggregation,conformational changes, temperature changes or areshear sensitive, may exhibit problems followingmicroencapsulation using the multiple emulsionmethod [72,73]. However, the addition of stabilizersand changes in process parameters have been usedsuccessfully to retain antigen integrity by this pro-cess [48,49,72,73,75–77]. Research efforts are un-derway by several groups to evolve conditions anduse selective excipients to maintain antigen integrityduring the solvent evaporation /extraction method[72,75–77].Fig. 4. A representative in vitro release profile of a model protein

For in vitro characterization, the integrity of the(ovalbumin) from PLG microparticles made by the solvent evapo-ration method. Each time point represents mean of samples. entrapped antigen should be evaluated by one or


more of the various antigen specific tests available, important to evaluate the potential of each proteinincluding SDS-PAGE, GP-HPLC, immunoassays, antigen for interaction with the polymer [89]. IfWestern blots and receptor binding assays [48– binding to the polymer occurs, excipients may be50,56–58,84]. These tests may provide some in- added to minimize these problems.formation about the nature of the antigen released With respect to possible scale-up, the solventfrom the microparticles and should show if gross evaporation process requires a lesser total volumedamage to the antigen has occurred during microen- than solvent extraction and this could be importantcapsulation. These tests are particularly useful in for large batches. Fig. 5 shows the microparticle sizeselecting the optimal excipients and conditions for and loading levels achieved for PLG microparticlesmaking the microparticles. with entrapped ovalbumin, prepared by solvent

Among a number of other issues, the potential for evaporation and scaled up ninefold over the ‘stan-protein-polymer interactions, or protein-protein inter- dard’ lab batch size of 400 mg of polymer. It shouldactions at high concentrations, also needs to be be clear that the scale-up was achieved with excel-evaluated for successful microencapsulation. Proteins lent reproducibility [58]. To allow the preparation ofwith high pI values (basic in nature) may not be microparticles for a human clinical trial, this processstable in the presence of lactic acid, which may be was eventually scaled up by a factor of 22-fold.generated during polymer degradation, and may be Nevertheless, higher batch sizes would require fur-more inclined to bind to the polymer structure. It is ther development work as the design of the scale-up

Fig. 5. Microparticle mean sizes (6S.D.) and mean total amounts (6S.D.) of ovalbumin entrapped in microparticles after the initial labprocess (400 mg of polymer) was scaled up 3 3 and 3 9. The process reproducibility is also shown for each batch size (n 5 4).


vessels and equipment remains a challenging task. s /o /w method involves the formation of a primaryHowever, several products containing entrapped suspension of solid antigen in the organic polymerpeptides and small molecular weight drugs in PLG solution, followed by addition to an external aqueousmicroparticles are currently available on the market phase to yield discrete microparticles. This method(Lupron Depot, Decapeptyl), suggesting full can be utilized to achieve higher loading levels of thescalability of this process for large batch sizes. antigen in the microparticles [50]. The method mayHence, both the solvent evaporation and extraction also reduce exposure of the antigen to the organicmethods show excellent reproducibility both at lab solvent, especially if the freeze dried antigen hasscales and pilot scales. Moreover, the processes are been stabilized through the addition of solid phasevery robust and amenable to manipulation to produce stabilizers [85]. It is recommended that this methodcustomized preparations. Table 3 lists some of the should be evaluated on a case by case basis, sinceparameters that can be manipulated to affect mi- many proteins (antigens) may be better entrappedcroparticle characteristics. using the w/o/w method.

