1
1. FITC-labelled polysaccharides
FITC-Dextran
Fluorescein isothiocyanate dextran
Chemical names:
Dextran(3´,6´dihydroxy-3-oxospiro(isobenzofuran-1(3H),9´-[9H]xanthen]-5(or 6)yl)carbamothioate.
Fluorescein isothiocyanate-dextran.
Fluoresceinyl thiocarbamoyl-dextran.
CAS number: 60842-46-8
Structure
Definitions
Mw; Weight average mean molecular weight
Mn; Number average mean molecular weight
DS; defines the number of substituent molecules per glycose molecule in the
polysaccharide chain
Properties
Selected dextran fractions prepared from native Dextran B512F are labelled with fluorescein
using a stable thiocarbamoyl linkage - the labelling procedure does not lead to any
depolymerisation of the dextran(1). The DS of FITC-dextrans ranges from 0.002-0.008 and at
these low levels of substitution, confer minimal charges to the dextran; which is important for
permeability studies.
2
FITC-dextran is supplied as a yellow/orange powder which dissolves freely in water or salt
solutions giving a yellow solution. The product also dissolves in DMSO, formamide and
certain other polar organic solvents but is insoluble in lower aliphatic alcohols, acetone,
chloroform, dimethylformamide. High molecular weight fractions should be dissolved by
slowly adding the powder to warm water (approx. 60°C) with vigorous stirring.
The dextran molecule at molecular weights greater than 5 000 Daltons behaves as a flexible
and extended coil in solution. Table 1. (below) shows the molecular dimensions at various
molecular weights. Dextrans and FITC-dextrans will exhibit Newtonian flow characteristics
i.e. the viscosity is independent of shear rate.
Dextran Mw
2 x 106
1 x 106
500 000
200 000
100 000
70 000
40 000
10 000
Stokes Radius Å
270
199
147
130
69
58
44.5
23.6
Radius of gyration Å
380
275
200
130
95
80
62
-
Table 1. Molecular dimensions of dextran.
Spectral data
Fig. 1. Fluorescence scan of FITC-dextran 70 in 0.025M borate pH 9.0 ( 10mg in 50 ml
buffer). Excitation 495nm; Emission 520nm.
3
FITC-dextran 70 -dependence on pH
10000
9000
Fluorescence intensity
8000
7000
6000
Fl
5000
4000
3000
2000
1000
0
0
2
4
pH
6
8
10
Fig. 2. Fluorescence (Emission 520nm) of FITC-dextran in the range pH 4-9
Storage and Stability
The stability of FITC-dextrans in vitro and in vivo is excellent - only at elevated pH(>9) and
elevated temperatures is there a risk for hydrolysis of the fluorescein label. Studies at 37° C
in rabbit plasma, muscle homogenate, liver homogenate and urine established that FITCdextrans are stable for at least 3 days. No changes in the Mw. and no release of fluorescein
moieties were noted. FITC-dextran is stable in 6% trichloracetic acid at room temperature for
3 days(2). Autoclaved FITC-dextran 70 solutions were stored at temperatures from 8 to 50°C
for periods up to 5 months. Only samples stored at 50°C showed a slight increase (1%) in free
amino-fluorescein. FITC-dextran was found to be stable at pH 4 but at pH 9, a considerable
(24%) decrease in fluorescence took place at 35°C over 1 month. Several studies(3) have
confirmed the in vivo stability of FITC–dextrans during the experiments(1-6 days) .
Applications
FITC-dextrans are primarily used for studying permeability and transport in cells and tissues
but may also be used for permeability studies of other materials (filters, gels etc). A particular
benefit is that measurements of the fluorescence provide quantitative data on the permeability
of healthy and diseased tissues in real time. Intravital fluorescence microscopy offers high
sensitivity and concentrations down to 1μg/ml can be detected in tissue fluids. Several models
have been developed for facilitating real time studies(4,5) FITC-dextrans have also been use
as a pH probe in cells(6,7). It has also been noted from polarization experiments, that the
lifetime of the excited state is similar to that before conjugation .
FITC-dextrans have proved valuable in the following fields of study (references selected from
an extensive list on each topic):
1.
2.
3.
4.
5.
Permeability studies on intestinal tissues(8-10)
Permeability studies of brain and nervous system(11-13)
Permeability studies on neoplastic tissues(14-16)
Permeability studies within the ocular chamber(17-19)
Permeability studies of renal tissues(20)
4
Fig. 3. Images taken from cheek pouch after infusion of FITC-dextran 150. The second image
shows the leakage of the microvasculature after subjection to histamine. (By kind permission
of E.Svensjö).
FITC-DEAE-dextran (FDD)
Chemical Names:
FITC-Diethylaminoethyl-dextran
Fluorescein-thiocarbamoyl-(O-2-diethylaminoethyl)-dextran
CAS number; not available
Structure
Properties
5
FITC-DEAE-dextrans are supplied as a yellow powder which is freely soluble in water or
electrolyte solutions. The rate of dissolution will depend on the particle size and structure and
it is advisable to add the powder slowly with vigorous stirring – for high molecular weight
products the water may be heated. The DS(FITC) lies between 0.001 - 0.008 and the limits for
nitrogen content are 3-5% - corresponding to about one DEAE-substituent per three glucose
units. The synthesis introduces two types of DEAE-substituents into the dextran chain(see
structure above). The predominant substituent is the single tertiary group but this reacts
further particularly at higher DS to yield a "tandem" group containing a quaternary amine as
well as the tertiary amine.
