Biomaterials, Artificial Cells and Immobilization Biotechnology
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
On the Perluorocarbon Enulsions of Second Generation H. Meinert, R. Fackler, A. Knoblich, J. Nader, P. Renter & W. Röhike To cite this article: H. Meinert, R. Fackler, A. Knoblich, J. Nader, P. Renter & W. Röhike (1992) On the Perluorocarbon Enulsions of Second Generation, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 805-818, DOI: 10.3109/10731199209119722 To link to this article: http://dx.doi.org/10.3109/10731199209119722
Published online: 11 Jul 2009.
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Date: 21 April 2016, At: 09:03
BIOMT., ART. CELLS a IWOB. BIOTECH.,
ON
TEE PERF-
zo(z-/,),
805-818 (1992)
-1oIIS
OF SECOND G W T I O N
8 . Ieinert, R. Fackler, A. Knoblich, J. Iader, P. Reuter and 1. R6hlke, University of Ulm, Dep. Chemistry of Bi-patible Caponnds,
D-7900 Ulm, Parkatr. 11, Federal Republic of Germany
Downloaded by [Universite Laval] at 09:03 21 April 2016
ABSTRACT Perfluorocarbon-emulsions of second generation were prepared by means of new perf luorocarbons (F-dimorpholines, F-dipiperidines and F-cyclohexylwrpholine) , acting both as oxygen carriers and as interfacial active compounds (IFACs). The stabilizing effect of these IFACs is interpreted and a new theory is introduced. Also new classes of fluorosurfactants were synthesized and tested for biocompatibility. In PFC mixtures compounds of the type R R (RF=C,F2m+1, RH=CnH2n+l) are acting as IFACs but also as anchor-groups for lipophflrc surfactants. INTRODUCTION
At the present time, two directions in the creation of oxygen-carrying blood substitutes are being intensively pursued: on the basis of emulsions of fluorocarbons IPFCs) and on the basis of modified hemoglobin [ l J . Fluorocarbons are practically insoluble in water. They can be brought into the blood stream only if emulsified. A fluorocarbon emulsion should possess a definite intravascular stability, which depends on the colloidal properties of the emulsion and, in the first place, on the size of the particles. The smaller the particles the longer the time they remain in the blood circulation system. The first fluorocarbon emulsion to reach the stage of clinical trials was "Fluosol-DA 20", developed in 1978 by Green Cross Corporation (Osaka, Japan) 121. The progress which need to he made over the first generation emulsions should concern the reliability (purity and reproducibility) of their specific components, fluorocarbons and surfactants, their oxygen transport capacity, their intravascular persistence, their shelf life, and the minimization of their side effects. These emulsions can be stored at roo. temperature over a period of days. By development and application of more effective surfactants emulsions can be prepared vith contents of PFCs up to 50 voll. These emulsions enable a better 02-delivery. For better oxygen-carrying capabilities, not only the PFC-content is important, but the increase in stabilities of these emulsions too. Emulsion system are thermodynamically unstable and over a period of time physical changes can occur [3]. In figure 1 we illustrate the two processes of coalescence and molecular diffusion degradation, respectively. Coalescence then 1s tne tormation of a droplet from two smaller droplets, the significant step occuring when the two dro2lets get close enough to allow a contact of the droplet phases. Now in a molecular diffusion process two droplets may form a single droplet if conditions are such that one droplet is allowed to grow while the other dissolves. Hence, in the molecular diffusion process, also called Ostwald ripening, the droplet phases are not required to ccme into contact. According to kinetics of particle growth Ostvald ripening is a second-order process while coalescence is a first-order process. Therefore coalescence can be excluded [,I. NEW SURFACTANTS AND BIOCOMPATIBILITY According to our knowledge, there are three ways to retard Ostwald ripening. The first way to solve this problem is by means of suitable surfactants.
805
Copyri6ht 6 1992 by Marcel Dekker, Inc.
HEINERT ET A L .
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806
0-0 STABILISATION AGAINST OSTYALO RIPENING BY FICUlg 1:
AN
Proeerrer of plyried leyrahtioi of mrlriou
IFAC
807
PERFLUOROCARBON EMULSIONS
Ia
2,268 2,268 1,838 1.546 4,192 3,192
m
IIa
rn ma
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m
3,320
Na lvb
3,029
0.07-10.0
NO.
