protein microparticles: protecting biopolymers with a hydrophobic coating

COLLOIDS AND g SURFACES Colloids and Surfaces B: Biointerfaces 9 (1997) 285 295

ELSEVIER

Silicone-modified starch/protein microparticles: protecting biopolymers with a hydrophobic coating Michael A. Brook a,,, Jianxiong Jiang a Philippa Heritage b, Brian Underdown b,1, Mark R. McDermott b a Department of Chemistry, McMaster University, 1280 Main St. W., Hamilton, Ont., L8S 4M1, Canada b Department qfPathology, McMaster University, 1200 Main St. W., Hamilton, Ont., L8N3Z5, Canada Received 9 August 1996; accepted 23 June 1997

Abstract

Silicone-coated starch/protein (human serum albumin, HSA) microparticles were prepared by precipitation of a starch/HSA/DMSO/water (water-in-oil) emulsion into acetone containing a silicone: the silicone polymer was either unfunctionalized (SiMe3 terminated, PDMS) or functionalized at its termini with Si(OEt)3 groups (TES-PDMS). The microparticles of approximate diameter 2-7 pm were highly hydrophobic with advancing contact angles /> 115°. Over several minutes, however, the contact angle decreased to ca. 40-70 °. Soxhlet extraction with water led to degradation of the microparticles, irrespective of the nature of their silicone coating, as evidenced by release of the protein from them. Intraperitoneal (IP) or gastric administration of the two different particles to mice, however, showed a clear difference between the two silicones. The microparticles coated with either PDMS or TES-PDMS led to very different immune responses. Oral administration of the microparticles prepared with functionalized silicone elicited a significant production of antibodies, whereas the particles prepared with the unfunctionalized silicone (PDMS) were only weakly active. By contrast, the IP results demonstrated that particles coated with PDMS elicited an immune response that was established much more rapidly than with the particles modified with TES-PDMS. It is proposed that the TES-PDMS forms a physically adhering film or covalent bond to the protein molecules, which serves to protect the microparticle from biological degradation in the gut and/or facilitates the microparticle/protein interaction with the immune system. © 1997 Elsevier Science B.V. Keywords: Silicone: Human Serum albumin: Starch; Microparticle; Protein release

1. Introduction

For m a n y ailments, the sustained delivery o f drugs (zero-order delivery) is often accompanied by better patient compliance and efficacy o f the * Corresponding author. Tel: (+ 1) 905 525 9140 ext. 23483; Fax: (+ 1) 905 522 2509; e-mail: [email protected] a Current address: Connaught Laboratories Ltd., 1755 Steeles Ave. W.. Willowdale, Ont. M2R 3T4, Canada. 0927-7765/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PH S0927-7765 (97)00036-2

drug. As a result, the development o f new strategies for the delivery o f small molecules, particularly oral delivery, is a field o f active interest. There are commercial and philanthropic reasons to deliver large molecules orally as well. For example, one could envisage oral vaccines comprised o f orally delivered proteins. However, the gastrointestinal tract is deleterious to the proteins typically used for vaccination. The stomach, in particular, is uniquely designed to degrade proteins t h r o u g h

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M.A. Brook et aL / Colloids Surfaces B: Biointerfaces 9 (1997) 2 8 5 ~ 9 5

enzymatic and acid-catalyzed protein hydrolysis. A viable oral vaccine preparation requires protection for the protein during transit in the gut. One approach to protecting the protein involves coating the material with a hydrophobic species that can provide a barrier which prevents protein contact with the gut fluids. The coating material must, however, be sufficiently porous or fragile to allow the protein to eventually present itself and elicit an immune response. Previous approaches of this type have used copolymers of glycolic and lactic acid [1]. We have chosen to examine the use of silicones, exceptionally hydrophobic materials [2], as protective coatings for proteins because of the analogy between polar protein surfaces and hydrophilic mineral surfaces such as silica. Silica is usually made hydrophobic by the action of a coupling agent, normally a silane reagent or functionalized silicone [3-5]. Alkoxysilanes crosslink in solution and, after migrating to the silica surface, covalently bond to it (1, Scheme 1) leaving a hydrophobic surface [6-8]. The analogous reaction should occur with polar organic surfaces. However, unlike silica, with which a disiloxane is formed, the transesterification reaction between an alkoxysilane and a biopolymer leads to a different alkoxysilane that remains susceptible to hydrolysis (2--+3, Scheme 2) [9,10]. An alkoxysilane is thus a "weak link" between the silane or silicone and the biopolymer. This weakness is an advantage in an oral delivery vehicle for a protein: following hydrolysis, the native protein can be presented to the mucosal immune system. Maintenance of protein immunogenicity and the rate of protein release from the siloxane matrix are the overriding challenges in such a system. Previous work with polysaccharides has shown

