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em đang cần tìm gấp tài liệu về các mã bộ ba của Valine và tARN-valine. mã bộ 3 nào được ưu tiên hơn và tRNA-valine nào phổ biến. ở EUKARYOTE NHÉ.THANHK
 
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Microbial resolution of 2-methylbutyric acid and its application to several chiral flavour compounds
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Effect of Size, Surface Charge, and Hydrophobicity
on the Translocation of Polystyrene Microspheres
Through Gastrointestinal Mucin
DANIEL A. NORRIS, PATRICK J. SINKO
College of Pharmacy, Rutgers, The State University of New Jersey, P.O. Box 789, Frelinghuysen Road,
Piscataway, New Jersey 08855
Received 27 March 1996; accepted 13 June 1996
ABSTRACT: Microspheres (MS) have been proposed for use as oral vaccine delivery
vehicles (VDV); however, due to poor and variable absorption their clinical utility is
limited. The effects of size, z-potential, and surface hydrophobicity on the translocation
(PT ) permeabilities of polystyrene (PS) MS with varying surface functional groups
(amidine, carboxyl, carboxylate-modified [CML], and sulfate) were determined through
gastrointestinal (GI) mucin. PT were determined, under steady-state conditions, using
a modified Ussing-type diffusion chamber and a mucin packet developed for use with
the Transwell-Snapwell system. PT followed the Stokes–Einstein relationship, demonstrating
the limited ability of larger MS (œ0.5 mm) to diffuse through the mucin layer.
PT also varied according to the surface characteristics. Even though the z-potential did
not correlate with the transport of MS through mucin, surface ionization appears to
be important in MS translocation. The PS–amidine MS were significantly less hydrophobic
and had a higher PT than that of the other MS, suggesting that hydrophobicity
is also a significant factor in MS transport through mucin. While these results
suggest that mucin may be a significant barrier to the oral absorption of vaccines and
VDVs in vivo, the rate-limiting barrier for the absorption of MS will be the intestinal
mucosa. q 1997 John Wiley & Sons, Inc. J Appl Polym Sci 63: 1481–1492, 1997
Key words: mucin; polystyrene microspheres; hydrophobicity; surface modification;
intestinal transport
INTRODUCTION tential as vaccine delivery vehicles (VDVs).2–6
Ideally, VDVs must protect the immunogen from
The weak immunogenicity, low intestinal per- GI inactivation, promote transport through biomeability,
and high presystemic clearance of logical barriers such as mucin and the intestinal
vaccines from the gastrointestinal (GI) tract re- mucosa, augment the immune response, and conquires
the use of prohibitively large and repetitive trol the kinetics of vaccine presentation to antigen
doses to elicit even a modest immune response1 presenting cells (APCs) thereby promoting an efmaking
mass immunization programs economi- fective immune response.
cally unfeasible. Controlled-release polymeric mi- The intestine not only allows for the absorption
crospheres (MS) have exhibited considerable po- of nutrients, electrolytes, and fluid, but also acts
as a barrier to prevent the free mixing of toxic
lumenal contents with the underlying interstitial
Correspondence to: P. J. Sinko and vascular fluids, thus preventing the absorp- Contract grant sponsors: Associated Institutes of Materials
Science; Corning-CoStar Corp. tion of potentially harmful substances. For VDVs
q 1997 John Wiley & Sons, Inc. CCC 0021-8995/97/111481-12 to be absorbed, they must pass through two barri-
1481
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1482 NORRIS AND SINKO
ers that are in series: the mucosa and the mucus
gel layer. The mucosal tissue acts as a barrier
since it restricts the penetration of vaccines, even
though it is an incomplete barrier to macromolecules
and particulate materials. Vaccine components
may enter the intestinal mucosa at several
possible sites, including the tips of villi where enterocytes
are extruded, across or between enterocytes,
or by specialized epithelial tissue composed
of membraneous (M) cells that cover lymphoid
aggregates known as Peyer’s Patches.7,8
Intestinal mucus, a high molecular weight glycoprotein
secretion, covers the mucosa with a continuous
adherent blanket. The primary function
of the mucus layer is to protect the gastrointestinal
mucosa from potentially harmful bacteria,
pathogens, or chemicals.9–11 Several investigators
have shown that mucin significantly decreases
the diffusion of small and large compounds such
as bovine serum albumin (BSA),12 lysozyme,12
tertiary amines, quaternary ammonium compounds,
13 and others.14 The thickness of the mucin
gel layer varies regionally throughout the GI
tract with thickness decreasing distally from 50
to 500 mm (Ref. 10) in the stomach to 16–150
mm (Refs. 15–17) in the colon. The primary gelforming
component of the mucus layer is mucin,
a highly heterogeneous glycoprotein, composed of
a protein backbone to which carbohydrate side
chains of various lengths are O-linked to serine Figure 1 Modifications to Snapwell ring and scheor
threonine amino acid residues11 which form a matic of Transwell-Snapwell diffusion chambers. The
mucin subunit. Several subunits joined by disul- 0.4 mm filter supplied with Snapwell rings is removed
fide bonds form mucin molecules as large as 2 and mucin is placed between two polycarbonate filters
of appropriate size (see text). Snapwell fits within the 1 106 D. The concentration of mucin in the mucus
gel layer also varies regionally. The concentration diffusion chamber and is sealed in place with rubber
of mucin is reported to be ‚ 50, ‚ 40, ‚ 30, and O-rings.
‚ 20 mg/mL in the stomach, duodenum, jejunumileum,
and colon, respectively.11,17,18