Compound
2,162 1,932 2,585 2,339 5,338 4,218 3,015 2,934
a.50
nemo1y.i.
*
-
+
+
+
+
+
+
11.
*
*
-
IIb
+
+
+
111a IIIb
-
IV. IVb
-
90,3
107,4 39,6 52.0 50,O 543
+
+
-
+
t
+
+
-
+
+
+
r
[blV/VlI i.a5 0.65
0.31
0.01
Molt4 Calla
HeLa cells
1. t
76,8 85,9 642 71.0 9,4 31,l 55.0 566
CONCENTRATION OF SURFACTANT 1.25 0.6s 0.31 0.01 a.50
Pluronic F60
Ib
73'2 73,2
+
+
+
+
+ +
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
-
+ +
+
+
-
-
+
-
-
+
+
+
+
-
-
Hecause, by their action a strong lowering of the interfacial tension between PFClwater is given. According to Lhe extraordinary surface activity, fluorosurfactants are especially suited as emulsifying agents. Fluorosurfactants are active even in very low concentrations; they form more stable emulsions and very small particles. But as a rule, an increase in interfacial activity is accmpanied by an increased interaction with the membranes and by this with an irreversible disturbtion of biological processes [a]. As a change in interfacial tension of PFC/water is the determing factor of emulsion stability, we put our interest in this fact. In the case of Pluronic F-68 the interfacial tension water/F-decalin is not lowered sufficiently. Therefore we synthesized new types of surfactants, consisting of a long CU chain between the perfluoroalkyl head and the hydrophilic tail. Some of their piySiCa1 propertie; are given in table I and 11. For a given perfluoroalkyl group - i.e. perfluorohexyl - the interfacial tension water/F-decalin is in a narrow range between the low values of 0.4 to 6 W/m, independant of spacer and hydrophilic group. However, the surface tension water/air
MEINERT ET AL.
808
Decalin I91 N-cyclohexylmorpholine[lo] Diaorpholinoethane N-cyclohexylpiperidine191 N-4-methylcyclohsxylpiperidine
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Dimorpholinobutane Tributylamine191
aa.0 31.0 36.0 36.0 38.0
3.4
13.0 14.0
12.1 12.0 8.0
6a.o
5.1
60.0
1.0 3.0 1.1
46.0
ia4.0
55.8
891.0
is structure depending. The longer the spacer and the hydrophilic group, the higher the surface tension water/air. By this the interfacial tension water/F-decalin is depressed strongly while the surface tension water/air must not be too low; like Pluronic. Thus, these surfactants are effective regarding the PFCs but moderate else and not damaging the cell membranes. Accordingly the surfactants are tested for hemlysis, complement activation and inhibited proliferation of HeLa cells and Molt4 cells after an exposure time of 48 hours (table 111). No complement activation is observed for these surfactants. Only surfactant Ib causes hemolyeis at concentrations higher than 0.35 (w/v). The compounds are tested at concentrations from 0.078 to 10 I(W/V). Surfactants Ia to IIb cause even at low concentrations inhibited proliferation either of HeLa cells or Wolt4 cells. Therefore they are not biocapatible. Whereas in the case of IIIa to IVb there are better results. Surfactants IIIa and IVa are at last as biocoapatible as Pluronic F-68 which inhibits proliferation of HeLa cells only at concentrations higher than 1.25 S(w/v). Therefore further biological tests are in progress. It seems, that a branched prolongator promotes biocompatibility of a surfactant more than an unbranched one.
PERFLUOROCARBONS-SELECTION CRITERIA Second way to retard degradation is by means of the perfluorocarbons.As has been found, the main route for the elimination of fluorocarbons from the organism is transpiration with the exhaled air. The rate of elimination of various fluorocarbons varies within wide limits - from a few days to several years [a]. It is well known, the elinination rate from the body also depends on the chemical nature or structure of the PFC-molecules and their solubilities in media of low polarity. The solubility of PFCs is conveniently estimated from the critical temperature of solution in n-hexane (CSTB) determined from temperature-composition phase diagrams. The CTSH is a measure of the relative solubility of the PFCs in lipids and characterizes the rate of their passage through the alveolar membranes. According to this, PFCs with CTSBs lower than 28 OC are usually considered lipohilic or oleophilic and thoee with CTSBs above 42 OC lipophobic or oleophobic [71. Well, as can be seen in table IV, a well-defined inverse correlation exists between the rate of elimination and the stability of the emulsions. lripophobic PFCs form more stable emulsions than lipophilic PFCs. The disadvantage, however is, that lipophobic PFCs have a longer retention time in the body.