that silane coupling agents do not confer much hydrolytic stability to the biopolymer matrix [ 11 ]. It seems possible, however, that a more hydrophobic species, for instance a long chain silicone polymer, could better protect a biopolymer [3]. Numerous examples exist of formulations in which starch and/or cellulose are combined with silicones for various reasons, including examples in which hydrophobization was explicitly sought. For instance, the reaction of silicones with starch under basic conditions, which should lead to starch-O-silicone linkages, has been reported [12]. Functional silicones bearing pendant amino groups have also been used to hydrophobize starch [13]. In both cases, hydrolytic stability is controlled either by physical adhesion 4 (latter case) or the covalent binding of a single bond 5 (Scheme 3). Situations such as these are not optimal. We have chosen to examine protein "coupling agents" that involve both multiple coupling sites, which bind more tenaciously to the substrate, and also highly hydrophobic silicone chains 6 (Scheme3). To determine the validity of this approach for protecting proteins from denaturation in the gut, we have prepared microparticles that are surface-modified by silicone polymers. The microparticles contain starch, both as a base material and diluent, and protein (human serum albumin, HSA). The silicones include both normal PDMS (Me3SiO(Me2SiO),SiMe3) and functionalized polymers PDMS-TES (7, (EtO) 3Si (CH2) 3SiO (Me2SiO) ,Si (CH2) 3Si (OEt)3, Scheme4). The particles were characterized by protein release rate and immunological viability as a function of the nature of the protecting silicone (functionalized or unfunctionalized).

(MeO)3Si~.~. ~,, OH A_ r yn I yN / S i O S i - O - S i -/~ , / Glass

R=

surface

R ~ ~ R R I o T[o 71o ~ o I o HO-Si" "Si[ "SUn'Si" " s f "H ~. ~ , ± I I / 0I 0I YI 0I 0i OH / "A,, / I Urn2 / - - S i ' O - S i - O ' S i - O'Si-O'Si-O-Si z

Me, PhH2N ' ' ~ ' ~ ' ' m ' ~

/

~

0~

0

0 O.v~-..~

Scheme 1. Modification of glass by silane coupling agents.

M.A. Brook et aL / Colloid~ Surfaces B: Biointerfaces 9 (1997) 285-295

287

Ro.R.o R ~'o[," Si ;.!,,~' ol I ,~;I

f f,-2- 7

RSi(OMe) 3 Biopolymer Surface (cellulose, starch, etc.)

OH 2 (RSiO312)n + Biopolymer

2 + MeOH

Scheme 2. Transesterification of a biopolymer surface.

\ o-s(

\ o-s(

.O.sl .o.i.o..

\ o-s °-s',

OH OH OH

O

4

\

o-s(

-s,

o

,a ~'

~s',.%-°~y.. 6°6

OH OH

6

5

R = silicone

Scheme 3. Approaches to protect biopolymers with silicones.

~Si(OEt) \/ \/ \/ H,.Si.O...{*-"Si.o...]l~nSi. H

PDMS-H

3 \/ \/ ~Si o~Si ~Si(OEt)3

\/

8

=- ( E t O ) 3 S i ~ S i . o H2PtCIs cat

7 PDMS-TES

Scheme 4. Preparation of PDMS-TES.

