Controlled Vaginal Delivery of Antibodies in the Mouse1

BIOLOGY OF REPRODUCTION 47, 133-140 (1992)

133

Controlled Vaginal Delivery of Antibodies in the Mouse1

MICHAEL L. RADOMSKY, KEVIN J. WHALEY, RICHARD A. CONE, and W. MARK SALTZMAN

Departments of Chemical Engineering and Biophysics, The Johns Hopkins University, Baltimore, MD 21218

ABSTRACT

Controlled delivery of monoclonal antibodies to the mucus secretions of the vagina might provide women with passive

immunoprotection against both sexually transmitted diseases and unwanted pregnancy. We have developed intravaginal devices

composed of poly(ethylene-co-vinyl acetate) (EVAc) that continuously release IgG antibodies for over 30 days into buffered

saline, and we have tested these devices in the vagina of mice. Polymeric devices containing either BSA (as a test reagent forproteins) or anti-hCG antibody, when inserted into the vaginas of mice, provided a continuous supply of either BSA or hCG-

binding antibodies to the vaginal mucus for 30 days. Antibodies released by the devices achieved high concentration in the

mucus within the lumen of the vagina, but did not significantly ascend into the uterine horns, as determined by epifluorescence

microscopy of fluorescently labeled mouse IgG and by immunohistochemical localization of rabbit IgG. Our results suggest that

long-term intravaginal delivery of functionally intact antibodies can be achieved with devices composed of EVAc.

INTRODUCTION

A century ago, von Behring and Kitasato first demon-

strated that systemic delivery of serum obtained from im-munized animals can protect against infection [1]. This ap-

proach to passive systemic immunization has proven highly

effective in many settings: currently, serum immunoglob-

ulins are administered to humans for treatment of chronic

immunodeficiency syndromes, prophylaxis against hepatitis

A, prevention and treatment of measles, postexposure pro-

phylaxis against hepatitis B, and prophylaxis against the de-

velopment of anti-Rh0 antibodies in Rh-negative women with

Rh-positive mates. Recently, it has been demonstrated that

antibodies delivered externally, not systemically, can pro-

vide significant protection to mucosal epithelia: oral deliv-

ery of antibodies protects animals [21 and humans [3] against

pathogenic bacteria, and nasal delivery of antibodies pro-

tects the upper respiratory tract against viral infections [41.We are now exploring whether antibodies delivered di-

rectly to the vagina can provide significant immunoprotec-

tion against sexually transmitted diseases (STh) as well as

pregnancy [51. The vagina is the primary infectious entry

site for many STD pathogens, as well as the entry site for

Sperm. Moreover, the vagina is well suited to accommodate

a device that provides sustained delivery of protective an-

tibodies. Importantly, vaginal delivery of antibodies for pas-

sive immunoprotection may not require the same level of

medical intervention as passive systemic immunization: the

user can readily insert and remove a vaginal device. Finally,

with the advent of the ability to manufacture human mono-

Accepted March 24, 1992.

Received January 14, 1992.

‘This work was supported by grants from the National Institutes of Health (GM-

43873 and BRSG S07 RR-07041) and National Science Foundation (BCS-9007762).

W.M.S. is a Camille and Henry Dreyfus Teacher-Scholar.

‘Correspondence: Professor W. Mark Saltzman, Department of Chemical Engi-

neering, The Johns Hopkins University, 3401 N. Charles Street, Room 42 New En-

gineering Building, Baltimore, MD 21218. FAX: (410) 516-5510.

‘Current address: Syntex Research, Palo Alto, CA 94303.

clonal antibodies, it may now be possible to use human

antibodies for repeated and sustained immunoprotection.

Human monoclonal antibodies are less likely to cause the

undesirable immunoreactions that occur with repeated sys-

temic immunizations with immunoglobulins obtained from

animals.

In this investigation we examined a new approach for

topical passive immunization: long-term, controlled release

of antibodies from a polymeric device in direct contact with

the vaginal mucosa! surface, the mucosal surface in need

of protection.