NEW THIRD COMPONENTS - THE CONCEPT OF IFACS A third way against Ostwald ripening was created by the Nottingham team Lowe, Sharma,
Bbllande and Davis.
809
PERFLUOROCARBON EMULSIONS
TAlU I PHYSICAL PROPERTIES OF PERFLUORIIATEO IFA-COIPOUIOS
(Meinert e t a l . )
0n F Y-(ff2)2-W n F 0
v
u
.0AF I-(ff2)3-I A F 0
u
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dimrp h olim -
U
n
A
u
v
19U
0 F I-(ff2),4F 0
dimrpholimPerflumodimrpholim-
a5
C14Fd202
A
n
v
u
0 F I-(Cr2)6-I F 0
PHYSICAL PROPERTIES OF F-OECALIN AN0 PERFLUORINATEO HSPO-AOOIrIVES
( S h e r m s er el..
Oxygen Transport
t o Tissue, Vol.
IX)
They proposed that such unstable emulsions may be stabilized with respect to this process by the addition of small amounts of a third PFC-component, a so-called higher boiling point oil (HBW) [ E l . Our research program dealed with these interestiog findings and in the following, some remarks are given on the results. In table V some of the physico-chemical characteristics of F-decalin and the interfacial active compounds (IFACs) used by us and the BBWs, used by Lowe and co-worders, are given. The stabilizing effect of these IFACs and of HBWs on emulsions of F-decalin is illustrated in figure 2. Changes in stability parameter Dt/DO plotted against storage time for different additives are given in this figure. The stability parameter i s defined by considering the ratio of the measured diameter after storage to that immediately upon preparation. Accordingly the additives differ in their stabilizing effect. Before introducing our own theory, the two previous theories should be reflected, which a~8U.e Ostwald-Ripening to be the coarsening mechanism of PFC-enulsions. Thus Davis et a1 [I11 explain the effect of the third component as follovs: The third component bas a lower vapour pressure than the predominant oil. This added
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STABILITY PARMETER Dt /Do
10
30
20
4 0 STORAGE
TIME [ D A Y S ]
Relative stability of perfluordecalin emulsions stabilized with 2% (w/v) IFAC (based on F-diaorpholines). 0 Control:
A n=2;
n=3; W n=4; V n = 5 ;
+
n=6
CI
0
9
4
2.0
a W
c
w
f
a < a >
c d $ 1.0 L
in
0
20
60
40
(A) F-MCAUW (E) F - O E C A U N / P E R F L I . M B W P (C) F M C A L I N / P E R F L W m (0) F - U E C " / P E W P (E) F - D E C A L I N / P € R F L U L U H W T H E N E
STORAGE TIME (DAYS)
PICULB 1 Emulslfled p a r f l u o r o c h a ~ w l sas phySlOhglC8l oxygen-tta~¶POrt f l u x l a : assesment of a novel formulatron. In: OXYGEN TRANSPORT TO TISSUE. VOL. Eds. S i l v e r . 1.1%.& S i l v e r . A . , pp. 97-108, Plenun. London
Ix,
811
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PERFLUOROCARBON EMULSIONS
ia
0 1
'
IS
30
45
STORAGE TIME (DAYS)
HEINERT ET AL.