2'. Experimental section 2.1. Apparatus, methods and materials

Advancing contact angles were measured on an NRL C.A. Goniometer (Ran6-Hart Inc.) with distilled water at ambient temperature. Optical densities (ODs) in the enzyme-linked immunosorbant assay (ELISA) were determined at 405 nm on a Titerteck Multiskan Plus from ICN Biomedicals.

viscosity and molecular weight are well known [14]. The termini that have been used in this paper are the following: X=Me3Si, PDMS; MezSiOH, PDMS-OH; Me2SiH, PDMS-H; Me2Si(CH2)3Si(OEt)3, TES-PDMS. The viscosity (in centistokes, cSt) of the polymer is then appended. Microparticles are described by their coating. For example, the microparticle coated with PDMS-TES-1000 is named MP-TESPDMS- 1000. 2.3. Materials

2.2. Nomenclature

The various silicone polymers used in this study differ in their molecular weights and terminal groups. To distinguish these, we refer to silicones (PDMS, XO(MezSiO),X) by their terminal groups (X) and viscosity. Silicones are typically sold by viscosity, and correlations between

PDMS-H (2-3cSt, 500cSt, 1000cSt and 10000cSt), PDMS (200cSt, 1000cSt and 5000 cSt), PDMS-OH (1000 cSt), allyltriethoxysilane 8, platinum catalysts HzPtC16, and Pt2[(HzC=CHMe/Si)20]3 were obtained from Htits-Petrarch Systems (now United Chemical Technologies, Bristol, PA) and used without fur-

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ther treatment. PDMS-TES was prepared by the HzPtC16-catalyzed hydrosilation of allyltriethoxysilane with PDMS-H [15]. Soluble potato starch and fluorescein isothiocyanate (FITC) were obtained from BDH. HSA (Fraction V, 96-99% Albumin), Aprotinin® and phosphatase substrate tablets were obtained from Sigma. Phosphatase-conjugated goat anti-mouse IgG or IgA was purchased from Southern Biotechnology Associates. Female BALB/c mice, age 6-8 weeks were purchased from Charles River Laboratories Inc., Montreal. Syringe filters (0.45 mm) were obtained from Millipore and microtiter plate wells from Costar. PE50 tubing was purchased from Becton Dickinson and Co.

prior to the final step. Particles could not be prepared in the absence of proteins. The water-in-oil emulsion was added with stirring (1100 rev min-1 with a magnetic stir bar) at room temperature to acetone (400 ml) containing a coating material which consisted of: (i) functionalized silicones PDMS-TES (from 0.25-4.0ml, depending upon the experiment), (ii) unfunctionalized silicones PDMS (0.25 4.0ml), or (iii) Tween 80 (2.0 ml, 1% in acetone). The acetone-precipitated starch/protein microparticles were centrifuged at 2000 rev rain ~ for 5 min at 4°C. The supernatent acetone was decanted and the residual microparticles were dried through evaporation of acetone in air for at least 3 days. The solids were then collected, weighed and stored.

2.4. The preparation of HSA-FITC

2.6. Surface properties." contact angles of the microparticles

Fluorescent HSA was prepared by grafting a fluorescein moiety to the protein. To HSA (0.1 g) in phosphate buffer (pH 8.9, 20 mL) was added FITC (2.0 mg, 5.1 pmol). The pH was adjusted to 9.5 with NaaPQ and the mixture was allowed to stir for 16 h at room temperature in the dark. The sample was dialyzed in 4 1 of distilled water at 4°C.

2.5. Preparation of controlled s&e starch~protein microparticles Microparticles were prepared as previously described [16]. A summary only is provided here. Soluble potato starch (1.0g) was suspended in DMSO (2.0 ml) with stirring. After heating (99°C) to form a solution, and cooling to 25°C, HSA (or HSA-FITC, 100.0 mg), dissolved in distilled water ( 1.0 ml) was added with vigorous stirring at 32°C (500 rev min- ~ with a magnetic stir bar or using a high speed mixer 30 000 rev min ~ with a brush as the agitating element). The starch/protein solution was added dropwise to vegetable oil (e.g. Crisco@, 30 ml) with continuous stirring (1300 rev min- ~ with a magnetic stir bar) to produce a water-in-oil emulsion. The higher the speed of the stirring and the longer the stirring time, the finer the emulsion droplet size. It was imperative to avoid large emulsion droplets