In this report, an Ussing-type diffusion cham- 0.01, 0.1, 0.2, 0.5, 1.0, and 2.0 mm yellow-green/
ber system was developed to investigate the bar- carboxylate-modified [CML], and 0.5 mm yellowrier
properties of mucin using model VDVs com- green/sulfate surface) were purchased from
posed of polystyrene (PS). The permeability of PS Molecular Probes (Eugene, OR). Nucleopore polyMS
of varying size and surface properties were carbonate filters were purchased from Corning-
determined and correlations were established Costar Corp. (Cambridge, MA). All other materidemonstrating
that the mucus layer may be a sig- als were purchased from either Sigma Chemical
nificant barrier to the intestinal absorption of (St. Louis, MO) or Fisher Scientific (Springfield,
VDVs. NJ) and used as received.
EXPERIMENTAL Modifications to Ussing-type Diffusion Chambers
Ussing-type diffusion chambers (Corning-Costar
Materials Corp.) were used with modified Snapwell rings to
PS MS of various sizes (0.5 mm yellow-green/ maintain a vertical layer of mucin (Fig. 1). The
amidine surface, 1.0 mm red/carboxyl surface, original filters were removed with a scalpel and
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TRANSLOCATION OF POLYSTYRENE MICROSPHERES 1483
replaced with Nucleopore polycarbonate filters of Table I Polystyrene Microsphere
varying pore sizes using a cyanoacrylate adhesive. Concentrations in Donor Chamber
A polystyrene plastic ring approximately 0.38mm
thick was then glued onto the Snapwell ring. The Particle Donor
modified Snapwell was filled with 50 Microsphere Diameter Concentration mL of recon-
Surfacea (mm) (particles/mL) stituted mucin (20, 30, or 50 mg/mL) and then
sealed with a second filter. Sulfate 1.0 5.0 1 107
Mucin was prepared by reconstituting gastric Amidine 0.5 1.0 1 108
mucin (porcine, crude type II, Sigma Chemical Carboxyl 1.0 5.0 1 107
Co.). Mucin was mixed with sodium phosphate/ CML 0.01 1.0 1 1013
sodium carbonate buffer (pH 6.5) in a mortar and CML 0.1 2.25 1 1010
triturated until completely wetted. The gel was CML 0.2 1.8 1 109
CML 0.5 6.5 1 108 then diluted to the appropriate concentration
CML 1.0 5.0 1 107 (20–50 mg/mL) and mixed well. Mucins from all
CML 2.0 2.5 1 106 mammalian species, including human and porcine
mucins, share considerable structural simi- a Amidine  amide group; CML  carboxylate-modified.
larities.9–11 Included in these shared properties
are the molecular weight and rheological properties
for native and reconstituted, purified mucins The Ability of the Mucin Packet to Maintain an
from gastric and small intestinal regions.19–21
Ionic (pH) Gradient
The donor chamber was filled with buffer at pH
6.5 and the receptor chamber with buffer at pH
Diffusion Chamber System Validation 7.4 with the mucin layer mounted between the
two half-chambers. The pH was then measured
The barrier properties of the mucin packet were on both sides of the diffusion chamber for 90 min
validated prior to the initiation of the uptake and using a Ag/AgCl electrode.
translocation experiments in order to confirm the
integrity of the system during the time course of Molecular Weight Distribution of Mucin the experiment. Three studies were performed:
the loss of mucin from the packet during the ex- Size-exclusion chromatography was used to charperiment,
the ability of the mucin packet to main- acterize the molecular weight distribution of retain
an ionic (pH) gradient, and the binding of constituted mucin. A low-pressure glass chromathe
MS to the chamber components. tography column (1.5 cm i.d.) was filled to a height
of 50 cm with Sepharose 2B size exclusion media.
One milliliter of mucin (1 mg/mL) was added to
Loss of Mucin from the Packet During the the column and eluted with a mobile phase con-
Experiment sisting of 0.2% NaCl and 0.02% sodium azide as
a preservative. The mobile phase was pumped at
The modified Snapwell rings were placed within a flow rate of 0.2 mL/min using a peristaltic pump
diffusion chambers and both sides (donor and and 2 mL fractions were collected and assayed for
receptor) of the chamber were filled with 7 mL mucin. Chromatograms were generated to deter-
0.01M sodium phosphate/sodium carbonate mine the molecular weight distributions for two
buffer at pH 6.5. The chambers were placed in mucin concentrations (50 and 30 mg/mL) at pH
prewarmed heating blocks and the gas lift sys- 6.5 and 2.5.
tem connected. The gas lift system continuously
bubbles 95% O2 : 5%CO2 into each half-chamber The Binding of MS to the Chamber Components to provide mixing during the experiment. After
90 min at 377C, the concentration of mucin The binding of MS to the acrylic chamber was
within the packet was determined using an es- determined by filling both sides of the diffusion
tablished method based on a periodic acid/ Schiff chambers with the MS suspension. The concenbase
reaction.22 The loss of mucin was evaluated trations used are given in Table I. Smaller MS are
using the filters with pore sizes of 1, 2, 3, 5, 8, less fluorescent than are larger MS and therefore
and 10 mm and physiologically relevant mucin require higher concentrations to produce detectconcentrations
20, 30, and 50 mg/mL.17 able levels during analysis. Samples were re-
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1484 NORRIS AND SINKO
moved at 0, 5, 10, 15, 30, and 60 min and assayed phosphate/sodium carbonate buffer (pH 6.5) and
using a Shimadzu RF-5000 spectroflourophoto- measuring the particle size for 90 min at 377C.
meter. Yellow/green fluorescent MS were assayed No agglomeration, as indicated by an increase in
at excitation l  490 nm and emission l  510 nm. effective particle size with time, was observed for
Red fluorescent MS were assayed at excitation l any MS during the experiment.
 585 nm and emission at l  610 nm.
To correct for MS binding to the receptor cham- Hydrophobic Interaction Chromatography (HIC)