An alternative modification of the above method is2.1.6. Alternative process to add the antigen and stabilizers in a lyophilized

A modification of the water-in-oil-in-water (w/o / form to the organic polymer solution and to sprayw) multiple emulsion method is represented by the this suspension into liquid nitrogen to freeze thesolid-in-oil-in-water (s /o /w) method [50,85]. The microsphere structure. The methylene chloride is

Table 3Process parameters in the PLG microencapsulation process which affect the quality and particle characteristics of the final product

Preparation step Parameters

Primary emulsion (w/o) Organic solventPolymer molecular weightPolymer monomer ratioPolymer concentrationSolvent volumeProtein concentrationAqueous to organic ratioHomogenizing speedType of homogenizerDuration of emulsificationTemperature of mixing

Secondary emulsion (w/o /w) Aqueous phase ratioHomogenization speedConcentration of emulsifierType of homogenizerDuration of emulsificationTemperature of mixing

Microparticle formation Solubility of solvent in aqueous mediumType of polymerDuration of mixingStirring speedsStirring temperature

Microparticle recovery Centrifugation forceNumber of washesTemperature of washingResidual solventFreeze drying equipment


then extracted with ethanol [40]. The main advantage calcium chloride solution in water (1–2%). Theof this method is that the process is carried out under resulting alginate microcapsules are collected bysubzero conditions and therefore, should be ideal for decanting the supernatant and washing with waterproteins that are thermolabile. Also, the solvent is [22]. Hence the preparation method is very simpleextracted under liquid nitrogen and the antigen and straightforward.would have limited exposure with the solvent under One of the advantages of using alginate microcap-ambient conditions. The main disadvantage of this sules for oral antigen delivery is the absence ofprocess for the application under discussion is that organic solvent during the microcapsule preparation.the microparticles prepared by this method are This could be important for some antigens, whichusually large in size and therefore, are appropriate might be denatured even by limited exposure toonly for parenteral administrations [40]. organic solvents. The ease of manufacture, lack of

Some of the vaccines that have been evaluated in sophisticated machinery, low cost of polymer andanimals through the oral route using biodegradable good safety profile all make this method an attractivemicroparticles include B. pertussis [7], ricin toxoid approach.[9], S. typhi [10], influenza virus [12] and rotavirus Typically, alginate microcapsules are character-[22]. Most of these oral formulations were effective ized for size, loading level, yield and antigen integri-in inducing antibody responses to the target antigens. ty. The size of alginate microparticles ranges from aHowever, the formulations have not been optimally few microns to about 30 microns [22]. The size isefficient and either large or multiple doses have often controlled by modifying the stirring speed and thebeen required. Perhaps the biggest limitation for oral sodium alginate concentrations. However, obtainingdelivery of vaccines in microparticles is the extent of microparticles in the 1–5-mm mean size range can beuptake of the particles, which is often low [34,38]. difficult. The loading levels of antigens have beenNevertheless, various strategies exist to enhance the reported to be between 0.5 and 2.0% w/w [22,23]. Itextent of uptake of microparticles. has been reported that alginate microcapsules are

taken up into the Peyer’s patches, if they areprepared with the appropriate small size [78]. How-

2.2. Preparation of alginate microcapsules ever, alginate microcapsules are relatively hydro-philic in comparison to PLG microparticles and

Sodium alginate chiefly consists of a sodium salt therefore, would be expected to be taken up to aof alginic acid, a linear glycuronan consisting of a lesser extent [78]. The alginate microcapsules canmixture of b-D-mannosyluronic acid and a-L- also offer protection to the antigen in the acidgulosyluronic acid residues. It is mainly used as a environment of the stomach [22].tablet disintegrant, viscosity enhancer and a suspend- Offit et al. [23] described a novel process for theing agent in oral and topical pharmaceuticals. It is microencapsulation of live rotavirus in microspheresalso used in sustained release oral dosage forms. It prepared from anionic polymers and amines, includ-has minimal toxicity associated with oral administra- ing sodium alginate and spermine hydrochloride.tion (oral LD50 in rats, . 5 g/kg). The main This aqueous based microencapsulation process al-advantages of using alginate microcapsules for oral lowed the preparation of microcapsules which wereantigen delivery is the low cost of polymer, good stable in the presence of simulated gastric acid, werebiocompatibility and ease of preparation. taken up into the GALT and induced enhanced

Upon interaction with calcium ions under stirring, immune responses in comparison to oral delivery ofalginate molecules crosslink to form microcapsules. unencapsulated live [23] or inactivated rotavirusThe formation of alginate microcapsules by ionic [24]. The same microsphere preparation process wasgelation represents an interesting approach to prepare subsequently used to encapsulate live reovirus fororal delivery systems for antigens [22–25]. A typical oral administration to suckling mice [25]. The micro-preparation method involves first mixing the antigen spheres were shown to be effective for the inductionwith sodium alginate solution (1–2%). This suspen- of active immunity, even in the presence of pre-sion is then added slowly along with stirring to a existing maternal antibodies [25].