Spectral data
Fig. 4. Fluorescence scan of FITC-DEAE-dextran in 0.025M borate pH 9.0 ( 10mg in 50 ml
buffer). Excitation 495nm; Emission 520nm.
Storage and Stability
The powder is stable at room temperature for more than three years providing it is stored in
well-sealed containers in the dark. In solution, FITC-DEAE-dextran should not be stored at
pH>8 for extended periods.
Applications
FITC-DEAE-dextrans will possess many of the properties shown by the parent DEAEdextrans but will also display fluorescence to permit traceability. Some of the applications of
DEAE-dextrans are given below:


As adjuvant for vaccines
Enhances uptake of protein and nucleic acids by cells - transfection techniques and viral
infectivity
For stabilisation of proteins (enzymes)
FITC-DEAE-dextrans have been used to study delivery of positively charged molecules into
nucleated cells via the perforin pore(21). The permeability of FITC-DEAE-dextrans, FITCdextran and FITC-dextran sulphate through nasal mucosa were compared in a study of
absorption promoters(22).
6
FITC-carboxymethyl-dextran (FCM-Dextran)
Chemical Names:
FITC-Carboxymethyl-dextran
Fluorescein-thiocarbamoyl-(O-carboxymethyl)-dextran
CAS number; not available
Structure
Properties
FITC-CM-dextrans are manufactured by reacting selected dextran fractions with an activated
carboxymethyl derivative in alkali whereby O-carboxymethyl groups are introduced along the
dextran chain. The carboxyl content is approximately 5% which is equivalent to about one
CM group for every five glucose units. Thereafter, fluorescein groups are introduced by
reaction with fluorescein isothiocyanate. The DS(FITC) lies between 0.003 - 0.008. FITCCM-dextrans are supplied as a yellow powder which is freely soluble in water or electrolyte
solutions. The products have a pronounced polyanionic character by virtue of the negatively
charged carboxyl groups attached. The solution properties of FITC-CM-dextrans are expected
to be comparable with those for CM-dextran(23-25). In neutral solutions, the carboxymethyl
substituents will repel each other leading to an expansion of the dextran coil. FITC-CMdextrans are insoluble in most organic solvents, for example, ethanol, methanol, acetone,
chloroform, ethyl acetate.
Spectral data
7
Fig. 5. Fluorescence scan of FITC-CM-dextran in 0.025M borate pH 9.0 ( 10mg in 50 ml
buffer). Excitation 493nm; Emission 519nm.
Storage and Stability
Prospective stability studies have established that CM-dextrans maintain their potency and
purity for at least 3 years(unpublished studies) and from our experience with FITC-dextrans,
we predict a similar stability for FITC-CM-dextrans. It is recommended that FITC-CMdextrans are stored in air-tight containers in the dark at ambient temperatures.
Applications
CM-dextran itself has been found to be biocompatible and is used as a starting material in
several pharmaceutical and diagnostic applications. The toxicity of FITC-CM-dextran is
likewise anticipated also to be low. The insertion of a carboxyl group in the dextran chain
provides further opportunities for immobilizing molecules with interesting biological
activity(pharmaceuticals, enzymes, diagnostic tracers) on to dextrans.The carboxyl moiety
may be used in many reactions, for example, esterification, amidation with amines, UGI or
Passerini reactions. Simple ion-binding reactions can also provide a range of derivatives
incorporating different cationic molecules(26,27). The carboxyl groups will also impart an
overall negative charge to the molecule, which may be valuable in gaining information on the
permeability characteristics of cell membranes and tissues(28). The application of FITC-CMdextran in studies of drug delivery systems has been reported(29).
FITC-polysucrose (FITC-Ficoll®)
Chemical Names:
Polysucrose(3´,6´dihydroxy-3-oxospiro(isobenzofuran -1(3H),9´-[9H]xanthen]-5
(or 6)-yl)carbamothioate
Fluorescein isothiocyanate-polysucrose
8
CAS number; not available
Structure:
Properties
Polysucrose fractions are labelled with fluorescein by a procedure similar to that described by
de Belder and Granath(1). The fluorescein moiety is attached by a stable thiocarbamoyllinkage and the labelling procedure does not lead to any depolymerisation of the polysucrose.
The DS of FITC-polysucroses lies between 0.001- 0.008 and at these low levels of
substitution confers minimal charges to the polysucrose.
FITC-polysucrose is supplied as a yellow powder which dissolves freely in water or salt
solutions giving a yellow solution. The product also dissolves in DMSO, formamide and
certain other polar organic solvents but is insoluble in lower aliphatic alcohols, acetone,
chloroform, dimethylformamide.
The polysucrose molecule behaves as a globular molecule in solution as is to be expected
from its structure. In Table 2 (below) a comparison of the Stokes radius of dextran and
polysucrose fractions reflects these differences in molecular flexibility. The molecule is best
regarded as an intermediate between a hard solid sphere and a flexible coil. Thus when
comparing polysucrose and dextran fractions of similar molecular weights, the molecular
dimensions of the polysucrose will always be smaller. Polysucrose solutions have very low
osmotic pressures compared to sucrose solutions of equivalent concentration. Thus a 10%
solution of polysucrose 70 has an osmolality of 3 mOs/kg compared to 150 for a 10% sucrose.
No detailed toxicity studies on FITC-polysucroses have been published. However,
polysucrose fractions (100 000 to 500 000) when administered intravenously at doses up to
12g/kg in experimental animals showed no toxic symptoms. Polysucrose exhibits excellent
9
biocompatibility with cells, virus, microorganisms etc. and has been used for many decades in
cell separation technology.