The advancing contact angles of distilled water on the silicone-grafted (PDMS-TES) microparticles were compared with those prepared from PDMS or Tween. Silicones have very high contact angles (typically > 100°). Starch, by contrast, was completely wetted (contact angle ~0°). The contact angle of HSA was dynamic: the initial angle of about 110° changed to 20 ° within about 6 min. As standards, the contact angles of the hydrophobic surfaces of silicone rubber and hexamethyldisilazane-treated silica were measured. These latter contact angles were always > 115 °. The hydrophilic glass surface was wetted by water with contact angles < 14°. A glass surface coated with PDMS-OH (1000 cSt) had an initial contact angle of 81+2 ° which, however, dropped to 55+5 ° within 30 min. A film of the microparticles at least 1 mm thick was prepared on a glass slide. The contact angles of the microparticle film were measured at 18°C. Similar to the PDMS-OH film, these contact angles were dynamic, showing lower values with time; the values are shown in Fig. 2. There were some problems associated with the determination of contact angles: (i) the contact angles out of the range of the apparatus (> 115°) were approximated to 115 ° unless otherwise specified; (ii) the contact angles

M.A. Brook et al. / Colloids Surjaces B: Biointerfaces 9 (1997) 285-295

of the particles obtained from acetone/Tween 80 solutions (30°~15 °) were difficult to establish, as the particles were easily wetted and the microparticles often floated on to the surfaces of the water beads. Within 6 min of placing the water drop on the microparticle film, the water bead was covered by the microparticles. 2.7. Protein release: extraction with water

Protein release was examined in two distinct ways. First, the microparticles (0.5g) were Sohxlet-extracted with D20 or H20 (90ml) for 195 h. The solid residues in both the Sohxlet cup and still pot (some very small particulate passed through the Sohxlet filter cup) were weighed at the end of the operations (Table 1); release was followed by 1H N M R and gravimetric analysis. It was not possible to establish whether the recovered protein and starch was covalently grafted to PDMS-TES. Second, the UV spectra of an aqueous suspension of microparticles prepared from HSA-FITC was followed with time at ambient temperature. Direct observation of the release of the protein from the microparticles was carried out by putting HSA-FITC-containing microparticles into water and assuming the contribution to the UV-absorbance by unreleased protein in the solid state was negligible. Thus, HSA-FITC-containing microparticles (about 0.21 g, 12 wt.% protein) were suspended in water (10 ml). The liquid in the UV cell was poured back into the flask after each measurement (Fig. 3). The release rates in solutions of different pH were similarly measured by suspending about 30 mg of HSA-FITC-containing microparticles in 3.0 ml water in a UV cell (Fig. 4).

Table 1 Results of the Sohxlet extractions of microparticles with water Silicone

PDMS-1000 PDMS-TES-1000

Amount released [starting] (wt.%)" Starch

Protein

DMSO

8.5 ]59.8] 17.7 [56.6]

1.4 [8.5] 6.5 [7.7]

14.1 ]13.9] 16.6 ]16.5]

a > 95% of the material was recovered.

289

2.8. Immunization protocol 2.8.1. Immunizations Groups of ten female BALB/c mice, age 6-8 weeks were used and allowed food and water ad libitum. Mice were inoculated in groups of five by intraperitoneal (IP) or intragastric (IG) adminisration, under ether anesthesia, on days 0, 7, 14 and 70. Animals received antigen intraperitoneally in phosphate-buffered saline (PBS, 250 ml, pH 7.4) or intragastrically in NaHCO3 (500ml, 0.2 M) using PE50 tubing. 2.8.2. Collection of serum and gut washes Individual blood samples were obtained via the retro-orbital plexus. Insoluble material was removed by centrifugation and sera were stored at - 7 0 ° C until used. To detect and quantify anti-HSA IgA in the intestinal lumen, mice were exanguinated and their small intestines removed and everted over capillary tubes. The everted intestines were incubated for 4 h in ice-cold enzyme inhibitor solution (5 ml) containing NaCI (150 mM), NaH2PO 4 ( 10 mM), ethylenediaminetetraacetic acid (5 mM), phenylmethylsulfonyl fluoride (2 mM), Aprotinin ® (0.05 U ml 1) and NaN3 (0.02%). Intestines were removed and the remaining solution (operationally termed gut washes, GW) were clarified by centrifugation (2×104xg, 15min) filtered through 0.45 mm syringe filters and stored at - 7 0 ° C until used. 2.9. Measurement of liSA-specific antibody responses