2.3. Preparation of grafted starch microcapsules oral administration [26]. Although starch microparti-cles are more hydrophilic than PLG microparticles

Starch is an additional natural polymer which has and therefore, should have limited uptake into thegreat potential to be used in oral antigen delivery Peyer’s patches, the surface grafting with a hydro-systems [26]. Starch is commonly used as an excipi- phobic polymer renders it more suitable for uptakeent in oral dosage forms, as a binder, diluent and than ungrafted microspheres [26]. Polyacrylstarchdisintegrant. Most oral tablet formulations have microspheres have also been shown previously tostarch in them in one form or another. Starch shows induce good immune responses following systemicno side effects in humans when taken orally and is a immunization [87].major component of many foods (mouse intraperi- The preparation method described for the formula-toneal LD50, 6.6 g /kg). Due to its inert properties, tion of starch microparticles involved the use oflow cost, proven safety and commercial availability, organic solvents. Therefore, this preparation processstarch seems to be an excellent natural polymer for is likely to have a similar effect on the antigen as thethe development of oral vaccine formulations. One preparation process for PLG microparticles. Only asuch approach has been described, involving the model antigen has so far been evaluated and furtherpreparation of starch microcapsules containing en- studies are necessary with more relevant antigens totrapped antigens, followed by the grafting of a allow a more thorough evaluation of this technology.hydrophobic polymer (polydimethylsiloxane orPDMS) onto the surface of the microcapsules. The 2.4. Preparation of polyamino acid microspherespolymer grafting was undertaken to enhance particlestability, increase in vivo half-life and to enhance Polyamino acid microspheres are formed by con-uptake into Peyer’s patches by making the microcap- densation of mixed natural amino acids, with asules more hydrophobic. stoichiometric excess of acidic amino acids, to form

Heritage et al. [26] developed a PDMS grafted microspheres under acid pH [29,30]. Thermallystarch microparticle formulation with entrapped condensed mixtures of amino acids have also beenhuman serum albumin as a model antigen. The used to fabricate oral particulate delivery system forantigen was entrapped in soluble potato starch antigens by Emisphere Technologies [29,30].(British Drug House) microparticles as follows, the Microspheres were prepared in an aqueous en-starch was dissolved in dimethylsulphoxide at 858C vironment. The derivatized a-amino acids wereand then cooled to 228C. To this mixture, 1 ml of a dissolved in water or dilute sodium hydroxide solu-10% antigen solution was added. This solution was tion. The antigen was dissolved in a 1% gum acaciathen emulsified in vegetable oil with stirring and and 2% b-cyclodextrin solution in 1.7 N citric acid.homogenization. To form the microparticles, the Both these solutions were incubated at 408C and theemulsion was slowly added to acetone containing proteinoid solution added to the antigen solution, theTween 80 under stirring. The microparticles were resulting suspension was mixed repeatedly to formcollected by filtration, washed with acetone and air- the microspheres. The microspheres were then col-dried. Replacing the Tween-80 with 3-(triethox- lected. The size of the microparticles was reported toysilyl)-propyl-terminated PDMS (hydrophobic poly- be in the range of 0.1 to 5 mm. The proteinoidmer for grafting) resulted in grafting of the silicone microparticles were characterized for size and load-polymer to the starch surface. The starch microparti- ing level. The antigens evaluated by this technologycles were evaluated for loading level of human were ovalbumin [30] and influenza [29]. Both theseserum albumin (HSA), size, yield and in vitro release antigens generated immune responses following oralprofile. The average size of the microparticles was immunization with proteinoid microparticles.about 4 mm and the antigen loading levels were The microparticle preparation method was carried5–6% w/w. The antigen entrapped into the starch out without the use of organic solvents and therefore,microparticles was shown to retain its integrity by might allow antigens to be entrapped with fullSDS-PAGE. HSA entrapped in PDMS starch mi- maintenance of integrity. However, the use of slight-croparticles induced immune responses in mice after ly low pH for microsphere formation might be a