Mw
2 x 106
1 x 106
500 000
200 000
100 000
70 000
40 000
10 000
Polysucrose
Å
140
106
74
55
49.5
40
-
Dextran
Å
270
199
147
95
69
58
44.5
23.6
Table 2. Comparison of molecular dimensions of polysucrose and dextran
Spectral data
Fig. 6. Fluorescence scan of FITC-polysucrose70 in 0.025M borate pH 9.0
(9.9mg in 50 ml buffer). Excitation 496nm; Emission 525nm. Measurements in biological
media may significantly affect the fluorescence intensity which may be enhanced or
depressed.
extran
Storage and stability
FITC-polysucrose powder when stored in air-tight containers at ambient temperatures is
stable for at least 6 years. The stability of FITC-polysucroses in solution has not been
investigated in detail. However, the stability of the thiocarbamoyl linkage between the
fluorescein moiety and polysucrose will be similar to that with dextran (see FITC-dextran).
FITC-dextran is stable at pH 4 for up to 1 month at temperatures up to 35°C but this is not to
10
be recommended for polysucrose based products owing to the lability of glycosidic linkages
in sucrose. Polysucrose itself can be autoclaved at neutral and slightly alkaline pH.
Applications
The available data on the two polysaccharides, polysucrose (Ficoll®) and dextran, for
assessing the glomerular permselectivity as compared to glomerular proteins has been
reviewed(30) . Polydisperse polysaccharides are excellent probes for measuring glomerular
permselectivity and are reproducible, reliable and elegant. The authors elaborate on the
various properties which may influence the results such as molecular size, shape, charge and
flexibility and assess their result in various pore models. Studies of the glomerular
permeability of Ficoll when infused intravenously showed that it has a cut-off at about 50Å
whereas dextran is excreted up to 60-70Å - this is explained by the greater flexibility of
dextran. The clearance of FITC-polysucrose in mice lacking endothelial caveolae was studied
in order to elucidate macromolecular transport pathways(31). The glomerular filter was
studied at different glomerular filtration rates using FITC-polysucrose 70 and 400 (also FITCinulin)(32) .
Studies on the glomerular filtration of dextran and Ficoll showed that the glomerular
membrane presented a much more restrictive barrier to Ficoll than to dextran(33) .
Interestingly, the values of the sieving coefficient for Ficoll approximated to those reported
for uncharged globular proteins. Glomerular sieving in rats, following surgery and muscle
trauma, was monitored using FITC-polysucrose 70/400(34). The rats were dosed with a
mixture of FITC-polysucrose 400 (960μg), FITC-polysucrose 70 (40μg) and FITC-inulin
(500μg) as a priming bolus. Glomerular permeability was studied in caveolin-1 knockout
mice using FITC-polysucrose 70/400(35) .
FITC-polysucrose 70 and albumin were infused in rats to explore the effects of temperature
and ammonium chloride on the fractional clearance. Polysucrose performs differently from
dextrans and was lower over the range 20-70 Å(36,37). FITC-polysucrose 70 was used to
evaluate whether the increase in clearance of native albumin after 9 weeks of diabetes was
due to reduced charge selectivity or to an alteration in the proportion of large pores(38).
FITC-DEAE-polysucrose
Chemical Names:
FITC-(O-diethylaminoethyl)-polysucrose
Fluoresceinyl-thiocarbamoyl-(O-diethylaminoethyl)-polysucrose
CAS number; not available
Structure:
11
Properties
FITC- DEAE-polysucrose is supplied as a yellow powder which is readily soluble in water or
buffer solutions and there is approximately one DEAE group for every five hexose groups.
The limits for the degree of FITC substitution are 0.001 to 0.008. The product possesses a
polycationic character. As depicted in the structural representation above, the DEAEsubstituents may be present either as a single unit or as a ‘tandem’ unit – the latter containing
a quaternary ammonium structure.
Spectral data
Excitation is best performed at 493 nm and fluorescence measured at 523 nm (see Fig.7).
Measurements in biological media may significantly affect the fluorescence intensity which
may be enhanced or depressed.
12
Fig. 7. Fluorescence scan of FITC-DEAE-polysucrose 70 in 0.025M borate pH 9.0
( 10mg in 50 ml buffer). Excitation 496nm; Emission 530nm. Measurements in biological
media may significantly affect the fluorescence intensity which may be enhanced or
depressed.
Storage and stability
FITC-DEAE-polysucrose is delivered as a dry yellow powder and should be stored in well
sealed containers at ambient temperatures in the dark. The pH of the product on delivery is
6.5 – 7.0 and should not be allowed fall below pH 5– the product should not be stored at pH >
7.0 for extended periods.
Applications
The product is used for studying the permeability of polycationic polymers relative to neutral
polymers in organs, tissues and cells.
FITC-CM-polysucrose (FITC-CM-Ficoll®)
Chemical Names:
FITC-(O-carboxymethyl)-polysucrose
Fluoresceinyl-thiocarbamoyl-(O-carboxymethyl)-polysucrose
CAS number; not available
Structure:
13
Spectral data
Excitation is best performed at 495 nm and fluorescence measured at 517 nm (see Fig.8).
Measurements in biological media may significantly affect the fluorescence intensity which
may be enhanced or depressed. CHECK
Fig. 8. Fluorescence scan of FITC-CM-polysucrose 70 in 0.025M borate pH 9.0
( 11mg in 50 ml buffer). Excitation 495nm; Emission 517nm.