a12
IFAC
F-DIIIORPROLINO- O.lk(w/v) 9 2' ETHANE 64 11 PROPANE 58 0 BUTANE 79 20 PENTANE 83 17 REXANE 100 13
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sI
TRANSCONDENSATION RATE [i0-28& 0.54(~/~) 1' *2 49
61 75 84
96
l.Ol(w/v) 2'
11 2 14 32 16
35
a
45
2 3 3 7
49 59 79
2.04(~/~) 1' m2 23 17
--
46 50
1
a 2 1 1 2
material reduces slightly the vapour pressure of the perfluorocarbon bubbles, as defined by Raoult's law. The lower boiling PFC-molecules now move from the smaller bubbles to the larger ones (figure 1). Thus the third component is enriched in the small bubbles. The reduced vapour pressure according to Raoult's law balances the higher vapour pressure of the small bubbles (Kelvin effect). According to Lore et a1 1121. the stabilizing effect of higher boiling point oils (HBPOs) on PFC-emulsions is directly related to boiling point and hence molecular weight of the HBPO. Other groups [13, 14, 15, 161 observed that the cube of the mean radius of the particles increases linearly with time. They called the change of the cube of the mean radius with time transcondensation rate and defined the transcondensation rate as given in eq. 1, d (0)'
8 u V, C D 9 R T
@I-=
d t
(eq. 1 )
a 18 the man radllrs of the particles. o 1s the interfacial tenmol, V ls the mlar volme of subn~iare of &dIspm€d rhse. and C awl D a r e the mlubflity nd diffusim cnefficlent of the dlsFelse* S I I t e t ~ in the
&re
"=-
1
5.b "a
(eq. 2 )
"b
&re
and I am the t-tim ot the lmilndual crapolents and and 5 are the vollne fracticm of the OcDpnents UI the asperse phase.
mapenrum sdiu.
These groups have proved that the third component influences the transcondensation rate. Therefore the comon transcondensation rate is represented as given in eq. 2. In our theory we assume Ostwald-Ripening to be the mechanism of emulsion degradation. We demonstrated this for a control emulsion consisting of 20 8(w/v) F-decalin and 4 8 (w/v) Pluronic F-68. A plot of the cube of the average radius of PFC bubbles versus storage time for our emulsions stabilized by IFACa and of our control emulsion is shown in figure 3. It can be seen that the graphs consist of two different linear parts with the tranacondensation rates wl and w 2. Each linear part obeys equation 1. In PFCs, the carbon skeleton is completely surrounded by fluorine atoms and the heteroatoms are masked by perfluoroalkyl groups. Because of this PFCs are chemically inert. PFCs were regarded as ideal liquids being fully miscible with one another. The curves of interfacial tension in figure 4 show a steep decline at the beginning. These findings lead, however, to the conclusion that IFACs are enriched at the interface F-decalin/vater forming a retaining film at the interface. This film reduces Ostwald-Ripening by hindering the transmission of F-decalin from the bubble into water. The closer the retaining film is packed the better is the stabilizing effect of an IPAC. Table VI shows that the transcondensation rate w (as seen in the second part of the growth curve) decreases as increasing amounts o? IF C re added. The transcondensabut F-dimrpholinoethane tion rate w seem to have a limit at about 1.5*10-h will probabfy reach this value only at bigher concentrations.
JIB,
813
PERFLUOROCARBON EMULSIONS
Y ImN 1 ml
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I
25.0
-.
20.8
-0.0
5.0
10.0
15.8
28.8
Pigire 1: Iitcrfacial teisioi of perfliorodecaliiliitcr vs coictitratioi of
2s.8
""""
pcrfliordiwr~loliioilk~ieo ($.-(CF~li-l~l
Decisive for the stabilizing effect of the IFAC is the time needed to form the retaining film. The shorter the time emulsions grow at transcondensation rate w1, the better is the stabilizing effect. Accordingly F-dimorpholinopropane and F-dimorpholinobutane are the most effective IFACs [171. SEHIFLUORINATED ALKANES Compounds of the type CmF CnH2 +1 (RpR ) [ l S l are also considered to be IFACs. They also stabilize PFC-emuiihlons h e a d y at low concentrations. Comparable to figure 3 the PFC-emulsions, stabilized with RFRH, grow with two different transcondensations rates (figure 5). But in contrary to the IFACs discusses befor, RFRHs are enriched in the interface in a different vay. Even at small concentrations RFRHs cover the PFC-bubbles completely with a monomolecular film. Accordingly the curves of the interfacial tension habe a deep decline at the beginning and reach a plateau at concentrations below 28 (figure 6 ) . Therefore RFRHS must be incorporated in the PFC/water interface in the way that R -chains are projecting into and the RH-Chains are jutting out of the PFC-bubbfes. Thus the PFC-bubbles act in their outer spheres like hydrocarbon-bubbles and the R -chains are used as anchor-groups for lipophilic surfactants. Thereby it is possihe to avoid the problems of fluorosurfactans. Therefore it can be concluded: In order to stabilize an emulsion against degradation by the effect of a third component. an IFAC-additive must be added, which even in small concentrations forms a strong retaining film and offers an acceptable half-life in the body.