problem for certain antigens. The physical charac- prepare antigen entrapped microcapsules. A typicalteristics of the microparticles are directly related to procedure for preparing antigen entrapped in CAPthe hydrophobicity of the individual polymer con- microcapsules involves the formation of the corestituents [30]. The uptake of these particles into the material first. This has been done by co-lyophilizingPeyer’s patches has been shown to correlate well the antigen with sucrose and mixing the resultingwith hydrophobicity and particle size [30]. The powder with additional sucrose. The mixture wasproteinoid particles appear to be targeted to the then passed through a stainless steel sieve to filterPeyer’s patches due to their sub-micron size. As far aggregates. The powder was then dispersed in 200as we are aware, this preparation method has been ml of paraffin oil, with an overhead stirrer. To thisutilized only at a lab scale and needs to be evaluated suspension 20 ml of a 10% (w/v) solution of CAP infurther with alternative antigens. The polymer acetone-ethanol (95:5) was added and stirred at 200(proteinoid) is not available commercially in bulk rev. /min for a few minutes. Once the microcapsulesquantities and due to its inherent nature, is likely to had formed, 75 ml of chloroform were added and thebe expensive in comparison to starch, PLG or other supernatant was decanted off. The resulting mi-materials. crocapsules were collected and air-dried under am-

bient conditions. Occasionally, a plasticizer e.g.2.5. Enteric coated microcapsules prepared by the diethyl phthalate or a wax coating (carnauba wax)coacervation phase separation method are also added to the formulation to improve pliabili-

ty and to reduce moisture uptake in the final formula-A number of antigens have been evaluated in tion.

clinical trials using enteric coated microcapsules, The CAP microcapsules formed by the aboveincluding mycoplasma hyopneumoniae [17,18], method are often large in size (50–300 mm) and arerabies antigen (ERA-H strain) [19], a vibrio anguil- irregular in shape, unlike PLG microparticles. Thelarum vaccine [20], an inactivated cholera vaccine objective for enteric coating of microcapsules is[79], a flu vaccine [80], a typhoid vaccine [81], BCG different from the objective during the preparation of[82] and a hepatitis B vaccine [83]. All of these 1–10-mm microparticle formulations. While PLG,antigens were accorded complete protection in the alginate, starch and proteinoid microparticles areacid environment of the stomach by the enteric primarily designed for enhanced uptake into themicrocapsules and were immunogenic after oral Peyer’s patches, for optimal antigen presentation, theimmunization. The mode of enteric protection uti- enteric coated microcapsules are designed to protectlized in these evaluations was either enteric coated the antigen from the acid environment in thegelatin capsules or cellulose acetate phthalate (CAP) stomach. After transit through the stomach and aftermicrocapsules. reaching the intestine, the enteric polymer dissolves

CAP is one of the most commonly used enteric and releases a ‘bolus’ of antigen into the gut.coating polymers. It has found wide applications in Interaction of the free soluble antigen with thethe pharmaceutical industry as a coating for tablets GALT can then occur.and capsules containing drugs that are labile at acidicpH, or induce local irritation in the stomach. The 2.6. Enteric coated microcapsules prepared bysame polymer was also thought to be a good spray dryingcandidate for preparing enteric microcapsules con-taining antigens to protect them from stomach acid An alternative approach to prepare CAP microcap-[17–21]. sules is to spray dry the antigen-polymer suspension,