Storage and stability
FITC-CM-polysucrose is delivered as a dry yellow powder and should be stored in well
sealed containers at ambient temperatures in the dark. Solutions may be kept at room
temperature or preferably in a refridgerator in the dark at pH 6-7 for several weeks.
14
Applications
FITC-CM-polysucrose has played an interesting role in elucidating the properties of the
glomerular membrane and it appears that despite the negative character of the membrane, the
permselectivity of the anionic Ficoll derivatives is greater than the neutral species(28,). Later
studies using FITC-CM-polysucrose, however, refuted these findings(39). The significant role
of solute charge in the sieving character of the glomerular membrane has been reexamined(40).
FITC-inulin
Chemical Names:
Inulin(3’,6’dihydroxy-3-oxospiro(isobenzofuran-1(3H) ,9’-[9H]xanthen]-5(or 6)yl)carbamothioate
Fluorescein isothiocyanate-Inulin
CAS number; not available
Structure:
Properties
An inulin fraction obtained from dahlia tubers is labelled with fluorescein by a procedure
similar to that described by de Belder and Granath(1) . The fluorescein moiety is attached by a
stable thiocarbamoyl linkage and the labelling procedure does not lead to any
depolymerisation of the inulin. The DS of FITC-inulin lies between 0.001-0.008 and at these
low levels of substitution, the effect of the charges are minimal.
FITC-inulin is supplied as a yellow powder which dissolves in water or salt solutions giving a
yellow solution. Dilute solutions (1-2%) remain clear on standing but more concentrated
(>10%) may form precipitates on standing, since inulin tends to form crystalline aggregates.
These precipitates will redissolve on heating. Temperatures up to 80° C may be employed
15
providing the solution is around neutral pH. The product also dissolves in DMSO, formamide
and certain other polar organic solvents but is insoluble in lower aliphatic alcohols, acetone,
chloroform, dimethylformamide, ethyl acetate.
The Mw of FITC-inulin as determined by SEC (Superose 6 + 12; dextran calibration) is
approx. 5000. Phelps(40) determined the Mw of inulin fractions from osmotic pressure data
and obtained a value of 5640.
Spectral data
Excitation is best performed at 490nm and fluorescence measured at 520 nm (see Fig.9).
Measurements in biological media may significantly affect the fluorescence intensity which
may be enhanced or depressed. The dependence of fluorescence on pH is depicted in Fig. 10).
Fig. 9. Fluorescence scan of FITC-inulin in 0.025M borate pH 9.0 ( 10mg in 50 ml buffer).
Excitation 492nm; Emission 519nm.
Fig. 10. Fluorescence of FITC-inulin in pH range 4-9; Emission 519nm.
16
Storage and stability
The stability of the thiocarbamoyl linkage between the fluorescein moiety and
inulin will be similar to that for dextran (see data-file for information on stability of FITCdextran). There are no prospective studies on the stability of FITC-inulin but retrospective
studies have shown that FITC-inulin powder when stored in air-tight containers at ambient
temperatures is stable for at least 6 years. Solutions of FITC-inulin should not be stored at
pH(< 5) or high pH(> 9) for prolonged periods particularly at elevated temperatures.
Applications
FITC-inulin has been shown to be ideal for studying glomerular filtration rate in experimental
animals as it is stable during filtration and renal passage and does not bind to plasma proteins
or penetrate the renal cells. Measurements of the fluorescence provide quantitative data on
transport and permeability of healthy and diseased tissues. Such studies can be performed in
real time by intravital fluorescence microscopy. The technique offers high sensitivity and
concentrations down to 1µg/ml can be detected in tissue fluids.
The tubular fluid to plasma concentration ratio was determined in rats following a bolus
injection of FITC-inulin in the femoral vein(41). The validity of the method as a measure of
glomerular filtration was established by comparisons with 51Cr-EDTA and [H3]-inulin.
A thorough investigation of the use of FITC-inulin for GFR studies was presented by Fleck in
1999(42). The rats were given 4mg/mL at a rate of 4ml/100g bodyweight per hour via the tail
vein or jugular vein. Dunn and coworkers(43) found excellent correlation between creatinine
clearance and FITC-inulin clearance in mice. Other studies using FITC-inulin for determining
glomerular filtration have been described(44,45).
FITC-inulin has been used for studying the permeability of intestinal epithelial cells(46,47).
Fluorescein hyaluronic acid (FHA-Se)
Chemical Names:
5-aminofluorescein-labelled hyaluronate
5-aminofluorescein-labelled hyaluronan
CAS number; not available
Structure:
17
Properties
Hyaluronic acid, a polysaccharide composed of alternating β(1-3) glucuronide and β(1-4)
glucosaminide units -derived from Streptococcus equi, is labelled with 5-amino-fluorescein
giving a yellow fibrous product that is soluble in water and electrolytes, however, the solid
requires prolonged gentle stirring – overnight – to dissolve(48). The degree of substitution lies
between 0.001 and 0.008. The molecular weight determined with a GPC system, calibrated
with dextran standards, gave Mw of 6.0 x 106.
Spectral data
Fig. 11. Fluorescence scan of FITC-hyaluronic acid in 0.025M borate pH 9.0 ( 12mg in 50 ml
buffer). Excitation 495nm; Emission 524nm.
Storage and stability
The dried product should be stored in air-tight containers at ambient temperatures in the dark.
A shelf-life of 5 years is proposed. No release of fluorescent material was noted when a
solution of the product was incubated at pH 7.5 at 37°C for one month(48).
Applications
Many applications of hyaluronan have appeared over the past years both in
medicine(particularly its indispensable contribution to eye surgery) and in cosmetics.