MEINERT ET AL.
8
/
r
I
/1
10
9
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6
4
1
1
0
10
to
.
I
.
STORAGE
30
.
.
.
I
.
.
TIME [DAYS]
FIG 5: Cube of naan diameter of partieels as function of time for emulsions of perfluorodeeslin stabilized srith Pluronic i68 and 1 : ur 2 Z l w / r ) of various RpRHs. 1. perfluorodecalin; 2. C#l$sH17
1221; 3 . C6FI3CgHI7 1 1 2 ) ;
4. CgF17CzH5 1211; 5 . CgF17C2Hg ( 1 2 ) ; 6 . C ~ O F ~ ~ C12:? ); H ~ 7 . C ~ O F Z ~ C(i11 ~H~
NBT PERFLUORCARBONS For that reason we have prepared several derivatives of wrpholine, piperidine and pyrrolidine via electrochemical fluorination [I91
.
As a part of our studies on PFC-oxygen-transport-eDulsions we have found that these perf luorinated compound6 exhibit favorable properties as IFAC-additives and as oxy-
gen-carrying agents in t e r m of both the emulsifiability, particle size stability and the phanadynamical properties. Within the same series of perf luoro-di-mrpbolinee or -di-piperidines by bridging the heterocycles with CF2-groups a continuous cbange in the wlecular weights and thereby in the pbysical properties is given (table VII - 1x1. Thus depending on the length of the CFZ-chain there only is a change of the distances of the polar centers but not in the positions of the heteroatoma each to the other. By the synthes; 7 of perf luoro-cyclohexylmorpholine, we have an excellent oxygencarrier, qualified for long-time stable emulsions (table X I .
a15
PERFLUOROCARBON EMULSIONS
60
Downloaded by [Universite Laval] at 09:03 21 April 2016
50
L L
FIG. 6: Interfacial tension of perfluorodecalin/uater vs concentration ot REX4 1. C ~ F ~ ~ C ~2. H CIOFz1C2AS; S ; 3 . CgF13CgH17
n F N-(ff,X-N
0
W
F 0
U
c 3 F N - (CFz),-N
F
n=0-6
FICUU 7: lerfliorocarbois apthesized ria BCF
816
MEINERT ET AL.
TllU VII Calculated Propertles Of Perfluorocarbons TSI
hr.
-CI
I 'C1
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-
"0
[
nW24hl
'
112
I dl
-
D1 m r p h o l 1nyl
0
20
4eo
125
21)
52
8.3
5
Dlmrpbol lnorrthana
1
31
510
144
15
50
2.9
I1 24
Dlmrpbol lnoethane
2
36
560
1e2
8
49
1.0
Dlmrpbol i n o p r o p n e
3
41
eio
178
4
47
0.3
55
Dl~rphollnohutnne
4
ae
660
194
3
4e
0. I
124
Dlmrphollnopentane
5
51
710
209
2
45
0.04
202
D l m r p b o l lrmhexane
e
56
780
224
1
43
0.01
043
Dlplperldyl
0
36
528
153
11
49
5.7
24
I
41
5713
170
6
48
2.0
55
Dlpl per Id1 no-tba
ne
Dlpl p r Id1 noetbnne
2
46
828
I06
3
46
0.7
124
Dlplerldlnopropane
7
51
678
202
2
45
0.2
282
Dlplperldlaobutane
4
56
?28
217
1
44
0.08
643
Dlplperldlnopentnne
5
51
778
231
1
43
0.03
i4ez
Dlplperldlnohornno
6
86
828
245
1
42
0.01
1327
TABLE VIII ECF of 0i.orpholino-derirmtivmi
mtmrting meteriml DrDduCtl
n n 0 F N - N FO
u u
n
n
OAN-CF2-N F 0
3
141
50
11
28
164
44
24
25
182
43
55
30
198
41
124
26
215
41
280
20
225
40
640
U
n
n
U
U
n
A
0 F N-ICF212- N F 0
0 FN-ICF,I,-N F 0
u
U
A
A
u
U
n
n
0 FN-ICF2Is-N FO
0 FN-ICF216-N FO U
U
celculetmd v m l u e i
TABLE IX ECF o f Oipipcridino-darirstirss starting matarial
CN-ltH2tn-N3
.sin
yimld
products
-N
s
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@-cF~-NTJ
-
02-aolub
bp
('t)
( 'I
n = 0
6
h a l f - l i r a elms* (days)
(rOl.X.37.C)
I
140-150
49
24
3
162-175
413
55
TllLK 1: ECF of rorpholinoc~clo~excie yield:
43 X
bp:
147.5
-
1413,5
12 Tarr. 37.C
p,:
oc
,
Product d l S t r i b u t l o n bp
("CI 141)
0 2 -solub. CSTH ( ~ o l . X . 3 7 ~ C I('C)
(151)'
47
excretion rate (days1
(50)-
11 (311'
13
(50)'
(413).
139
A
\
2 X
'
0 FN-C6F,3
(149)
U
* calculated ralues
for campariaon: bp
0 -solub.
(OC)