An ELISA was used to detect and quantify HSA-specific antibodies in individual serum and GW (Figs. 5 and 6). Duplicate serial dilutions of serum and GW were examined using microtiter plate wells incubated with HSA (100ml, 10 mg ml- 1 in TBS) followed by incubation with gelatin (150 ml of 0.1% in TBS, blocking buffer). Anti-HSA antibodies were quantified by incubating wells with alkaline phosphatase-conjugated goat anti-mouse IgG or IgA ( 100 mt, heavy chain specific) diluted in blocking buffer. After washing, diethanolamine buffer (100 ml of 1.0 M, pH 9.8),

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containing MgC12 (50 mM) andp-nitrophenylphosphate (1.0 mg ml - 1, 5 mg phosphatase substrate tablets; Sigma), were added to each well and ODs were determined at 405 nm. Normal mouse serum (NMS) or normal gut wash (NGW) pools, prepared from untreated animals, were used to establish baseline mean OD values. The results were expressed as reciprocal end-point titers representing the greatest serum or GW dilutions giving OD values exceeding three standard deviations above NMS or NGW mean values.

3. Results

3.1. Preparation of particles The preparation of microparticles is summarized in Section 2 [17]. Essentially, an aqueous mixture of DMSO/starch and HSA were emulsified in vegetable oil. Droplet size was controlled by the degree of stirring and sonication to which the emulsion was exposed. When the emulsion was precipitated into acetone containing Tween, PDMS or TES-PDMS [17], starch/protein (/silicone) microparticles of diameter 2-7 lam precipitated (SEM, Fig. 1) and were harvested by

centrifugation. It should be noted that stable silicone-coated particles could not be made in the absence of protein.

3.2. Microparticle surface structure." contact angle The advancing contact angle of silicones is extremely high when compared with other organic species; values are typically greater than 115° [2]. Thus, changes in contact angle serve as a sensitive probe for diagnosing changes in silicone content. The contact angles of water droplets were measured on films comprised of microparticles coated with PDMS or PDMS-TES. Somewhat surprisingly, these values were not static. A plot of the changes of contact angle with time are shown in Fig. 2. We ascribe the dynamic nature of the contact angle values to three factors: (i) swelling of the protein-starch particle exposes the underlying biopolymers, which have significantly lower contact angles, to the water drop, (ii) hydrolysis of the protein-O-silicone linkage exposes a hydrophilic protein surface (see below), and (iii) migration of the particles from the glass surface to the water-droplet/air interface exposes the underlying glass surface to the water. The time frame of the contact angle change is comparable with the protein release rate (see below). By contrast, commercial Me3Si-functionalized silica particles were found to have a high contact angle (> 115°) which did not change appreciably over the same time period. The silica particles with their higher density

¢

105~

PDMS-200

- - B - - PDMS-1000 ~

PDMS.-5000 )(

PMDS-TES-3

X( PMDS-TES-500 PMDS-'nES-1000 I

35 0

5

10

15

20

PMDS-TES-10000 Tween 80

!

Time (rain)

Fig. 1. Scanning electron micrograph of PDMS-TES/HSA starch microparticles (bar = 1 pm).

Fig. 2. Plot of contact angles with time for microparticles coated with different silicones. The contact angle of the Tween-coated particles dropped from 50 ° to 25 ° over 5 min.

M.A. Brook et al. / Colloids Surfaces B: Biointerfiwes 9 (1997) 285-295

were found to be much less mobile in the presence of the water droplet than the starch microparticles. To a small extent, the rate of change of contact angle is dependent upon the silicone molecular weight; the contact angle values for higher molecular weight materials changed somewhat more slowly (Fig. 2). A second factor which contributes to the rate of change is the thickness of the microparticle film which was relatively difficult to control.