Coacervation phase separation is a commonly used resulting in the preparation of microcapsules ofmethod for the preparation of microparticles for around 1–20 mm [21]. Although this approach is lessantigen delivery [17,19]. This method is used with a complex than the coacervation method, it usuallyvariety of polymers, ranging from cellulose deriva- requires a larger batch size and a larger amount oftives to synthetic polyesters. The method has been antigen (25–500 mg). Even with lab scale sprayingcommonly used with enteric coating polymers to equipment, the yields with small batches are fairly


low (25–50%), since the majority of the polymer Eudragit L-30D needs to be established with relevantsticks to the sides of the spraying chamber. With antigens and both organic and aqueous coatings maylarger scale batches, these losses tend to become be equally effective.minimal and the yield of microcapsules improves. A typical method of coating was described by

A typical preparation run involves suspending the Wong et al. [20], the antigen (lyophilized vibrioantigen in a sucrose solution, along with poly-L- anguillarum bacteria) was suspended in a 70%lysine, and adding the CAP solution. The mixture is ethanol solution and coated onto 0.9-mm sugar beadsthen atomized through a Buchi spray dryer and the (NPS) using an Aercoat Strea-I spray coater. Eud-resulting microcapsules recovered [21]. The mi- ragit L-30D was then spray coated in an aqueouscrocapsules are characterized for size, antigen load- medium to yield a final Eudragit content of 15% foring level, CAP content and antigen release in simu- a 10% antigen loading level. The enteric coatedlated gastric fluid. One of the antigens evaluated in formulation induced immune responses in salmonidvivo with spray dried enteric CAP microcapsules fish after oral administration. In an alternative ap-was a rotavirus vaccine [21]. The vaccine was proach the antigen solution is also coated onto theaccorded complete protection in the acid environ- NPS in an aqueous phase without the use of organicment of the stomach and was immunogenic follow- solvent, followed by aqueous enteric coating usinging oral delivery. Eudragit L-30D [86].

2.7. Coating of non-pareil seeds with acrylicresins 2.8. Preparation of granules for oral delivery

An alternative technology for producing an oral Granulation is one of the oldest procedures avail-particulate antigen delivery system involves coating able to provide bulk to small doses of activethe antigen onto non-pareil seeds (NPS) and coating ingredients for oral administration. Granules werethe seeds with enteric polymers [20,21]. The NPS are popular as an oral dosage form by themselves a fewusually in the size range of 300 mm to 3 mm. The decades ago, but these days they are used mainly forantigen coating of NPS is carried out in a fluidized compression into tablets or filled into gelatin cap-bed coating apparatus, in which the seeds are sus- sules. Granulation allows the powdered drug topended on an air bed and the antigen coating is display good flow properties for tablet compression,applied from the top or bottom. The common adds bulk to attain a specific weight per dose andprocedures involve either suspending the antigen in a offers sustained release properties if required.volatile solvent like ethanol or in an aqueous solution Granules containing entrapped antigens are anotherat moderately elevated temperatures. alternative particulate delivery system for oral ad-

Polymethacrylates are copolymers of methacrylic ministration of vaccines. Granules are prepared byacid and acrylic esters. These are referred to as blending excipients such as cellulose, starch, sucrose

TMEudragit , which are manufactured commercially and gelatin at variable ratios [21]. The antigenby Rohm Pharma GmbH. The three main types of solution is added to the blend and mixed thoroughlyEudragits are Eudragit L, S and L-30D. These to accord uniform distribution of the antigen. Thepolymers differ in their methacrylic acid content and wet mass is passed through a series of sieves and theviscosity. They are mainly used in oral dosage forms granules are dried under vacuum at 48C. The an-as film coating agents, enteric polymers or sustained tigen-containing granules are then coated with Eud-release polymers. The Eudragits can be used either in ragits to provide enteric protection. The granulessolution, dissolved in organic solvents like iso- could also be compressed under a hydraulic press topropanol, or they can be sprayed as an aqueous form tablets for in vivo evaluation. Granules andsuspension (L-30D). The use of L-30D enteric granulated tablet formulations are mainly designedpolymer may be a better way of coating the NPS, for enteric protection only. The main advantage ofsince this would avoid exposing the antigens to this process is that it can be easily scaled-up from aorganic solvents [86]. However, the advantages of few mg of granules or tablets, to hundreds of grams


of granules or thousands of tablets, using commercial lenge. Following challenge, orally vaccinated ani-scale machines. mals survived longer and had less symptoms of