ber, an empirical correction factor was deter-
mined. MS of known concentration, equal to the The relative hydrophobicity of PS MS was deterexpected
amount transported, were added to the mined using hydrophobic interaction chromatogreceptor
chamber every 2 min. Samples were col- raphy based on a previously described method.23
lected from the receptor chamber and assayed at Briefly, columns were prepared using Pasteur pipettes
(5 3
4 in. L 1 1
4 30, 60, 90, and 120 min. These concentrations in. i.d.) containing a small
were then compared to the amount of MS added amount of glass wool with ethyl, pentyl, hexyl, or
to the chamber. The empirical correction factor octyl agarose beads by gravity feed to a column
was calculated by the following general equation: height of 3 cm (1.2 mL of agarose suspension).
The columns were then washed with at least 10
mL of 0.6M NaCl buffered to pH 7.4 with 0.001M
ECF(MS, time) 
Ctheoretical
Cexperimental
(1) sodium phosphate to remove the residual suspending
solvents. One hundred microliters of miwhere
ECF is the empirical correction factor crosphere suspension (0.02% solids) were added
which is a function of both the MS and the sample to the top of the column and eluted by gravity flow
time, C with 5 mL 0.6M NaCl followed immediately by 5 theoretical is the concentration of MS added to
the chamber, and C mL of 0.1% Triton X-100. The eluate was collected experimental is the experimentally
determined MS concentration at each time point. and assayed for MS. HIC was performed eight
The ECF was multiplied by the experimentally times for each surface.
determined transport concentrations to deter-
mine the actual amount of MS which transported Contact Angle
within each time point.
Two milligrams of MS, suspended in water, were
cast onto a microscope slide and evaporated to
Microsphere Surface Characterization dryness. The MS were scraped off the slide, collected,
and dissolved in 1 mL of xylenes (mixture
Zeta Potential of o-,m-, and p-xylene). The polymer solution was
The z-potential was determined at pH values then cast onto a coverslip and the solvent evaporanging
between 2.0 and 10.0 using a BI-Zeta Plus rated to dryness to obtain a smooth film. The con(
Brookhaven Instrument Corp.). MS were diluted tact angle was then determined using distilled
to a concentration of 0.002% solids in 0.001M so- water and a Rame-Hart NRL Model 100 goniomedium
phosphate/sodium carbonate buffer; the pH ter. Ten measurements were made on each cast
was adjusted using 0.01N HCl or 0.01N sodium film.
hydroxide.
Microsphere Transport
Confirmation of Particle Size
Snapwell rings were prepared as described to conThe
particle-size range was confirmed using a BI- tain mucin at 30 mg/mL. The Snapwell rings were
Zeta Plus instrument. MS suspensions (2% sol- inserted into the diffusion chambers which were
ids) were diluted 2 mL to 3 mL in 0.01M sodium then placed in heating blocks prewarmed to 377C.
phosphate/sodium carbonate buffer (pH 6.5). The MS suspensions were added to the donor side and
particle size was measured for 2 min, five times blank buffer was added to the receptor side. The
for each MS. All cuvettes and solutions were donor concentrations used are shown in Table I.
rinsed and/or filtered to remove dust prior to mea- Diffusion chambers were removed serially every
surement. The agglomeration of MS was deter- 30 min for 2 h. The mucin layer was completely
mined by diluting MS suspensions (2% solids) to removed and diluted to 3mL in sodium phosphate
the concentrations shown in Table I using sodium buffer. Since mucin did not fluoresce at the wave-
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TRANSLOCATION OF POLYSTYRENE MICROSPHERES 1485
Statistical Methods
Statistical analyses were performed using BMDP
New System (BMDP Statistical Software, Los
Angeles, CA). All values are reported as mean
{ SEM. Differences are considered to be significant
at a  0.05. A least significant difference test
was performed to determine specific differences
between means on any ANOVA with a › 0.05.
RESULTS
Diffusion Chamber System Validation
The results of the validation studies are shown in
Figures 2–4. The three concentrations of mucin
evaluated in these studies (20, 30, and 50 mg/mL)
correspond to the physiological concentrations of
mucin found in the colon, jejunum-ileum, and
stomach, respectively.11,17,18 Figure 2 The percent mucin remaining within the Filters with pore
packets after 90 min, 377C, pH 6.5. Mucin packets were sizes less than 5 mm retained at least 85% of the
prepared containing (m) 50 mg/mL mucin, (.) 30mg/ mucin for 90 min for all three initial mucin conmL
mucin, and (l) 20 mg/mL mucin placed between centrations (Fig. 2). When the pore size of the
polycarbonate filters of varying pore size. filter was greater than 5 mm, a significant amount
lengths used, corrections for background fluorescence
were not necessary. The donor chamber was
removed and refilled with a fresh MS suspension
to maintain a constant donor chamber concentration
every 30 min. The samples were assayed as
previously described.
The translocation permeability, PT, was calculated
using the equation
PT 
VR
AC0
dCR
dt
(2)
where dCR/dt is the slope of the regression line
of the receptor concentration vs. time plot; A, the
surface area of the mucin layer (1.03 cm2); C0, the
donor concentration of MS; and VR, the volume of
the receptor chamber (7 mL).
Figure 3 Maintenance of a barrier to pH by mucin
packet. pH remains constant beginning at 30 min when
Nonlinear Regression Methods H/ diffusion between donor and receptor chambers is
Scientist for Windows (MicroMath Scientific Soft- prohibited by a physical barrier: (m) line A donor side,
line F receptor side). pH changes significantly when ware, Salt Lake City, UT) was used to fit the ex- donor and receptor chambers are separated by a packet