3.3. Protein release Protein release from the particles by Sohxlet extraction with water showed that the PDMS-TES coating released the protein more easily than PDMS (Table 1). An independent assessment of the degree to which the starch/protein particle surface was affected by exposure to silicone polymers could be made by examining the rate and degree of protein release upon exposure to water. Utilizing a ftuorescein-labelled protein (HSAFITC, see Section 2), the concentration of protein in the aqueous supernatent could be readily assessed by UV spectroscopy (Fig. 3). The effect of molecular weight of the silicone could be more clearly seen in this experiment; unlike the contact angle data, the question of "film thickness" does not arise. An increase in molecular weight of the silicone retards the rate of protein release. There is, however, no obvious difference between PDMS- and PDMS-TES-coated microparticles. The silicone, even low molecular weight silicone, clearly provides a hydrophobic barrier on the surface of the particle, as evidenced by the slower protein release when compared with 120

291

uncoated particles. It should be noted that the UV activity indicates that the protein is exposed to/swollen with water and liberated from the particle structure. However, it does not allow one to discriminate between protein bonded to or free from silicone.

3.4. Effect of pH Since the stability of the silicone is affected by pH [18], and the protein is also sensitive to pH, we examined protein release over a relatively wide pH range. The results, shown in Fig. 4, show surprisingly little effect of the pH on the protein release from either type of coated microsphere.

3.5. Immunological results As an independent means of examining the effect of the silicon on the protein, the in vivo immunological effect of the protein was examined. Thus, microparticles were introduced to mice via oral and ??intraperitoneal administration (for complete experimental details and discussion, see Ref. [16]). Groups of five animals were immunized on days 0, 7, 14 and 70 with various doses of HSA: (i) incorporated in PDMS-TES-grafted microparticles, (ii) PDMS-coated microparticles, (iii) silicone-free microparticles, or (iv) in 0.2M NaHCO3. Following IP introduction of the three microparticles, antibody assays were done on days 28, 42 and 63. The increased titer of the PDMS60 _

s0

¢,0 -,B--pH 5.5 I ,t pH6.8 I

~- 30

100 : e Tween80 --B-. ~ 1 0 0 0

6O

J. PDMS-TBS-3 )( PIDMS-TES-1000

40 20

a.

20

)(

....

pH 8.4 I

10 0m 0

i 100

----i 200

T i m e (min)

0 0

100

200

300

400

Time (min)

Fig. 3. Effect of microparticle coating on protein release.

Fig. 4. pH dependence of protein release from microparticles. The weights of the microparticles MP-PDMS-TES-1000 in the pH experiments were: pH 1.5, 30.8 mg; pH 5.5 29.1 mg, pH 6.8 29.6 mg and pH 8.4 29.0 mg; all in 30 ml aqueous solution.

M.A. Brook et al. / Colloids Surfaces B: Biointerfaces 9 (1997) 285-295

292

modified particles is seen at an early stage (Fig. 5). The lower titer of the TES-PDMS and unmodified particles shows up somewhat later. By contrast, with oral administration the TES-PDMS-modified particles showed a strong antibody titer, whereas the PDMS or unmodified particles elicited no such response (inset Fig. 6).

4. Discussion

4.1. Nature of the particles

In the initial stages of preparation of the microparticle, a homogeneous solution of water, DMSO, 300000 250000 200000

•

SD

-.U-,-PDMS

100oo/o A~AAB 150000 50000 0

20

40

•

SD

J.

FDMS-TES

60

Day==

Fig. 5. Antibodies raised to HSA following IP immunization with silicone-coated starch microparticles (each point represents average of five animals). Standard deviation (SD) of the response is shown by the additional points above and below the line.

45OO 4000 3500 3000 2500 20OO 1500 1000 5O0

o

0

.

~ •

SD

-.I-- PDMS •

SD

4. RDMS-TES

20

,:

40

HSA and starch was dispersed in vegetable oil to give a water-in-oil emulsion. Of the components present in this solution, the protein is best able to stabilize the oil-water interface, a feature which is characteristic of proteins [19,20]. Upon exposure of the droplet to acetone/silicone, the protein exposed at the surface is best able to interact with the silicone (Scheme 5). We attribute the inability to make stable particles in the absence of protein to this interaction; starch cannot react/interact with the silicone sufficiently quickly to stabilize the particle droplet prior to aggregation. 4.2. P D M S