Materials

MATERIALS AND METHODS

Poly(ethylene-co-vinyl acetate) (EVAc; Elvax 40W, Du-

Pont, Wilmington, DE) was washed for 48 h in water and

48 h in acetone in a Soxhlet extractor (Baxter, Columbia,

MD) to remove impurities. Solid particles containing the

protein to be released were prepared prior to incorpora-

tion into EVAc matrices for use in four different experi-

mental groups (Table 1). For the vagina! rings used in groups

A and B, BSA (Sigma Chemical Co., St. Louis, MO) was mixed

24:1 with fluorescein isothiocyanate-conjugated BSA (FITC-

BSA, Sigma) and lyophilized to create solid particles. The

particles were crushed and sieved to less than 74 p.m. For

the vagina! rings used in group C, 10 mg of tetramethyl-

rhodamine isothiocyanate-labeled mouse IgG (TRITC-mouse

IgG, Jackson Immunochemicals, West Grove, PA) in buff-

ered saline solution was mixed with 4.8 mg of mouse

monoclonal antibody against hCG (anti-hCG, BioDesign,

Kennebunkport, ME) and 1000 mg of Ficoll (70,000 Mr,

Sigma). The resulting solution was lyophiized, crushed, and

sieved to obtain particles less then 100 p.m. For the vaginal

rings used in group D, polyclonal rabbit lgG (50 mg, Jack-

son Immunochemicals) was mixed with TRITC-mouse IgG

(8 mg, Jackson Immunochemicals). The solution was ly-

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134 RADOMSKY ET AL.

TABLE 1.

Group(n)’ Insertion Method

Active agent per

deviceInert agent per

deviceTotal mass;

thickness

A

(12) With collars 800 �g FITC-BSA 19 mg BSA 54 mg; 1.8 mm’

B

(6) With suture 200 �g FITC-BSA 5 mg BSA 15 mg; 1.6 mmbC With suture 30 �sg mouse 6 mg Ficoll and 12 mg; 1.3 mmb�

(6 + 3 control9 anti-hCG saltsaD With suture 80 �g mouse 2.6 mg Ficoll and 8 mg; 1.1 mm”(8 + 2 control) TRITC-lgG and

500 p.g rabbit lgGsalts

(n) = number of animals in group.

‘Ring-shaped device with 7 x 5-mm surface area.bRing�shaped device with Outer diameter -4 mm and inner diameter -1.5 mm.cCOntrOl animals received a blank ring containing inert agents but no active agent.5Antibodies were obtained in buffered salt solutions; those salts remained after lyophilization.

‘Polymer rings were coated with a pure layer EVAc containing 4 small pinholes.

ophilized, and the powder was crushed to obtain 50-500-

p.m solid particles.

Female mice (C57BL/6, 8 wk, 15-20 g, Harlan Sprague

Dawley, Indianapolis, IN) were housed individually and had

unlimited access to tap water and food.

Fabrication of Intravaginal Rings

Solid particles containing BSA or antibody were added

to a 10% (w/v) solution of EVAc in methylene chloride.

Sufficient solid particles were added so that the resulting

polymer matrix was 35% or 50% solid powder by weight.

The solution was mixed well and poured onto a prechilled

glass mold (2 X 2 cm, -80#{176}C). After solidification (-10

mm), the slab was maintained at atmospheric pressure at

-20#{176}Cfor 48 h and under vacuum at 25#{176}Cfor an additional

48 h. Rings or slabs were cut from the resulting matrix to

the desired size with a razor blade and/or cork borer.

Composition and size of the devices are provided in Table

1.

To extend the period of release from a small ring and

to obtain a more constant antibody release rate, some of

the rings were coated with a pure layer of EVAc. These rings

were first pierced with two 30-gauge hypodermic needles

and then cooled by immersion in liquid nitrogen. The cooled

ring was briefly dipped into a 20% solution of EVAc in

methylene chloride and allowed to dry overnight in a vac-

uum desiccator to remove the methylene chloride. Upon

removal, the hypodermic needle left a small pore through

which water had access to the polymer matrix. This simple

fabrication technique mimics inwardly-releasing hemi-

spherical geometry that is known to produce a nearly con-

stant rate of release [6].

Release of Antibodies from Polymers into a

Well-Stirred Solution

The rate of protein release was determined by immers-

ing the ring in PBS, incubating at 37#{176}C,and periodically

exchanging the PBS. FITC-BSA and IgG were detected in

the exchanged solutions by total protein assay (Pierce,

Rockford, IL); anti-hCG was detected by total protein assay

and also by an ELISA sensitive only to intact (undegraded)

antibody.