perimental data to the Stokes–Einstein equation containing buffer: (.) line C donor side, line D receptor
by nonlinear regression. PT values were weighted side. pH remains constant when donor and receptor
by 1/SEM. The point at which the second deriva- chambers are separated by a mucin packet: (l) line B
tive of the fitted curve approached zero was used donor side, line E receptor side). (*) indicates slope is
as the size cutoff for MS transport through mucin. significantly different from 0 between 30 and 90 min.
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1486 NORRIS AND SINKO
of mucin diffused out of the packet (Fig. 2). Based
on these results, all subsequent experiments were
conducted with filters possessing pore sizes less
than or equal to 5 mm. A mucin concentration of
30 mg/mL was utilized in order to simulate intestinal
mucin concentrations.
The ability of the mucin layer to maintain an
ionic gradient is shown in Figure 3. The mucin
layer maintained a pH gradient between pH 6.5
and 7.4 better than the control (buffer placed between
the filters) during the time course of the
experiment.
To characterize potential structural changes in
mucin during the time course of the experiment,
size exclusion chromatography (SEC) was performed.
Analysis of mucin composition was performed
prior to and after completing the experiment.
Typical chromatograms are shown in Figure
4. At pH 6.5, the amount of mucin (initial
 20, 30, and 50 mg/mL) retained in the 5 mm
filter insert was approximately 85% (only 50 mg/
mL data shown). The molecular size range of the
retained mucin after the experiment [Fig. 4(b)]
was found to be similar to control chromatograms
[Fig. 4(a)], indicating that mucin loss was not
attributable to a particular molecular weight fraction.
The total mucin lost is consistent with that
reported in Figure 2.
The binding of MS to the diffusion chambers
[Fig. 5(a)] was dependent upon size. Over 90
min, 40–70% of the MS remained suspended in
the diffusion chambers. To properly determine the
steady-state translocation permeability using
Fick’s Law, it was necessary to maintain a constant
donor MS concentration. To overcome this
problem, 4 mL were removed from the donor
chamber and replaced with 4 mL of fresh MS suspension.
As shown in Figure 5(b), using this
method, the concentration of MS in the donor
chamber at 90 min was constant and not signifi- Figure 4 Size exclusion chromatograms for mucin recantly
different from the initial concentrations. constituted at 50 mg/mL: (a) mucin before diffusion experiment; (b) mucin retained within packet after a
diffusion experiment. One milligram of mucin was
added to a 50 cm Sepharose 2B column and eluted with Microsphere Surface Characterization 0.2% NaCl/0.02% sodium azide at a flow rate of 0.2
Zeta ( z)-Potential mL/min. Two milliliter fractions were collected and analyzed
by periodic acid/Schiff base assay.
The zeta (z)-potential is the potential difference
between the charge on a surface and the bulk of
the solution in which it is submerged. The values boxyl, CML, and sulfate) studied and are shown
obtained for z-potential are dependent upon the in Figure 6. The positively charged surface, amidionic
strength and pH of the buffer as well as upon ine, had less negative z-potential values comthe
temperature at which the measurement is pared to the other MS surfaces. The carboxylatetaken.
The pH– z-potential profiles were charac- modified surface exhibited a moderately charged
terized for the four MS surfaces (amidine, car- surface at pH values œ 6 when compared to the
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TRANSLOCATION OF POLYSTYRENE MICROSPHERES 1487
Figure 6 z-Potential of unmodified PS MS (0.002%
solids) diluted in 0.001M sodium chloride/sodium carbonate
buffer (ionic strength  0.007) determined by
laser light scattering using a BI-ZetaPlus instrument.
Electrical field applied and reversed five times: (m) PS–
sulfate; (l) PS–amidine; (j) PS–carboxyl; (.) PS–
CML.
modified surface appeared to have a much lower
pKa (‚ 2.5). A pKa of 6.0 was estimated for the
amidine surface.
Contact Angle
The distilled water contact angle gives an indication
of the hydrophobicity of the cast film. The
results of the contact angle measurements are
shown in Table II and Figure 7. Using this
method, differences in contact angles could not be
discerned. It is hypothesized that, by dissolving
the MS in mixed xylenes before casting the films,
Figure 5 (a) The binding of MS to diffusion chambers Table II Contact Angles and Hydrophobic
over time, pH 6.5, 377C: (m) 0.01 mm; (.) 0.1 mm; (l) Interaction Chromatography Results
0.2 mm; (j) 0.5 mm; (l) 1.0 mm. (b) Donor chamber
concentrations using donor suspension replacement: Polystyrene Contact HIC %
(white) percent of initial concentration at t  0 min, Microsphere Angle Retained,
(black) percent of initial concentration at t  90 min, Surfacea (7) Pentyl Agarose
pH 6.5, 377C.
Sulfate 71.3 { 0.61 85.91 { 6.19
Amidine 70.6 { 1.95 70.42 { 9.17b
other surfaces. Using these pH profiles, it was Carboxyl 73.7 { 0.75 93.66 { 0.34
possible to estimate the approximate pKa value CML 73.5 { 5.02 91.93 { 2.33
for each surface. The carboxyl and sulfate sur- a Amidine  amide group; CML  carboxylate-modified. faces demonstrated a reduction in z-potential at b Indicates statistical significance, P value ›.05 after AN3.5
and 5.0, respectively, while the carboxylate- OVA followed by least significant difference analysis.
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1488 NORRIS AND SINKO
sulfate (85%), carboxyl (93%), and CML MSs
(91%). The HIC results are shown in Figure 7.
Microsphere Translocation
The calculated translocation permeabilities, PT,
for MS of different surfaces with similar size are