Fluorescein-labelled hyaluronic acid may be used as a probe for following the fate of
hyaluronan in vitro. A FITC-labelled hyaluronic preparation greatly enhanced the
visualisation of the permeation of the substrate through skin(49). Other applications of
fluorescein labelled hyaluronic acid have appeared(50-53)
2.
TRITC-Dextran
Chemical names:
TRITC-labelled polysaccharides
18
Tetramethyl-rhodamine isothiocyanate-dextran
Dextran 3´, 6´bis(tetramethylamino) -3-oxospiro(isobenzofuran-1(3H),9´-9H]xanthen]-5(or
6)- yl)carbamothioate.
Tetramethyl-rhodamine B thiocarbamoyl-dextran
CAS number; not available
Structure:
Properties
TRITC-dextrans are prepared from selected dextran fractions by coupling to
tetramethylrhodamine B isothiocyanate (mixed isomers). The DS( TRITC) ranges from 0.001
to 0.008. At these low degrees of substitution, the charge contribution from the tertiary aminogroups on the rhodamine moiety is negligible. All batches are checked for molecular weight,
loss on drying and free TRITC.
Spectral data
Excitation is best performed at 550nm and fluorescence measured at 572 nm (see Fig.12).
Studies in our laboratories have shown that the fluorescence from a TRITC-dextran solution
shows only slight changes over the range pH 3-9 (see Fig.13) This is of interest when making
quantitative measurements. Measurements in biological media may significantly affect the
fluorescence intensity which may be enhanced or depressed.
19
TD40_20239(EM)
300
200
100
0
300
400
500
600
700
800
nm
Fig. 12. Fluorescence scan of TRITC-dextran 40 in 0.025M borate pH 9.0
(10mg in 50 ml buffer) Excitation 550nm; Emission 572nm.
TRITC-dextran 70- dependence on pH
450
Fluorescence intensity
400
350
300
250
fl
200
150
100
50
0
0
2
4
pH
6
8
10
Fig.13. Dependence of fluorescence intensity(em. 572nm) of TRITC-dextran with pH.
Storage and stability
The stability of TRITC-dextrans has not been investigated in detail but it is presumed to be
similar to that of FITC-dextrans since both cases the substituents are linked via a
thiocarbamoyl unit. Only at elevated pH (>9) and elevated temperatures is there a risk for
hydrolysis of the thiocarbamoyl linkage (see FITC-dextran). TRITC-dextran solutions(pH 67) can be stored at room temperature in the dark for several weeks. The dry powder when
stored in well-sealed containers in the dark has a shelf life of 5 years.
Applications
TRITC-dextrans are primarily used for studying permeability and transport in cells, vessels
and tissues. Measurements of the fluorescence can be made quantitatively and in real time by
intravital fluorescence microscopy. The technique offers high sensitivity and concentrations
20
down to 1μg/ml can be detected in tissue fluids. A further important property is that TRITCdextran does not bind to artery walls(54,55).
The microvasculature of the hamster cheek pouch has proved to be a useful model for
studying plasma leakage resulting from various inflammatory conditions. The cheek
pouches are examined by intravital fluorescence microscopy using suitable filter (490/520nm)
and images are captured with a digital camera(see Fig. 14). A 5% TRITC-dextran 150
solution in normal saline is administered i.v. (approx. 100mg/kg bodyweight)(56-58). For
more information on these techniques see Thorball(3). TRITC-dextrans have been used
extensively for permeability studies in tissues and cells and only a few references selected at
random are given(59-65).
Fig. 14. TRITC-dextran 150 injected in a hamster cheek pounch 15min after
histamine challenge. (by kind permission of E.Svensjö).
TRITC-polysucrose (TRITC-Ficoll®)
Chemical names:
Tetramethyl-rhodamine isothiocyanate-polysucrose
Polysucrose 3´,6´bis(tetramethylamino) -3-oxospiro(isobenzofuran-1(3H),9´-9H]xanthen]5(or 6)- yl)carbamothioate.
Tetramethyl-rhodamine B thiocarbamoyl-polysucrose
CAS number; not available
Structure:
21
Properties
TRITC-polysucrose is a derivative of polysucrose – a polymer synthesized by cross-linking
sucrose with epichlorohydrin. TRITC-polysucrose is prepared by reacting polysucrose with
TRITC under similar conditions to those used for TRITC-dextrans. TRITC-polysucrose is
readily soluble in water and salt solutions over a wide range of pH. Polysucrose is more
sensitive to acid than dextran so that care must be taken when working at acid pH. It is
supplied as a red powder which is freely soluble in water.
Spectral data
22
Fig. 15. Fluorescence scan of TRITC-polysucrose 70 in 0.025M borate pH 9.0
(11mg in 50 ml buffer) Excitation 522nm; Emission 552nm.
Storage and stability
TRITC-polysucrose powder when stored in air-tight containers at ambient temperatures is
stable for at least 6 years. The stability of TRITC-polysucroses in solution has not been
investigated in detail. The stability of the thiocarbamoyl linkage between the
tetramethylrhodamine moiety and polysucrose will be similar to that with dextran (see
TRITC-dextran). However, low pH storage is not recommended for polysucrose-based
products owing to the lability of glycosidic linkages in sucrose. Polysucrose itself can be
autoclaved at neutral and slightly alkaline pH.
Applications
TRITC-polysucrose has similar applications to those described for FITC-polysucrose but has
certain advantages. As mentioned earlier, the fluorescence of tetramethylrhodamine is less
dependent on pH than FITC-labels. Also the longer emission wavelength avoids background
interference in experimental environments.