Total protein assay. Briefly, 50 p.! of Coomassie blue

dye was mixed with 100 pA of sample in a well of a 96-well

microplate. The optical density was determined on a mi-

croplate reader (Thermomax, Molecular Devices, Menlo Park,

CA) at 595 nm and compared to the optical density of stan-

dard solutions prepared from unlyophilized antibody or BSA

(0.1-5.0 p.g/ml). The detection limit for this method was

0.5 p.g/ml.

Anti-hCG EIJSA The concentration of anti-hCG in an-

alyte solutions was determined by ELISA: (1) Antigen so-

lution (100 p.1, 250 lU/mi hCG, Sigma) was added to each

well of a 96-well plate (Nunc-Immuno Plate Maxisorp, In-

termed, Naperville, IL) and incubated 1 h at 37#{176}C.(2) The

remaining adsorption sites were blocked by the addition of

1% BSA in PBS, 200 p.1/well, and incubation for 1 h at 37#{176}C.

(3) A 100-pA sample containing anti-hCG, either an un-

known or a standard solution, was added to each well and

incubated 1 h at 37#{176}C.(4) A solution of goat anti-mouse

antibody conjugated to horseradish peroxidase (HRP; con-

jugate-GAM-HRP, BioRad, Richmond, CA) was added to each

well (100 p.1/well) and incubated 1 h at 37#{176}C.The plate

was thoroughly washed after each of steps 1-4 outlined above

(3 cycles of 200 p.1 0.04% Tween 20/well). (5) A peroxide

substrate solution (2,2 ‘-azino-di-[3-ethyl-benzthiazoline-6-

sulphonic acid] and hydrogen peroxide, ABTS Peroxidase

developer solution, BioRad) was added to each well (100

p.1/well). The optical density in each well at 405 nm was

monitored with a microplate reader for 10 mm after ad-

dition of the substrate. The detection limit for an undiluted

sample was 0.1-1 ng/ml, but the detection limit for a mu-

cus sample was -10 ng/ml because mucus samples re-

moved from the vagina were so small that they had to be

diluted 50-100 times for performance of the assay.

Anti-hCG speqflc activity. Antibody concentrations in

PBS were determined by both ELISA and total protein assay.

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‘-s--- Ovary

Cervix

Intravaginal Ring

CONTROLLED VAGINAL DELIVERY OF ANTIBODIES 135

Since the total protein assay gives the total mass of antibody

and the ELISA measures the mass of intact antibody still

capable of binding antigen, specific activity was defined as

the mass of antibody determined by ELISA per mass of an-

tibody determined by total protein assay.

Release of Antibodies from Polymers into the

Vaginal Secretions

To study the long-term delivery of antibodies directly into

the vagina, we fabricated small polymer devices and in-

serted them into the vaginas of mice. The performance of

the device was monitored by determining the concentra-

tion of released agents in mucus samples withdrawn from

the vagina.

In preliminary experiments we tried a number of device

designs including rings, discs, and thin fiber meshes of dif-

ferent sizes. The vaginal opening was gently stretched with

stainless steel forceps to allow easy entry of the mntravaginal

device to be tested. However, we found that during groom-

ing, unrestrained mice could easily remove any type of vag-

inal insert, because mice have extremely flexible spines and

long tongues and teeth. Therefore, in group A animals, we

used a stiff Elizabethan-style collar to prevent the removal

of the device during normal grooming. A flexible polypro-pylene disk (52 mm o.d., 15 mm i.d., 0.5 mm thick) was

made with a single radial cut. On the edge of the inner

circle, a small-diameter tube (2.2 mm) cut lengthwise was

attached to the inner circle to protect the skin of the neck.

The disk was placed on an anesthetized mouse and over-

lapped at the radial cut to form a cone-shaped collar. The

collar was fixed by a single staple through the overlapped

disk. The collars of the mice reduced the animals’ flexibility

and ability to groom, but still allowed for normal eating

and drinking. If a ring was expelled from the vagina during

an experiment, the animal was not considered in any sub-

sequent analysis.