shown in Table III and Figure 8. Significant differences
in the calculated PT of the MS were observed.
The PT of the PS–amidine MS was an
order of magnitude greater than was the PT for
the other surfaces (5.36 1 1004 vs. ‚ 3 1 1005
cm/min). The PT for the PS–CML MS was 2.63
1 1005 cm/min. The PT for the PS–sulfate and
PS–carboxyl MS were, respectively, 30 and 110%
higher than the PT for the PS–CML MS but were
Figure 7 Percent MS retained on pentyl-agarose hy- not significantly different due to the variability in
drophobic interaction chromatography columns (5 3 the measurements.
4
1 The diffusion of particles through mucin should 1
4 in.) eluted with 0.6M NaCl followed by 0.1% Triton
X-100. Distilled water contact angle on cast films of MS follow the Stokes–Einstein equation:
dissolved in xylenes, 10 measurements, five on left side
of sessile drops, five on right side. (Black) PS–sulfate;
D 
kT
6hpr
(white) PS–amidine; (gray) PS–carboxyl; (white- (3)
striped) PS–CML; (gray-striped) unformulated PS. (*)
Percent PS–amidine on HIC only significant difference
after ANOVA and least significant difference analysis. where k is the Boltzmann constant; T, absolute
temperature; h, the viscosity of the medium; and
the surface character of the MS was apparently
abolished since only the surface groups of the MS
were functionalized. Therefore, by dissolving the
MS and casting a film, a significantly higher fraction
of unfunctionalized PS was exposed on the
surface of the film. As seen in Figure 7, the cast
films’ hydrophobicities are not significantly different
than the control (nonfunctionalized PS) supporting
this hypothesis.
Hydrophobic Interaction Chromatography (HIC)
Hydrophobic interaction chromatography allows
the hydrophobicity of the MS to be evaluated
without physically modifying the MS. Greater MS
hydrophobicity will increase the percent of MS
retained on the column. Preliminary experiments
determined that the pentyl agarose media was the
most efficient for detecting hydrophobic differences
between the MS (data not shown). The HIC
results run on pentyl agarose media are shown in Figure 8 Translocation permeabilities for PS MS.
Table II. Significant differences in the percent MS Translocation permeabilities calculated by eq. (2) (see
retained were found between the surfaces. The text). Permeabilities were determined through mucin
least retained MS were the amidine (70%). The packets containing mucin at 30 mg/mL and polycarbopercent
of amidine MS retained was also signifi- nate filters with 5 mmpores, pH 6.5 donor and 7.4 recepcantly
different from the percents retained for the tor, 377C.
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TRANSLOCATION OF POLYSTYRENE MICROSPHERES 1489
Table III Translocation Permeabilities
Polystyrene Reported Diameter Actual Diameter
Microsphere Surfacea (mm) (mm) PT (cm/min)
Sulfate 1.0 1.08 3.46 1 1005 { 1.15 1 1005
Amidine 0.5 0.88 5.36 1 1004 { 1.15 1 1005
Carboxyl 1.0 1.12 5.54 1 1005 { 3.83 1 1005
CML 0.01 0.07 2.54 1 1004 { 3.83 1 1005
CML 0.1 0.14 1.11 1 1004 { 6.47 1 1005
CML 0.2 0.27 4.57 1 1005 { 1.62 1 1005
CML 0.5 0.49 7.85 1 1006 { 6.93 1 1006
CML 0.5 0.58 1.06 1 1005 { 3.60 1 1006
CML 1.0 1.07 2.63 1 1005 { 8.31 1 1006
a Amidine  amide group; CML  carboxylate-modified.
r, the radius of the diffusing particle. Since P (per- meability coefficient, P, is directly proportional to
meability) is equal to D/h, where D is the aqueous D, by the equation P  D/h, and the diffusing
diffusion coefficient (cm2/s) and h is the length of particles are spherical, the translocation permethe
diffusion pathway, the Stokes–Einstein equa- ability should also follow the Stokes–Einstein
tion can be rewritten as equation. The translocation permeabilities were
fitted to the Stokes–Einstein equation. This is
P  shown in Figure 9 and demonstrated the limited
kT
6hprh
(4)
ability of MS larger than 0.5 mmto diffuse through
the mucin layer. These results indicate a sharp
The translocation permeabilities (PT ) for five decrease in PT as the MS diameter increases to
different sizes of CML MS were determined. For approximately 0.3 mm. A gradual decrease is then
these studies, all MS had CML surface functional observed as the diameter is increased to 0.5 mm
groups and the size was varied from 0.1 to 1 mm. where no further significant decrease in PT is ob-
The results are shown in Figure 9. Since the per- served as the diameter increases. At MS diameters
greater than 0.5 mm, the second derivative of
the fitted curve approaches zero, which suggests
a size range cutoff for MS larger than 0.5 mm.
Figure 10 shows PT plotted as a function of the
hydrophobicity of the MS. The amidine MS, which
have the highest PT, also have the lowest hydrophobicity.
The sulfate, CML, and carboxyl MS,
which were retained equally on the pentyl agarose
column, also show similar PT.
As shown in Figure 11, the z-potential at ‚ pH
7.0 vs. PT shows that the z-potential may be a
valuable indicator in determining the PT. The
highest PT was associated with the MS that had
the most positive z-potential (PS–amidine). For
the other MS, the PT gradually decreased as the
z-potential became more positive.
Figure 9 Translocation permeability of PS MS as a DISCUSSION
function of particle size. Translocation permeabilities
calculated by eq. (2). Permeabilities were determined
through mucin packets containing mucin at 30 mg/mL It is well established that the major component of
and polycarbonate filters with 5 mmpores, pH 6.5 donor the mucus gel layer coating the intestinal memand
7.4 receptor, 377C. Curve was fitted to eq. (4), branes is mucin, a highly heterogeneous glycoproweighted
by 1/SEM. tein of molecular weight 1–2 1 106 D.10,11 The
/ 8e70$$ut10 01-06-97 00:49:39 polaa W: Poly Applied
1490 NORRIS AND SINKO
indicate that the PT for PEG-4000 through the