(v0l.X.37~C)
2
CSTH
I'C)
eacretion rate *
P,
(Tarr,37*C)
(days)
FOC
142
42
22
13
7
FTBA
177
41
so
2
I20
818
MEINERT ET AL.
On ECF of rorpholino-cyclohexene(1) as main products F-cyclohexylborpholine and F-nhexylwrpholine are formed.. In a side reaction splitting of the rorpholine ring takes place. The synthesis of new perfluorocarbona, acting both as oxygen carriers and as IFAC-additives, and of new classes of surfactants is our contribution to the preparation of PFC emulsions of second generation and to the understanding of their physicochemical properties. ACKNOWLEDGEMENTS
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We thank KaliChemie AG, Uannover, FRG and Hoechst AG, Gendorf, FRG for support of chemicals. All cytotoxic testing8 were performed by H. Northoff and L. Haidmann, DRKBlutspendezentrale Ulm, 7900 Ulm, FRO. This work has been carried out with financial support of the Bundesministerium fllr Forschung and Technologie, Federal Republic of Germany. REFERENCES [I] K.C. Lowe, Blood Substitutes, Ellis Horwood, Chichester, 1988 121 The Green Cross Corporation, FC-43 Emulsion, Technical Information Ser. No. 3, Osaka, 1976 [31 W.I. Uiguchi and J. Nisra, J. Pharm. Sci. 51, 459 (1962) [41 A.S. Kabal'nov, Yu.D. Aprosin, O.B. Pavlova-Verevkina, A.V. Pertsov and E.D. Shchukin, Kolloidnyi Zhurnal, 48, 27 (1986) 151 E. Dellacherie, P. Labrude, C. Vigneron and J.G. Riess, CRC Crit. Rev. in Therapeutic Drug Carrier Systems, Vol. 3, Issue I, 41 (1987) [61 J.G. Riess, Artificial Organs, 8, 44 (1984) [71 G.Ya. Rozenberg and K.N. Nakarov, Zhurnal Vses. Khim. Ob-va ia. D.I. Nendeleeva, 30, 27 (1985) [8] S.K. Sharma, A.D. Bollands, S.S. Davis and K.C. Lore, Oxygen Transport to Tissue, IX, I.A. Silver and A . Silver, 1987 p. 97ff; S.K. Sharaa, K.C. Lowe and S.S. Davis, Biomat., Art, Cells, Art, Org., 16 (1988) 447 [9] K. Yamanouchi, N. Tanaka, Y. Tsuda, K. Yokoyaba, S. Awazu and Y . Kobayashi, Chem. Pharm. bull, 33, 1221 (1985) 1101 U. Neinert and J. Blader, in Instrumentalized Analytical Chemistry and Computer Technology (W. Gllnther, J.P. Natthes and H . 4 . Perkampus, eds.) GIT Verlag, DarMtadt, 1990 [111 S.S. Davis, H.P. Round and T.S. Purewal, J. Coll Interface Sci., 80. 508 (1981) [12] S.K. S h a m , A.D. Bollands, S.S. Davis and K.C. Lowe, in Oxygen Transport to Tissue, IX (I.A. Silver ans A. Silver, eds.), 1987 [131 A.S. Kabal'nov. A.V. Pertsov, Yu.D. Aprosin and E.D. Shchukin, Kolloidnyi Zhurnal, 41, 1048 (1985) [141 A.S. Kabal'nov, Yu.D. Nakarov, L.L. Gervits, A.V. Pertsov and E.D. Shchukin, Kolloidnyi Zhurnal, 48, 393 (1986) [151 0.6. Pavlova-Verevkina, Yu.D. Aprosin, L.V. Novopashina and N.I. Afonin, Kolloidnyi Zhurnal, 49, 178 (1987) [161 A.V, PertBOV, A.S. Kabal'nov and E.D. Shchukin, Kolloidnyi Zhurnal, 46, 1172 (1984) [171 U. Neinert and J. Nader. J. Fluorine Chem., 51, 335 (1991) [la] U.P. Turberg and J.E. Brady, J. Am. Chem. SOC., 110. 7797 (1988) [191 8. Neinert. R. Fackler. J. Nader, P.Reuter and W. Rbhlke, J. Fluorine Chem., 51, 53 (1991)
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
On the Perluorocarbon Enulsions of Second Generation H. Meinert, R. Fackler, A. Knoblich, J. Nader, P. Renter & W. Röhike To cite this article: H. Meinert, R. Fackler, A. Knoblich, J. Nader, P. Renter & W. Röhike (1992) On the Perluorocarbon Enulsions of Second Generation, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 805-818, DOI: 10.3109/10731199209119722 To link to this article: http://dx.doi.org/10.3109/10731199209119722
Published online: 11 Jul 2009.