A TRITC-polysucrose 70 has been used in studies of the renal endothelial barrier(66,67).
Antibody response to thymus-independent antigens has been studies with the aid of TRITCFicolls(68).
Tetramethyl-rhodamine hyaluronic acid (TR-HA)
Chemical names:
Tetramethyl-rhodamine hyaluronic acid
Hyaluronan, 6´bis(tetramethylamino) -3-oxospiro(isobenzofuran-1(3H),9´-9H]xanthen]-5(or
6)- yl).
Tetramethyl-rhodamine B hyaluronan
CAS number; not available
Structure:
23
Properties:
Hyaluronic acid, a polysaccharide composed of alternating β(1-3) glucuronide and β(1-4)
glucosaminide units -derived from Streptococcus equi, is labelled with aminotetramethylrhodamine giving a red product that is soluble in water and electrolytes. The DS
lies between 0.001 and 0.008. The Mw determined with a GPC system, calibrated with
dextran standards, gave a value of 6.0 x 106.
Spectral data
Fig. 16. Fluorescence scan of TR-HA in 0.025M borate pH 9.0
(12mg in 50 ml buffer) Excitation 552nm; Emission 576nm.
Applications
Tetramethyl-rhodamine hyaluronic acid(TR-HA) has similar applications to those described
for fluorescein hyaluronic acid(see earlier section) but has certain advantages. As mentioned
earlier, the fluorescence of tetramethylrhodamine is less dependent on pH than FITC-labels.
24
Also the longer emission wavelength avoids interference from background images in
experimental environments. Invasive growth into brain tissue employing TR-HA and 2photon imaging has been described(69).
References.
1. A.N. de Belder and K. Granath, Preparation and properties of fluorescein labelled
dextrans, Carbohydr Res, 30(1973), 375-378.
2. P. Kurtzhals, C. Larsen and M. Johansen, High performance size-exclusion
chromatographic procedure for the determination of fluoresceinyl isothiocyanate
dextrans of various molecular masses in biological media. J Chromatogr, 491(1989), 117127.
3. N. Thorball, FITC-dextran tracers in microcirculatory and permeability studies
using combined fluorescence Stereo Microscopy, Fluorescence Stereo-microscopy and
electron microscopy, Histochemistry, 71(1981), 209-233.
4. E. Svensjö, The hamster cheek pouch as a research model in inflammation.
Chapter 30 In: David Shepro (Editor), Microvascular Research - Biology and
Pathology, 2006, 195-207, Elsevier Academic Press.
5. J. Jonsson, K.E. Arfors, and H.C. Hint, Studies on relationships between blood
and lymphatic systems within the microcirculation, 6th Europ Conf Microcirculation, Aalborg.
1970, 214-218 (Karger, Basel 1971).
6. M.J. Geisow, H.P. D´Arcy and M.R. Young, Temporal changes of lysosome and
phagosome pH during phagolysosome formation in macrophages; studies by fluorescence
spectroscopy, J Cell Biol, 89(1981), 645-652.
7. S. Ohkuma and B. Poole, Fluorescence probe measurement of the intralysosomal
pH in living cells and the perturbation of pH by various agents, Proc Natl Acad Sci USA,
75(1978), 3327-3331.
8. T.Mochizuki, H.Satsu, M.Totsuka and M.Shimizu, Transepithelial transport of
macromolecular substances in IL-4 treated human intestinal T84 cell monolayers,
Biosci Biotechnol Biochem, 73(2009), 2422-6.
9. B. Lei, W. Zha, Y. Wang et al., Development of a novel self-microemulsifying
drug delivery system for reducing HIV protease inhibitor-induced intestinal
epithelial barrier dysfunction, Mol Pharm, 7(2010), 844-53.
10. F. Berthiaume, R.M. Ezzell, M. Toner M et al., Transport of fluorescent dextrans
across the rat ileum after cutaneous thermal injury, Critical Care Medicine.
22(1994), 455-64.
11. D. Hultström, FITC-dextrans in neurobiological research, Acta Universitatis
Upsaliensis, 438, Almquist and Wiksell, Uppsala, 1982 and references cited
therein.
12. Y. Olsson, E. Svensjö, K.E. Arfors, Fluorescein labelled dextrans as tracers for
vascular permeability studies in the nervous system, Acta Neuropathol, 33(1975),
45-50.
13. W. Shougang, B. Baseri, J.J. Choi et al., Delivery of fluorescent dextrans through
the ultra-sound induced blood-brain barrier opening in mice. Ultrasonics
Symposium, 2008, IUS2008, 1702-1705.
14. L.E. Gerlowski and R.K. Jain, Microvascular permeability of normal and
neoplastic tissues, Microvasc Res, 31(1986), 288-305.
15. Li T, Hao Q, Wang X et al., The effect of focused ultrasound on the
physiochemical properties of Sarcoma 180 cell membrane, Sheng Wu Yi Xue
Gong Cheng Xue Za Zh., 26(2009), 941-6.
25
16. F. Chen, M. Hou, F. Ye et al., Ovarian cancer cells induce peripheral mature
dendritic cells to differentiate into macrophage-like cells in vitro. Int J Gynecol Cancer,
19(2009), 1487-93.
17. E. Mannermaa, M. Reinisalo, V.P. Ranta et al., Filter cultured ARPE-10 cells as
outer blood-retinal barrier model, Eur J Pharm Sci, 40(2010), 289-296.
18. M. Shivanna, G. Rajashekhar and S.P. Srinivas, Barrier dysfunction of the
corneal endothelium in response to TNF alpha, Invest Opthalmol, Vis Sci, 51(2010), 1575-82.