intestinal mucosa is 8.58 1 1005 cm/min.30 Based
on the work of Donovan et al., the PT for the larger
PS MS should be no greater than the permeability
for PEG-4000. Therefore, the PS MS would have,
at best, a PT of 8.5 1 1005 cm/min through intestinal
tissue. The PS–CML MS PT through mucin
were fitted to eq. (4) and the PT calculated for a
molecule approximately the size of PEG-4000.
The PT was estimated to be 7.94 1 1004 cm/min,
which is 10 times greater than the PT observed
for PEG-4000 through intestinal tissue. This suggests
that GI mucus may not be the rate-limiting
barrier to the intestinal uptake of microparticles
and that the target size of orally administered
VDVs should be less than 1 mm.
The second factor investigated was surface
charge. For these studies, the size of the MS were
Figure 10 Correlation of translocation permeabilities fixed at 1.0 mm or, for the PS–amidine surface,
in Figure 8 to percent MS retained on pentylagarose which was not commercially available at 1.0
media (Fig. 7): (l) PS–amidine; (j) PS–sulfate; (m) mm, 0.5 mm particles were selected. Based on
PS–CML; (.) PS–carboxyl. the Stokes–Einstein relationship, which demonstrated
that the PT for particles above 0.5 mm was
relatively independent of size, the difference in
viscoelastic and gel-forming properties of purified size would not bias the results. The PS–carboxyl,
mucin are comparable to native mucus gels.17,18 –CML, and –sulfate surfaces are negatively
Intestinal mucus functions as a barrier main- charged MS, while the PS–amidine MS is positaining
a pH difference between the GI lumen and tively charged. The oligosaccharides surrounding
the mucosal surface.19–21 The role of mucus in the mucin protein core contain high levels of five
drug and vaccine absorption from the GI tract has monosaccharides: fucose, galactose, N-acetylganot
been extensively studied. It was the objective
of this investigation to determine the importance
of mucus as a barrier to vaccine and microparticle
absorption. Three fundamental parameters were
studied: microparticle size, surface charge, and
surface hydrophobicity using commercially available
PS MS.
MS with diameters as large as 10 mm are reported
to be taken up into M-cells3 ; however, only
MS less than 5 mm are reported to be transported
out of the M-cell. VDVs between 1 and 10 mm are
readily phagocytosed by APCs such as macrophages24–
28 with an optimal size less than 5 mm.
As shown in Figure 9, the PS–CML MS PT’s followed
the Stokes–Einstein relationship demonstrating
the limited ability of MS larger than 0.5
mm to diffuse through the mucin layer. A similar
phenomenon was demonstrated for the gastrointestinal
absorption of various poly(ethylene glycol)
s (PEGs) by Donovan et al. who showed that
the percent absorption of PEG decreased to less Figure 11 Correlation of translocation permeabilities
than 2% as the molecular weight was increased to in Figure 8 to z-potential (Fig. 6) at pH 7: (l) PS–
1500 and remained constant for larger size PEG amidine; (j) PS–sulfate; (m) PS–CML; (.) PS–carmolecules.
29 Preliminary data in this laboratory boxyl.
/ 8e70$$ut10 01-06-97 00:49:39 polaa W: Poly Applied
TRANSLOCATION OF POLYSTYRENE MICROSPHERES 1491
lactosamine, N-acetylglucosamine, and sialic fusion of MS through mucin since MS that were
acid.11 These are neutral or negatively charged retained more than 85% on HIC were also less
sugars which impart an overall negative charge permeable.
to the mucin molecule. The MS can be ranked The current results suggest that particle size,
according to the PT (PS–CML › PS–sulfate surface ionization, and surface hydrophobicity
› PS–carboxyl ! PS–amidine). For the purpose play an important role in the diffusion of model
of this discussion, the MS are placed into two vaccine microparticles through GI mucus. Using
groups: MS with relatively low PT (PS–CML, PS– a physiologically relevant mucin diffusion system,
sulfate, and PS–carboxyl) and MS with relatively this investigation also demonstrated that mucin
high PT (PS–amidine). The pKa values deter- is expected to be a significant barrier in the oral
mined from Figure 6 indicate that the surface absorption of vaccine microparticles; the mucin
functional groups on PS–CML, PS–sulfate, and barrier, however, will only represent 10% or less
PS–carboxyl MS (low PT ) are almost completely of the total resistance to MS uptake.
ionized (œ99%), while the surface of the PS–
amidine MS (high PT ) is only 9% ionized. While An American Foundation for Pharmaceutical Educait
appears that there is a relationship between tion Fellowship and New Jersey Center for Biomaterithe
surface ionization and PT, further study is als and Medical Devices Summer Fellowship (D.A.N.),
required to quantify these effects. The results the Hoechst Celanese Innovative Research Award
shown in Figure 11 indicate that z-potential may (P.J.S.), the Associated Institutes of Materials Science,
not be a significant factor in determining the PT and Corning-CoStar Corp. are acknowledged for supof
PS MS. On the one hand, since PS MS are porting these studies. The following are also gratefully
significantly more hydrophobic than are other acknowledged: Prof. Joachim Kohn (Department of
MS,28 hydrophobic factors may have overwhelmed Chemistry, Rutgers University) for the use of the goniometer
used for contact angle measurements and Dr. the effects of surface charge in the current study. Glen Leesman (Ribi ImmunoChem Research, Inc.) for
On the other hand, Saitoh, et al.13 showed that performing the particle agglomeration studies.
the charge of the species does not affect the diffusion
of quaternary and tertiary amine compounds
through mucin.
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Prevalence of chromosomal abnormalities in couples with recurrent miscarriage