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Date: 21 April 2016, At: 09:03
BIOMT., ART. CELLS a IWOB. BIOTECH.,
ON
TEE PERF-
zo(z-/,),
805-818 (1992)
-1oIIS
OF SECOND G W T I O N
8 . Ieinert, R. Fackler, A. Knoblich, J. Iader, P. Reuter and 1. R6hlke, University of Ulm, Dep. Chemistry of Bi-patible Caponnds,
D-7900 Ulm, Parkatr. 11, Federal Republic of Germany
Downloaded by [Universite Laval] at 09:03 21 April 2016
ABSTRACT Perfluorocarbon-emulsions of second generation were prepared by means of new perf luorocarbons (F-dimorpholines, F-dipiperidines and F-cyclohexylwrpholine) , acting both as oxygen carriers and as interfacial active compounds (IFACs). The stabilizing effect of these IFACs is interpreted and a new theory is introduced. Also new classes of fluorosurfactants were synthesized and tested for biocompatibility. In PFC mixtures compounds of the type R R (RF=C,F2m+1, RH=CnH2n+l) are acting as IFACs but also as anchor-groups for lipophflrc surfactants. INTRODUCTION
At the present time, two directions in the creation of oxygen-carrying blood substitutes are being intensively pursued: on the basis of emulsions of fluorocarbons IPFCs) and on the basis of modified hemoglobin [ l J . Fluorocarbons are practically insoluble in water. They can be brought into the blood stream only if emulsified. A fluorocarbon emulsion should possess a definite intravascular stability, which depends on the colloidal properties of the emulsion and, in the first place, on the size of the particles. The smaller the particles the longer the time they remain in the blood circulation system. The first fluorocarbon emulsion to reach the stage of clinical trials was "Fluosol-DA 20", developed in 1978 by Green Cross Corporation (Osaka, Japan) 121. The progress which need to he made over the first generation emulsions should concern the reliability (purity and reproducibility) of their specific components, fluorocarbons and surfactants, their oxygen transport capacity, their intravascular persistence, their shelf life, and the minimization of their side effects. These emulsions can be stored at roo. temperature over a period of days. By development and application of more effective surfactants emulsions can be prepared vith contents of PFCs up to 50 voll. These emulsions enable a better 02-delivery. For better oxygen-carrying capabilities, not only the PFC-content is important, but the increase in stabilities of these emulsions too. Emulsion system are thermodynamically unstable and over a period of time physical changes can occur [3]. In figure 1 we illustrate the two processes of coalescence and molecular diffusion degradation, respectively. Coalescence then 1s tne tormation of a droplet from two smaller droplets, the significant step occuring when the two dro2lets get close enough to allow a contact of the droplet phases. Now in a molecular diffusion process two droplets may form a single droplet if conditions are such that one droplet is allowed to grow while the other dissolves. Hence, in the molecular diffusion process, also called Ostwald ripening, the droplet phases are not required to ccme into contact. According to kinetics of particle growth Ostvald ripening is a second-order process while coalescence is a first-order process. Therefore coalescence can be excluded [,I. NEW SURFACTANTS AND BIOCOMPATIBILITY According to our knowledge, there are three ways to retard Ostwald ripening. The first way to solve this problem is by means of suitable surfactants.