Alternatively, to keep the ring in place for longer du-

rations and to permit the animals full freedom of move-

ment, the devices were inserted and held in place with a

single suture through the vaginal wall (Fig. 1). Each animal

was anesthetized with methoxyflurane (Metafane, Pitman-

Moore, Inc., Washington Crossing, NJ). The abdomen was

shaved and the peritoneum exposed with a 1-cm midline

incision. A small amount of excess fat was removed from

the right uterine horn and cervix. Ten centimeters of non-

absorbable nylon suture (Braunamid, B. Braun Melsungen

AG, Germany) was firmly attached to the EVAc ring (4.5

mm o.d., 1 mm id., 1-1.6 mm thick) containing FITC-BSA,

anti-hCG, or IgG intended for implantation. The suture was

then threaded through a hypodermic needle (22 gauge).

The needle was inserted through the external vaginal open-

ing until it penetrated the vaginal wall near the cervix, and

the suture was pulled through the wall into the abdominal

opening; the needle was then removed. A smaller second

ring (3 mm o.d., 0.5 mm id., 1.5 mm thick) of pure EVAc

AnchoringRing

Vagina

FIG. 1. Position of vaginal rings in the mouse reproductive tract. The

EVAc/protein ring was retained in the vagina by a single suture secured

to another small ring of EVAc. This diagram reflects the actual size of the

rings used in group C and D animals. Dimensions of the rings are providedin Table 1.

was secured to the peritoneal end of the suture to anchor

the ring containing the test agents securely in the vagina.

Excess suture material was trimmed from both ends, and

the abdominal incision was then closed with three to five

7.5-mm stainless steel clips. The steel clips were removed

7-10 days after surgery. Once the ring was stitched in place,

the behavior of the mice and the vaginal secretions did not

reveal any signs of irritation.

Mucus sample preparation and analysis. At various

times after insertion of the polymer ring, each animal was

anesthetized, and a mucus sample was obtained by intra-

vaginal lavage with 20 pA of PBS. Rings were left in place

during the lavage procedure. To obtain enough volume for

ELISA, the mucus samples were then diluted 100 times in

PBS and mixed well by repeated pipetting of the diluted

solution. The diluted mucus sample was added directly to

the wells of the pretreated ELISA plate and analyzed for

anti-hCG as described above.

Mucus samples with FITC-BSA were analyzed directly af-

ter the lavage procedure with no further sample prepara-

tion. The concentration of FITC-BSA was determined by

drawing a small amount (5-20 p.1) of lavage solution into

a flat glass capillary tube (50 x 3 x 0.3 mm, Vitro Dynamics,

Rockaway, NJ) and placing the tube in the beam of an

inverted epifluorescence microscope (Diaphot; Nikon, Tokyo,

Japan). FITC-BSA concentration was quantified by compar-

ing unknowns to standards prepared identically in PBS. The

microscope was equipped with a video camera (NC-70, Dage-

MTI, Michigan City, IN) interfaced with a computer (386/

20e, Compaq, Houston, TX) and image processing hard-

ware and software (Data Translation, Marlborough, MA). The

detection limit for FITC-BSA in this assay system was 0.1

mg/mi of lavage fluid.

Antibody localization by immunofluorescence and im-

munohistochemistry. Polymer rings containing rabbit an-

tibodies and fluorescently labeled mouse antibodies were

inserted into the vaginas of mice. At various intervals fol-

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FIG. 2. Controlled release of BSA and anti-hCG from EVAc polymersinto buffered water. (a) Release of FITC-BSA (white squares) and anti-hCG

(white circles) from EVAc polymer vaginal rings into well-stirred buffer water.For BSA, the cumulative mass released from an EVAc matrix (n = 3, 15

mg total matrix mass, 5 mg BSA) into 5 ml of 0.2 phosphate-buffered waterat 37CC is plotted versus time. FITC-BSA was detected by quantifying flu-orescence intensity in a flat glass capillary tube. For anti-hCG, the cumu-lative mass of anti-hCG released from a coated EVAc matrix (n = 3, 12 mgtotal matrix mass) into 1 ml of 0.2 M phosphate-buffered water at 37’C isplotted versus time. Anti-hCG was detected by ELISA. (b) Release (white

circles) and specific activity (white triangles) of anti-hCG from EVAc slabs.In a separate experiment using larger polymer slabs instead of rings, themass of the antibody released from each of three slabs (16 mg total matrixmass, 5.5 g FicoIl, 140 �g anti-hCG, 1 x 4 x 6 mm) was determined bytotal protein assay and ELISA. Specific antibody activity-mass of antibodydetermined by ELISA divided by mass determined by total protein assayis shown as a function of time (not cumulative). In all cases, error barsindicate standard deviations.