Hatem Elghezal M.D.a, , , Samir Hidar M.D.b, Soumaya Mougou M.D.a, Hedi Khairi M.D.b and Ali Saâd M.D., Ph.D.a
 aDepartments of Cytogenetics and Reproductive Biology
bGynaecology and Obstetrics, Farhat Hached University Teaching Hospital, Sousse, Tunisia


Received 2 October 2006;
revised 27 November 2006;
accepted 27 November 2006.
Available online 23 February 2007.

Chromosome abnormalities affect 6.93% of Tunisian couples with recurrent miscarriage.

Article Outline

References
Recurrent miscarriage is defined as three or more consecutive pregnancy losses before 24 weeks of gestation (1) and (2). It affects approximately 2% of fertile couples and constitutes an important cause of sterility. The causes are heterogeneous and multiple but are identified only in 30% to 60% of the cases (3).
Parental chromosome abnormalities represent an important etiology of recurrent miscarriage; studies published elsewhere have shown a prevalence of chromosomal anomalies that varies from 2% to 8% of couples who are affected by recurrent miscarriage. However, in many of the reported series, the number of studied couples is insufficient to determine a precise prevalence of these anomalies in the studied populations (3), (4), (5) and (6).
To the best of our knowledge, no published data exist regarding the association of chromosomal aberrations and recurrent miscarriage in North African populations. The aim of our study is to determine the prevalence of chromosomal anomalies in a large series of Tunisian couples with recurrent miscarriage.
A total of 1,400 couples (2,800 patients) who presented to our service from January 1989 to December 2005 for recurrent miscarriage (range of number of miscarriages, 3 to 9) were offered chromosomal analysis. In all cases, karyotypes were performed from peripheral blood lymphocyte culture, and cytogenetic analysis was performed by using R-banding. Fifteen metaphases were systematically studied, and if any mosaicism was suspected, the number of analyzed metaphases was enlarged to 50. Chromosomal abnormalities have been reported in accordance with the current international standard nomenclature (7). Parental karyotyping is part of the recommended systematic investigation of recurrent miscarriage (1) and (2); we therefore did not require the approval of the local institutional review board.
Ninety-seven chromosome anomalies were detected among the 1,400 studied couples (6.93%). This prevalence appears to be in the range of previously reported studies in other populations (3), (4), (5) and (6).
Moreover, there are very large published series that have reported that the frequency of chromosomal abnormalities in the general population is only about 0.5%, and there is a general agreement that this frequency is not influenced by ethnicity (8) and (9).
In our series, women were more frequently affected then men, with a prevalence of 5.21% and 1.71%, respectively (P<.001). No couples presented an abnormal karyotype in both partners (Table 1).

Table 1.
Demographic and anomaly data.
AbortionsCouplesPaternal chromosome anomaliesMaternal chromosome anomaliesTotal of chromosome anomalies, n (%) 3716172845 (6.28) 440232629 (7.21) 515821113 (8.23) 661055 (8.20) ≥763235 (7.94) Totals1,40024 (1.71)73 (5.21)97 (6.93)
Full-size table

Note: All data are n; data in parentheses are percentages.
Elghezal. Cytogenetics of recurrent miscarriage. Fertil Steril 2007.

View Within Article





Women with recurrent miscarriage show a high incidence of X-chromosome aneuploidies.
Of the 32 patients with a detected X-chromosome aneuploidy, 20 were found to have a 45,X/46,XX mosaicism, 8 have 45,X/46,XX/47,XXX mosaicism, 2 have nonmosaic 47,XXX, and 2 have complex mosaicism 45,X/46,XX/47,XXX/48,XXXX. However, the association between X-chromosome aneuploidies and recurrent pregnancy loss is unclear, and loss of X-chromosome copy particularly may involve a diminished ovarian reserve and many anatomical uterine anomalies that explain the occurrence of recurrent miscarriages (10) and (11).
Among 57 balanced chromosomal anomalies, 37 (64.91%) were detected in women. These data can be explained by the high incidence of sterility in males with balanced chromosomal anomaly, as reported in a previous study (12). Indeed, balanced chromosomal anomalies involve meiotic blocking of the spermatogenesis, but ovogenesis usually is conserved and produces gametes with a high risk of presenting unbalanced forms of the chromosomal anomaly (4).
As reported in the literature (4), reciprocal translocations are the most frequent balanced chromosomal anomalies that are detected in couples with recurrent miscarriage in our North African population. Indeed, in our study, reciprocal translocations were detected in 2.07% of couples, followed by inversions (1.14%). Robertsonian translocations were observed only in 0.86% of couples. The strong prevalence of reciprocal translocations compared with Robertsonian translocations would be a result of the difference in the segregation modes of these anomalies. Indeed, by using fluorescent in situ hybridization, reciprocal translocations were shown to generate between 18.4% and 72.1% of unbalanced gametes. In contrast, Robertsonian translocations produce an imbalance in only 11.6% to 17.8% of produced gametes (13), (14) and (15).
In our study, the prevalence of chromosomal aberrations among women decreases significantly when they are >38 years of age (58 anomalies in 912 women <38 years of age [6.36%] vs. 15 anomalies in 488 women who were ≥38 years of age [3.07%]; P<.01). This relative decrease is probably caused by a sample bias. Women who are ≥38 years of age will more likely have recurrent abortions as a result of nondisjunction rather than of other chromosomal abnormalities; further, more other factors such as endocrine factors (16) may more frequently lead to recurrent pregnancy loss, and therefore, the relative prevalence of chromosomal abnormalities in these women will decline (4) and (5).
In our study, prevalence of chromosomal anomalies does not appear to be connected with the number of the abortions, as shown in Table 1 (P=.67). However, when a history of repetitive abortions, malformative syndrome, or mental retardation is found in the family of one of the two parents, the risk of finding a structural chromosomal anomaly is significantly higher; indeed, 28 structural anomalies (6.80%) were found in 412 couples who presented a family history of at least one of these pathologies, whereas among the 988 couples without a family history, only in 29 (2.94%) were anomalies detected (P<.001).
In conclusion, as a result of their incidence, chromosomal anomalies should be investigated as a part of recurrent-miscarriage diagnosis to allow better counseling, especially in the case of young couples who have a family history of abortive disease, malformative syndrome, and/or mental retardation. A chromosomal anomaly finding in one of the two parents makes it possible to evaluate the prognosis of future pregnancies because the risk of miscarriage in couples with reciprocal translocations is approximately 25%–50%, and with Robertsonian translocations it is approximately 25%. This identification will also allow genetic counseling, and couples in whom one partner is found to have a chromosome abnormality should be counseled regarding the recurrence risk for miscarriage and should be offered prenatal genetic studies in all future pregnancies to detect possible unbalanced chromosome abnormalities in the fetus.