A live rotavirus vaccine has been evaluated using pneumonia than unimmunized animals.the granulation approach [21]. The granulation pro- This approach is designed mainly to deliver thecess resulted in only a 0.3 log loss in virus infectivi- antigen preferentially into the intestine, followingty. However, enteric coating led to an additional 1.61 gradual swelling of the hydrogel. The formulationlog loss in infectivity of the virus, due to solvent provides protection for the entrapped antigen andexposure. Although a primary coating of hydroxy releases active material in the intestine. The hydrogelpropylmethyl cellulose (HPMC) onto the antigen preparation process does not involve the use ofentrapped granules, prior to enteric coating reduced organic solvents and absorbs antigen by swellingthe infectivity loss only to 0.78 logs. under mild conditions. Therefore, this method should

maintain antigen integrity during formulation.2.9. Preparation of poly(methacrylic acid)hydrogels 2.10. Polyanhydride co-polymers for oral antigen

deliveryPolymers which swell on exposure to water are

used to form hydrogels, either as microcapsules or Bioadhesive polymers adhere to epithelial mem-implants. The swelling phenomenon leads to a branes and deliver active ingredients in vivo usingsustained release of entrapped active ingredients over the adhesion properties of the polymer. Sometime. Polymethacrylic acid is a polymer which has bioadhesive polymers are synthetic e.g. polycar-been used to form hydrogels for controlled drug bophil, while others are naturally occurring e.g.release. Polymethacrylic hydrogels have also been chitosan and hyaluronic acids. The bioadhesionevaluated to deliver antigens to the lower gastroin- properties of polymers could be utilized for an oraltestinal tract (GIT) of cattle and sheep [28]. The pH antigen delivery through an extended retention timedependent solubility of the hydrogels ensures that of antigen in the gut and more effective delivery tosignificant amounts of antigen are not released in the the GALT.rumen (first stomach) but are released in the lower Polyanhydride co-polymers of fumaric and sebacicGIT. acid are one category of polymers which show

Poly(methacrylic acid) hydrogels were prepared bioadhesive properties [74]. Almost 65% of mi-by crosslinking a 40% solution of methacrylic acid croparticles made from this polymer adhered sponta-with a 0.8% solution of N,N9-bisacrylamide. Am- neously to everted intestinal sacs and showed extend-monium persulphate and sodium bisulfite were used ed retention times in vivo [74]. These polymers haveas reaction initiators. The solutions were purged with recently been evaluated as an oral delivery systemnitrogen and polymerized at 608C for 18 h. The gels for plasmid DNA (pCMV/b galactosidase) and drugswere then cut into cylindrical discs of about 5 mm [74]. The microspheres were prepared by a phasediameter and 3 mm length. The antigen was incorpo- inversion method, in which the drug-polymer solu-rated into the hydrogels by incubating them in an tion in DCM was poured under stirring into petro-antigen solution and upon swelling, the antigen leum ether and the microspheres were collected bydiffused into the hydrogel matrix. The hydrogels centrifugation, washed and dried under vacuum.were placed into the antigen solution for 2 days to However, this preparation method involved organicaccord maximum incorporation. Once the antigen solvents, similar to the PLG microparticle and CAPhad been absorbed, the hydrogels were dried at 378C microcapsule preparation methods.in an incubator to a hard glassy state. The hydrogelformulations are attractive for administration to largeanimal models, since the discs can be easily adminis- 3. Conclusionstered. The antigen evaluated with these hydrogels inpigs was a pasteurella haemolytica vaccine, which is A number of polymeric formulations for the oralmeant to provide protection against pulmonary chal- delivery of vaccines have recently been evaluated.