136 RADOMSKY ET AL.

lowing insertion, animals were killed under anesthesia by

cervical dislocation. The reproductive tract was dissected,

and individual organs (vagina, cervix, and uterine horns)

were quickly frozen by immersion in liquid nitrogen. Tis-

sues were stored in freezer vials (Nunc Cryotubes, In-

termed) in a liquid nitrogen freezer until sectioning. A small

piece of tissue was immobilized in tissue freezing medium

(Triangle Biomedical Sciences, Durham, NC) at -20#{176}C.Cross

sections (10 p.m thick) were cut from each tissue segment

on a cryomicrotome (Microm, Heidelberg, Germany),

mounted on glass slides pretreated to increase tissue ad-

herence (Vectabond, Vector Laboratories, Burlingame, CA),

and photographed immediately with an epifluorescence

microscope. The same sections were immediately treated

to permit detection of rabbit antibody by immunohisto-

chemical localization (Vectastamn elite ABC kit, Vector Lab-

oratories). A biotinylated anti-rabbit antibody was added to

the tissue section, and incubation followed with an avidin

and biotinylated HRP macromolecular complex. The sub-

strate (3,3’-diaminobenzidine, Vector Laboratories), which

precipitates a black substance in the presence of HRP, was

added to the tissue section.

RESULTS

Both FITC-BSA and anti-hCG were released continuously

from small EVAc rings for 30 days when the rings were

immersed in PBS (Fig. 2a). Fifty percent of the FITC-BSA

and 100% of the anti-hCG was released over this period.

In a separate experiment, anti-hCG release from a larger

polymeric device was detected by both total protein assay

and ELISA; the specific activity of the antibody (i.e., the frac-

tion of released molecules capable of binding antigen) was

determined as a function of time. Although most of the anti-

hCG was released from polymer matrix slabs in the first 10

days, a significant fraction of the antibody released from the

polymeric device was still capable of binding to hCG even

after 30 days of incubation (Fig. 2b).

To demonstrate the feasibility of controlled release di-

rectly into the vagina, polymer devices containing BSA were

inserted in mice restrained with collars (group A, Table 1)

or inserted with a single retaining suture (group B). Intra-

vaginal polymers in group A animals were frequently ex-

pelled: 10 days after insertion only 5 of 12 collared animals

retained the polymeric device, and only 2 of 12 animals

retained the device to Day 16. Two animals died during the

experiment (one each on Days 3 and 10). Use of a single

retaining suture, but no collar, dramatically reduced the in-

cidence of expulsion. One group B animal removed the

suture holding the device in place and expelled the device

from the vagina on Day 5; all other polymers were retained

for the entire experimental period. In both groups, the flu-

orescence intensity in mucus samples obtained by lavage

followed a strikingly similar pattern (Fig. 3a), suggesting

that neither the collar restraint nor the single suture influ-

I I I I I

5 10 15 20 25 3 0

enced the performance of the polymer devices in the va-

gina.

Polymer rings containing mouse anti-hCG were inserted

into the vaginas of 6 mice and secured with a single suture

(group C, Table 1). To provide a more prolonged release

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5 10 15 20 25 30

Time (days)


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FIG. 3. Controlled release of BSA and anti-hCG from EVAc polymers

into mouse vaginas. The composition of the polymers used in each ex-perimental group is provided in Table 1. Top panel: Intravaginal concen-tration of FITC-BSA following release from an inserted polymeric device

(groups A and B). The concentration of FITC-BSA was determined by quan-tifying fluorescence intensity in lavage fluid collected into a flat glass cap-

illary tube. Each point represents the mean of 2-12 animals (group A, whitesquares) or 6 animals (group B, white circles); error bars indicate standarddeviation. Bottom panel: Intravaginal concentration of antibody followingrelease from an inserted polymeric device (group C). The concentration ofanti-hCG was determined by ELISA and is shown as a function of time.