References


1 American College of Obstetricians and Gynecologists, Management of recurrent early pregnancy loss. ACOG practice bulletin, American College of Obstetricians and Gynecologists, Washington, DC (2001).

2 Royal College of Obstetricians and Gynaecologists, The management of recurrent miscarriage, Royal College of Obstetricians and Gynaecologists, London, United Kingdom (1998).

3 M.D. Stephenson, Frequency of factors associated with habitual abortion in 197 couples, Fertil Steril 66 (1996), pp. 24–27.

4 M. De Braekeleer and T.N. Dao, Cytogenetic studies in couples experiencing repeated pregnancy losses, Hum Reprod 5 (1990), pp. 519–528. View Record in Scopus | Cited By in Scopus (72)

5 M. Goddijn, J.H.K. Joosten, A.C. Knegt, F. Van Der Veen, M.T.M. Franssen and G.J. Bonsel et al., Clinical relevance of diagnosing structural chromosome abnormalities in couples with repeated miscarriage, Hum Reprod 19 (2004), pp. 1013–1017. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (19)

6 M. Sugiura-Ogasawara, Y. Ozaki, T. Sato, N. Suzumori and K. Suzumori, Poor prognosis of recurrent aborters with either maternal or paternal reciprocal translocations, Fertil Steril 81 (2004), pp. 367–373. Article | PDF (76 K) | View Record in Scopus | Cited By in Scopus (34)

7 F. Mitelman, ISCN—an international system for human cytogenetic nomenclature, Karger, Basel, Switzerland (1995).

8 A. Hernandez, M.C. Reynoso, F. Soto, D. Quinones, Z. Nazara and A. Rolon et al., Aneuploidies, chromosome aberrations and dominant gene mutations detected in 113,913 consecutive newborn children in Mexico, Mutat Res 232 (1990), pp. 23–29. Abstract | PDF (446 K) | View Record in Scopus | Cited By in Scopus (1)

9 J. Nielsen and M. Wohlert, Chromosome abnormalities found among 34,910 newborn children: results from a 13-year incidence study in Arhus, Denmark, Hum Genet 87 (1991), pp. 81–83. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (275)

10 L. Tarani, S. Lampariello, G. Raguso, F. Colloridi, I. Pucarelli and A.M. Pasquino et al., Pregnancy in patients with Turner's syndrome: six new cases and review of literature, Gynecol Endocrinol 12 (1998), pp. 83–87. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (61)

11 P.L. Kuo and H.R. Guo, Mechanism of recurrent spontaneous abortions in women with mosaicism of X-chromosome aneuploidies, Fertil Steril 82 (2004), pp. 1594–1601. Article | PDF (111 K) | View Record in Scopus | Cited By in Scopus (5)

12 H. Elghezal, S. Hidar, R. Braham, W. Denguezli, M. Ajina and A. Saad, Chromosomes abnormalities in 1000 infertile males with non-obstructive sperm disorders, Fertil Steril 86 (2006), pp. 1792–1795. Article | PDF (72 K) | View Record in Scopus | Cited By in Scopus (5)

13 H. Honda, N. Miharu, O. Samura, H. He and K. Ohama, Meiotic segregation analysis of a 14;21 Robertsonian translocation carrier by fluorescence in situ hybridization, Hum Genet 106 (2000), pp. 188–193. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (32)

14 R.H. Martin and E.L. Spriggs, Sperm chromosome complements in a man heterozygous for a reciprocal translocation 46,XY,t(9;13)(q21.1;q21.2) and a review of the literature, Clin Genet 47 (1995), pp. 42–46. View Record in Scopus | Cited By in Scopus (37)

15 H. Carp, B. Feldman, G. Oelsner and E. Schiff, Parental karyotype and subsequent live births in recurrent miscarriage, Fertil Steril 81 (2004), pp. 1296–1301. Article | PDF (157 K) | View Record in Scopus | Cited By in Scopus (24)

16 V.J. Vitzthum, H. Spielvogel, J. Thornburg and B. West, A prospective study of early pregnancy loss in humans, Fertil Steril 86 (2006), pp. 373–379. Article | PDF (152 K) | View Record in Scopus | Cited By in Scopus (2)
 
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