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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50- +4+ + +
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9-0U)
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++10 15 20 25
Time (day)30
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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100-
1
1
1
5 10 15 20 25 30
Time (days)
CONTROLLED VAGINAL DELIVERY OF ANTIBODIES 137
�10-
I()
0.1
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
I 50O�tm I
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Vagina Uterine Horn
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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
+++
++
+10 a� a. rr� ____Cervix
++++
+++
++
+10
+++
++
+/0
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.
CONTROLLED VAGINAL DELIVERY OF ANTIBODIES 139
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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