19. S.L. Lightman, L.E. Caspers-Velu, S. Hirose et al., Angiography with fluorescein-labelled
dextran in a primate model of uveitis. Arch Ophthalmol, 105(1987), 844-848.
20. W.I. Lencer, P. Weyer, A.S. Verkman et al., FITC-dextran as a probe for
endosome fuction and localization in kidney, Amer J Physiol, 258(1990), C309-317.
21. S.E. Stewart, S.C. Kondos, A.Y. Matthews et al., The perforin pore facilitates the delivery
of cationic cargoes, J Biol Chem, 289(2014), 9172-81.
22. N. Uchida, Y. Machia, Y. Machida et al., Influence of bile acids on the permeability of
insulin through the nasal mucosa of rabbits in comparison with dextran derivatives, Int J
Pharmaceut, 74(1991), 95-103.
23. K. Gekko, Solution properties of dextran and its ionic derivatives, ACS Symposium Series,
150(1981), 415-438.
24. K. Gekko and H. Noguchi, Selective interaction of calcium and magnesium ions with
ionic dextran derivatives, Carbohydr Res, 69(1979), 323-326.
25. O.Smidsröd and A.Haug, Estimation of the relative stiffness of the molecular chain in
polyelectrolytes from measurements of viscosity at different ionic strengths, Biopolymers,
10(1971), 1213-27.
26. P. Rongved and J. Klaveness, Water soluble polysaccharides as carriers of paramagnetic
contrast reagents for magnetic resonance imaging; Synthesis and relaxation properties,
Carbohydr Res, 214(1991), 315-323.
27. S.W Zheng, M. Huang et al., RGD-conjugated iron oxide magnetic nanoparticles for
magnetic resonance imaging contrast enhancement and hyperthermia, magnetic resonance
imaging contrast enhancement and hyperthermia, J Biomater Appl, 28(2014), 1051-1059.
28. D. Asgiersson, D. Venturoli, B. Rippe and C. Rippe, Increased glomerular permeability
to negatively charged polysucrose relative to neutral polysucrose in rats, Am J Physiol Renal
Physiol, 291(2006), F1083-9.
29. C. Martin, E .Dolmazon, K. Moylan et al., A charge neutral tuneable polymersome
capable of high biological encapsulation efficiency and cell permeation, Int J Pharmaceutics,
481(2015), 1-8.
30. D. Venturoli and B. Rippe, Ficoll and dextran vs. Globular proteins as probes for
glomerular permselectivity; effects of molecular size, shape, charge and
deformability, Am J Physiol Renal Physiol, 25(2005),77-84.
31. B.I. Rosengren, A. Rippe, C. Rippe et al. Transvascular protein transport in mice
endothelial caveolae, Am J Physiol Heart Circ Physiol, 291(2006), H1371-7.
32. C. Rippe, D. Asgeirsson, D. Venturoli et al., Effects of glomerular filtration rate on
the Ficoll sieving coeffcients in rats, Kidney, 69(2006), 1326-32.
33. J.D. Oliver III, S. Andersson, J.L.Troy et al., Determination of glomerular
size-selectivity in the normal rat with Ficoll, J Am Soc Nephrol, 3(1992), 214-22.
34. J. Axelsson, I. Mahmutovic, A. Rippe et al., Loss of size selectivity of the
glomerular filtration barrier in rats following laparotomy and muscle trauma.
J Physiol Renal Physiol. 297(2009), F577-82.
35. G. Grände, C. Rippe, A. Rippe et al., Unaltered size selectivity of the glomerular
filtration in caveolin-1 knockout mice. Am J Physiol Renal Physiol, 297(2009), F257-62.
26
36. M. Ohlsson, J. Sörensson and B. Haraldsson, Glomerular size and charge
selectivity in the rat as revealed by FITC-Ficoll and albumin, Am J Physiol Renal
Physiol, 279( 2000), F84-91.
37. C. Rippe, A. Rippe, O. Torfvit et al., Size and charge selectivity of the glomerular
filter in early experimental diabetes in rats. Am J Physiol Renal Physiol, 293(2007),
F1533-8.
38. J. Axelsson, K. Sverrisson, A. Rippe et al., Reduced diffusion of charge-modified,
conformationally intact anionic Ficoll relative to neutral Ficoll across the rat glomerular
filtration barrier in vivo, Am J Physiol Renal Physiol, 301(2011), F708-12.
39. N. Ferrell, Basal lamina secreted by MDCK cells has size- and charge-selective
properties, Am J Physiol Renal Physiol, 300(2011), F-86-90.
40. C.F. Phelps, The physical properties of inulin solutions, Biochem. J., 95(1965), 41-47.
41. M. Sohtell, B. Karlmark and H. Ulfendal, FITC-inulin as a kidney tubule marker,
Acta Physiol Scand, 119(1983), 313-6.
42. C. Fleck, Determination of the glomerular filtration rate (GFR); Methodological
problems, age-dependence, consequences of various surgical interventions, and
the influence of different drugs and toxic substances, Physiol Res, 48(1999), 267-279.
43. S.R. Dunn, Z. Qi, E.P. Bottinger et al., Utility of endogenous creatinine clearance as
a measure of renal function in mice, Kidney Int, 65(2004), 1959-67.
44. Z. Qi, I. Whitt, A. Mehta et al., Serial determination of glomerular filtration rate
in conscious mice using FITC-inulin clearance, Am J Physiol Renal Physiol, 286(2004),
F172-7.