Each point represents the mean of 6 animals; error bars indicate standard

deviation.

of the incorporated antibody, the ring was coated with a

layer of pure EVAc with 4 small pinholes. The polymers

stayed in place in all of the 6 animals for the 30-day du-

ration of the experiment. The concentration of anti-hCG in

the mucus secretions was readily detected for the entire 30

days (Fig. 3b). In control animals with rings containing no

anti-hCG, none of this antibody was ever detected in the

mucus samples (n = 3, data not shown).

To determine antibody distribution in the mouse repro-

ductive tract following release, rings containing both TRITC-

mouse IgG and rabbit IgG were inserted into the vaginas

of 8 mice and each was retained with a single suture (group

D, Table 1). Animals were killed after a fixed time (1-9

days), and TRITC-mouse IgG and rabbit IgG were detected

in tissue sections by epifluorescence microscopy and im-

munohistochemistry, respectively. TRITC-mouse IgG and

rabbit IgG had identical patterns of distribution in the tis-

sue sections (Fig. 4): antibody was detected within the lu-

men of the vagina, but not within the uterine horns. Also

within the vagina, the antibody was restricted to the lumen

and did not appear to penetrate the epithelial cell layer.

Control photographs, taken of tissue sections from mice with

no device and with a device containing no antibody, re-

vealed no epifluorescence or immunohistochemical signal.

Figure 5 summarizes the antibody distribution in the tis-

sues studied at 1, 3, 6, and 9 days after insertion of the

polymeric ring containing antibodies.

Animals with inserted polymeric devices showed no vis-

ible weight loss and remained in good health. The animals

remained physically active throughout the entire experi-

ment. At necropsy, the tissue surrounding the single stitch

through the vaginal wall showed little or no signs of scar

tissue or bleeding.

DISCUSSION

Polymeric devices of EVAc can release biologically active

compounds with a range of physicochemical properties at

reproducible rates for extended periods of time. Impor-

tantly, a 10-mg EVAc device can release biological macro-

molecules, like proteins and polysaccharides, at rates rang-

ing from ng/day to mg/day [7-10]. EVAc is biocompatible

[11] and has been approved for human use in the eye [12]

and in the uterus [13]. EVAc implants for treatment of car-

diovascular disease [14], immunization [15], insulin replace-

ment in diabetics [16], and treatment of chronic brain dis-

eases [9, 17, 18] have been studied in appropriate animal

models.

Proteins are released from EVAc polymers by diffusion

through the water-filled pore network that is formed as the

dispersed protein particles dissolve [8, 19]. To create a con-

nected network of pores, which permit diffusion in the

polymer device, the total loading of particles in the poly-

mer must be greater than -35% [8]. For polymers of the

size studied here, these high loadings produce release rates

of approximately 0.1-1.0 mg total protein/day (see, for ex-

ample, the release of BSA in Fig. 2a). To reduce the rate of

anti-hCG release, we used methods similar to those de-

scribed previously [9, 20] for the release of microgram

quantities of growth factors. Solid particles containing an-

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138 RADOMSKY ET AL.

3 days following implantation10 j�t.m cross sections

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FIG. 4. Local distribution of antibody following release from a poly-meric device containing rhodamine-labeled mouse IgG and unlabeled rab-

bit IgG (group 0). Cross sections (10 �m) were cut from the vagina (left)and uterine horn (right) with a cryomicrotome. Untreated sections were ob-served with phase contrast optics (top panels) and epifluorescence (middlepanels). Sections were further treated for immunohistochemical detection

of rabbit lgG and observed by light microscopy (bottom panels).

Uterus

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+++

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Treated

Control

0 �. .�

Time (days)

FIG. 5. Distribution of antibodies within tissues of the reproductive tract

(group D). A qualitative measure of signal intensity, presence of either flu-

orescence or a black precipitate representing level of antibody, was deter-mined in 10-�m-thick frozen tissue sections at various times after insertionof an antibody-loaded vaginal polymeric device. Open symbols (white squares

and circles) indicate animals treated with antibody-containing vaginal rings,and closed symbols (black squares and circles) indicate animals treated with

control polymer rings containing no antibody. All observations were madeat either 1, 3, 6, or 9 days following insertion of the polymer device. In thefigure, some values were offset slightly in time to permit visualization ofall the data. Key: (++++) strong, localized signal; (+++) average, local-ized signal; (++) weak, localized signal; (+/0) weak, diffuse signal or nosignal.