The microparticles coated with PDMS cannot form a covalent bond between the biopolymer and silicone. Instead, the silicone, known for low surface energy [21], can wet out the protein surface [22]. Hydrolytic challenge, in which the microparticles were exposed to a water droplet (contact angle measurement) or suspended in bulk water, showed that the protein was, however, able to escape from the microparticle. There are two possible types of protein: the first will have been exposed to silicone (9, Scheme 5) and the second will not, by virtue of being in the particle core. It has not yet been possible to distinguish between these two types of protein; both could lead to the observed UV absorbances and 1H N M R signals. However, the mouse immunoassay shows that little viable protein is present following oral administration, as evidenced by a feeble immune response. This suggests that if there is a protein-PDMS association, it is insufficiently strong to survive the relatively brutal biological conditions found in the gastrointestinal tract; both free and silicone-associated proteins are degraded. PDMS thus acts to provide an efficient, but only temporary, hydrophobic barrier to the protein/starch microparticle.

i

60

8O

n=ys

Fig. 6. Antibodies raised to HSA following IG immunization with silicone-coated starch microparticles (each point represents average of five animals). Standard deviation (SD) of the response is shown by the additional points above and below the line.

4.3. PDMS- TES

The comments made above are equally valid for the functionalized silicone PDMS-TES. However, in addition to the relatively weak association between protein and silicone, a covalent bond

M.A. Brook et al. / Colloids Surfaces" B: Biointerfaces 9 (1997) 285-295

oil O Protein

293

Silicone

Starch

Siliconized Microparticle

Water-in-oil emulsion ~ ' / "

OH 2

+ Normal PDMS

9

PDMS-TES

10

Scheme 5. Possible hydrolysis routes of the microparticles.

between the two polymers can arise (10, 0 , Scheme 5) via transesterification or, less likely, transamination processes (11 ~12, Scheme 6). To the extent that such bonds form, they will provide an additional stability to the interface between the two polymers. Alternatively, or subsequently, the protein-silicone covalent bonds can hydrolyze to give a physically adhering, cross-linked silicone film (12~13, Scheme 6). The observed extraction, contact angle, and protein release phenomena can all be explained by a combination of the factors already described for PDMS and this additional bonding.

The two silicones are best differentiated by the differences in biological activity. PDMS-TES clearly provides some protection to the protein in the gastrointestinal tract. Similarly, the very long induction time for an immune response, following IP immunization with PDMS-TES-grafted microparticles, may be a consequence of the silicone"protected" protein only slowly undergoing deprotection to liberate free protein. The observed IG and IP immune responses could be indicative of the silicone behaving as an adjuvant in addition to its protective role [23]. The possible application of these silicone/protein Annealed Film

Covalently Bonded Silicone HO-Si-O

OH

OH OH Starch 11

OH NH2

L

OH OH

I

(EtO)3Si_Silicone

OH NHa

Starch

I

v

OH OH

I

OH NH2

Starch

12 Scheme 6. Covalent versus physical adhesion of proteins and silicons.

13

I

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M.A. Brook et al. / Colloids Surfaces B." Biointerfaces 9 (1997) 285-295

systems in the area of oral vaccines is intriguing. As part of our continuing efforts in this area, we shall endeavor to examine the exact nature of the bonding between the silicone and protein to see if indeed the functionalized silicones are behaving as coupling agents on the protein surface.

5. Conclusion Silicones hydrophobize biopolymer surfaces comprised of protein and starch. Increasing the silicone molecular weight and thickness of the silicone layer affords better protection to the biopolymer. Functionalized (PDMS-TES) and unfunctionalized (PDMS) silicones behave remarkably similarly in physicochemical analyses which, at first glance, would suggest similar protein/silicone interactions in both cases. However, their behavior in a biological environment is quite different. Oral administration of the microparticles to mice demonstrated either that more viable protein leaches from a protective PDMS-TES shell and/or that the modified protein which interacts with the immune system possesses enhanced immunogenicity. In contrast, proteins associated with PDMS are not protected following IG immunization. This suggests a stronger interaction between the two polymers in the former case, possibly involving a covalent bond between the two polymers.

Acknowledgments We gratefully acknowledge the financial support of Connaught Laboratories, the Natural Sciences and Engineering Research Council of Canada, the Ontario Technology Fund and the Ontario University Research Incentive Fund.

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