The objectives in preparing these formulations are as An alternative approach to the oral delivery offollows; to protect antigens from degradation due to vaccines using polymers involves the encapsulationgastric acidity and intestinal proteases, to target of vaccines into enteric coating polymers. Theantigens to the Peyer’s patches for enhanced uptake, objective of this approach is simply to protect theor to extend the duration of antigen retention in the antigen against the low pH in the stomach. Thisintestine due to bioadhesion. In recent years, consid- approach has the advantage of being well establishederable effort has been expended on the microen- in the pharmaceutical industry, since many drugs arecapsulation of antigens into PLG polymers. These currently administered using enteric coating technol-microparticles have been prepared with the appro- ogy. In a number of clinical trials, this approach haspriate dimensions to allow their uptake into the also been applied to the oral delivery of vaccines,Peyer’s patches ( , 10 mm). In addition, PLG poly- with some success. Nevertheless, it appears likelymers offer protection for encapsulated antigens in the that enteric coating alone may not allow the success-harsh environment of the gut. In a number of animal ful development of oral vaccines, co-administrationmodels, PLG microparticles have induced potent of adjuvants may also be needed. Although to someimmune response to entrapped antigens and protec- extent, this may be dependent on the nature of thetive immunity. Several similar approaches are now antigen being administered. Enteric coated formula-being applied, involving the microencapsulation of tions appear particularly attractive for the oral deliv-antigens into a range of alternative polymers. These ery of mucosal adjuvants like cholera toxin, whichalternative approaches are being applied mainly are susceptible to acid denaturation [17,18].because PLG microencapsulation is not ideal for To date, most of the polymeric formulations forsome antigens due to the use of organic solvents oral delivery of vaccines have been evaluated only induring the preparation of microparticles. However, mice, although some studies have been performed indirect comparisons of the alternative approaches with larger animal models [18,20,28]. With respect to thethe PLG approach are not usually undertaken. More- scale-up and manufacture of oral polymeric formula-over, it is sometimes not entirely clear how or why tions, the granules and tablets stand out as ap-the newer approaches might be advantageous, since proaches which would present minimal problems.exposure to solvents often remains as an element of Standardized tablet and granule formulations havethe microencapsulation process. In addition, signifi- been used in the pharmaceutical industry for overcant progress has been made with PLG recently, five decades and are prepared by highly sophisticatedallowing the stabilization of a number of proteins in automated machines. PLG microparticles have alsomicroparticles during the preparation process [75– been successfully scaled-up and several commercial77]. Overall, the approaches which involve the products are available on the market. However, theseencapsulation of antigens into small microparticles microparticle formulations have a much larger size( , 10 mm), suffer from the same significant limita- than would be appropriate for the development oftion, the extent of uptake of particles into the Peyer’s oral vaccines. To meet individual antigen require-patches is low [32–34,38]. The extent of uptake is ments and high cost-effectiveness, PLG microen-dependent on a number of parameters, including capsulation needs in depth evaluation at pilot scalesparticle hydrophobicity and particle size. Hence, before large scale-ups are undertaken.more hydrophobic particles e.g. PLG and poly- In summary, although several alternative ap-anhydrides would appear to be attractive in com- proaches are currently available for the developmentparison to more hydrophilic particles e.g. alginates. of polymeric oral vaccine delivery systems, very fewThe optimal particle size for vaccine delivery is of these approaches have been evaluated in anycurrently unknown and conflicting reports have depth. Among the available approaches, only PLGappeared in the literature [32–34,38]. More work is microparticles have been looked at in some detail, inneeded to define the optimal size for each individual a number of independent labs. This approach hastype of microparticle, although particle size must be been explored with a number of different antigensconsidered in relation to additional issues, including and many interesting observations have been madeantigen load and release rates. [31–49]. Nevertheless, it is still unclear if this


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The preparation and characterization of polymeric antigen delivery systems for oral administration