tibody and Ficoll, an inert polysaccharide, were formed priorto fabrication of the polymer matrix. In preliminary studies

using polymers prepared from solid particles of IgG andFicoll, we determined that the rates of IgG and Ficoll re-

lease were identical [21]. The addition of other agents, like

polysaccharides, may also stabilize the proteins within the

polymer by preventing the aggregation that normally oc-

curs in the presence of small quantities of water [22]. In

this case, anti-hCG retained its antigen-binding capability

after release from the polymer (Fig. 2b). Of course, the ELISA

technique used to quantitate antigen binding does not ruleout the possibility of antibody aggregation. Since it could

affect function of the released antibody, aggregation will be

more completely examined in future experiments.

We designed intravaginal rings to deliver antibodies di-

rectly to the vaginas of mice. Torus-shaped vaginal rings,

intermediate in size between a diaphragm and cervical cap,

have been used to deliver contraceptive steroids in humans__________ _____ _____ [23, 24]. In the present study, we attempted to duplicate the

geometry of vaginal rings considered acceptable for human

use [25] at a reduced scale appropriate for insertion into

female mice. Biologically active antibody molecules were

detected in the vaginas of mice for 30 days following in-

sertion of an antibody-loaded polymer ring. The antibody

2? concentration reached a maximum 1-2 days after insertionof the polymeric device and decreased -=10-fold over the

remainder of the experiment (Fig. 2b); over the same pe-

nod the release rate from the device, as measured in buff-

ered saline, also decreased, as indicated by the greatly re-

Cl) _____ _____ duced slope at 30 days (Fig. la). This suggests that devices

releasing antibody at a more constant rate will provide nearly

constant mucus levels of protective antibodies. In future

Cl) studies involving larger animals, and therefore larger poly-

mer rings, it may be possible to achieve nearly constant

antibody levels in the vagina for an extended period. For

topically applied antibodies, the degree of control over re-

lease rate that will be required for a safe and effective de-

vice is not yet known. However, since the titer (or concen-

tration) of specific antibodies in the serum of immunized

___ animals varies considerably, we anticipate that the working

range for vaginally delivered antibodies may also be quite

large.

After release from the polymeric device, antibodies were

found in large quantities throughout the lumen of the va-gina but were not detectable in other tissues of the repro-

ductive tract (Fig. 4). Since mucus normally flows from the

uterus out through the introitus of the vagina [26], mucus

flow may prevent vaginally delivered antibody from as-

cending into the uterus through the cervix. We have pre-viously shown that antibody molecules can diffuse -1-2

mm in a day through unstirred human midcycle cervical

mucus [27]. Since diffusion is the slowest possible mecha-

nism for antibody movement in mucus, there appears to be

sufficient time for antibody molecules to penetrate the rel-

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140 RADOMSKY ET AL.

atively “unstirred” mucus layer and reach the underlying

epithelia cell layer.

As determined by epifluorescence microscopy and im-

munohistochemistry, mouse IgG and rabbit IgG were not

absorbed across the epithelia in the vaginas of the mice.

Many compounds do cross the vaginal epithelia [28]: ste-

roids in humans [24], fluorescently labeled HRP and equine

and bovine proteins in mice [29], and bovine antibody in

baboons [30] have been shown to cross the stroma of the

vagina and cervix. The transport of peptides and proteins

across other epithelial tissues has also been demonstrated

[31-34]. In our case, mouse and rabbit antibodies may not

have been detected in the vaginal epithelia because if only

a small fraction (-1%) of antibodies present in the vagina

are absorbed, the concentration present in the tissue will

be lower than the limit of detection of the assay system.

Polymeric devices of EVAc can provide a continuous, long-

term supply of biologically active antibodies. We have ap-

plied that technology to achieve high levels of antibody well

distributed on a mucosal surface that is frequently in need

of protection against infection and unwanted pregnancy.

ACKNOWLEDGMENTS

We thank Timothy E. Hoen for his expert surgical assistance and animal care,

and Jong Han Park and Melody A. Swartz for technical assistance.

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