45. J.N. Lorenz and E. Gruenstein, A simple, non radioactive method for evaluating
single-nephron filtration rate using FITC-inulin, Am J Physiol, 276(1999), F172-7.
46. N.T. Baker and L.L. Graham, Camphylobacter fetus translocation across Caco-2
cell monolayers, Microb Pathog. 49(2010), 260-72.
47. M. Neunlist, F. ToumiF, Oreschkova T etal., Human ENS regulates the intestinal
epithelial barrier permeability and a tight junction-associated protein ZO-1 via
VIPergic pathways. Am J Physiol Gastrointest. Liver Physiol., 285(2003), G1028-36.
48. A.N. de Belder and K.O. Vik, Preparation and properties of fluorescein-labelled
hyaluronate, Carbohyd. Res., 44(1975), 251-257.
49. J-E. Yang, E-S. Kim, J.H. Kwon et al., Transdermal delivery of hyaluronic acid – human
growth hormone, Biomaterials, 33(2012), 5947-5954.
50. Y. Mitsui, M. Goto, T. Yamada et al., Hyaluronic acid inhibits mRNA expression of proinflammatory cytokines and cyclooxygenase-2/prostaglandin E2 production, Paper no. 209,
54th AM of Orthopaedic Society.
51. M. Yokoo, Y. Miyahayashi, T.Nagamuna, Identification of hyaluronic acid-binding
proteins and their expressions in porcine cumulus-oocyte complexes during in vitro
maturation, Biol Reprod, 67(2002), 1165-71.
52. D .Cheng, W. Han, K. Song et al., One-step facile synthesis of hylauronic acid
functionalized fluorescent gold nanoprobes sensitive to hyaluronidase in urine specimen from
bladder cancer patients, Talanta, 130(2014), 408-14.
53. M. Yoneda, S. Shimizu, Y. Nishi et al., Hyaluronic acid-dependent change in the
extracellular matrix of mouse dermal fibroblasts that is conducive to cell proliferation, J Cell
Sci., 90(1988), 275-286.
54. B.A. Walsh, A.E. Mullick, C.E. Banka et al., Estradiol acts separately on the
LDL-particle and artery walls to reduce LDL-accumulation, J Lipid Res.
41(2000), 134-141.
55. G. Gardner, C.E. Banka, K.A. Roberts et al., Modified LDL-mediated increase in
27
endothelial-layer permeability are attenuated with 17B-estradiol, Arterscler Thromb Vasc
Biol., 19(1999), 854-861.
56. D. Hultström and E. Svensjö, Intravital and electron microscopy study of
bradykinin induced vascular permeability changes using FITC-dextran as a tracer,
J. Pathol., 129(1979), 125-133.
57. E. Svensjö, The hamster cheek pouch as a research model in inflammation.
Chapter 30 In: David Shepro (Editor), Microvasular Research-Biology and
Pathology. p. 195-207, 2006, Elsevier Academic Press.
58. E. Svensjö, E.M. Saraiva, M.T. Bozza et al., Salivary gland homogenates of
Lutzomyia longipalpis and its vasodilatory peptide maxadilan cause plasma
leakage via PAC1 receptor activation, J Vasc Res, 46(2009), 435-446.
59. J. Jonsson, K.E. Arfors and H.C. Hint, Studies on relationships between blood and
lymphatic systems within the microcirculation, 6 Europ.Conf.Microcirculation
Aalborg, 1970, 214-218 (Karger, Basel 1971).
60. A.E. Mullick, B.A. Walsh, K.M. Reiser et al., Chronic estradiol treatment attenuates
stiffening, glycoxidation and permeability of rat carotid arteries, Am J Physiol Heart Circ
Physiol., 281(2001), H2204-H2210.
61. M.J. Geisow, Fluorescein conjugates as indicators of subcellular pH, Exp Cell
Res, 150(1984), 29-35.
62. S. Ohkuma and B. Poole, Fluorescence probe measurement of the intralysosomal
pH in living cells and the perturbation of pH by various agents, Proc Natl Acad Sci, 75(1978),
3327-31.
63. T. van der Wijk, S.F. Tomassen, A.B. Houtsmuller et al., Increased vesicle
recycling in reponse to osmotic cell swelling. Cause and consequence of
hypotonicity-provoked ATP release, J Biol Chem, 278(2003), 40020-5.
64. M. Kauppi, A. Simonsen, B. Bremner et al., The small GTPase Rab22 interacts
with EEA1 and controls endosomal membrane trafficking, J Cell Sci, 115(2002), 899-911.
65. M. Mairhofer, M. Steiner, U. salzer et al., Stomatin-like protein-1 interacts with stomatin
and is targeted to late endosomes, J Biol Chem., 42(2009), 29218-29.
66. M.P. Hutchens, T. Fujiyoshi, R. Komers et al., Estrogen protects renal endothelial barrier
from ischemia reperfusion in vitro and in vivo, Am J Physiol Renal Physiol, 303(2012),
F377-F385.
67. M. Ikeda, K. Schenning, S. Anderson et al., Estrogen administered after cardiac arrest and
cardiopulmonary resuscitation is renoprotective, www.epostersonline.
om/asa2014/node/1059.
68. L. Amlot, D. Grennan and J.H. Humphrey, Splenic dependence of the antibody response
to thymus-independent (TI-2) antigens, Eur J Immunol, 15(1985), 508-512.
69. A. Pusch, A. Boeckenhoff, T. Glaser et al., CD44 and hyaluronan promote invasive
growth of B35 neuroblastoma cells into the brain, Biochim Biophys Acta, 1803(2010), 261274.