805
Copyri6ht 6 1992 by Marcel Dekker, Inc.
HEINERT ET A L .
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806
0-0 STABILISATION AGAINST OSTYALO RIPENING BY FICUlg 1:
AN
Proeerrer of plyried leyrahtioi of mrlriou
IFAC
807
PERFLUOROCARBON EMULSIONS
Ia
2,268 2,268 1,838 1.546 4,192 3,192
m
IIa
rn ma
Downloaded by [Universite Laval] at 09:03 21 April 2016
m
3,320
Na lvb
3,029
0.07-10.0
NO.
Compound
2,162 1,932 2,585 2,339 5,338 4,218 3,015 2,934
a.50
nemo1y.i.
*
-
+
+
+
+
+
+
11.
*
*
-
IIb
+
+
+
111a IIIb
-
IV. IVb
-
90,3
107,4 39,6 52.0 50,O 543
+
+
-
+
t
+
+
-
+
+
+
r
[blV/VlI i.a5 0.65
0.31
0.01
Molt4 Calla
HeLa cells
1. t
76,8 85,9 642 71.0 9,4 31,l 55.0 566
CONCENTRATION OF SURFACTANT 1.25 0.6s 0.31 0.01 a.50
Pluronic F60
Ib
73'2 73,2
+
+
+
+
+ +
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
-
+ +
+
+
-
-
+
-
-
+
+
+
+
-
-
Hecause, by their action a strong lowering of the interfacial tension between PFClwater is given. According to Lhe extraordinary surface activity, fluorosurfactants are especially suited as emulsifying agents. Fluorosurfactants are active even in very low concentrations; they form more stable emulsions and very small particles. But as a rule, an increase in interfacial activity is accmpanied by an increased interaction with the membranes and by this with an irreversible disturbtion of biological processes [a]. As a change in interfacial tension of PFC/water is the determing factor of emulsion stability, we put our interest in this fact. In the case of Pluronic F-68 the interfacial tension water/F-decalin is not lowered sufficiently. Therefore we synthesized new types of surfactants, consisting of a long CU chain between the perfluoroalkyl head and the hydrophilic tail. Some of their piySiCa1 propertie; are given in table I and 11. For a given perfluoroalkyl group - i.e. perfluorohexyl - the interfacial tension water/F-decalin is in a narrow range between the low values of 0.4 to 6 W/m, independant of spacer and hydrophilic group. However, the surface tension water/air
MEINERT ET AL.
808
Decalin I91 N-cyclohexylmorpholine[lo] Diaorpholinoethane N-cyclohexylpiperidine191 N-4-methylcyclohsxylpiperidine
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Dimorpholinobutane Tributylamine191
aa.0 31.0 36.0 36.0 38.0
3.4
13.0 14.0
12.1 12.0 8.0
6a.o
5.1
60.0
1.0 3.0 1.1
46.0
ia4.0
55.8
891.0
is structure depending. The longer the spacer and the hydrophilic group, the higher the surface tension water/air. By this the interfacial tension water/F-decalin is depressed strongly while the surface tension water/air must not be too low; like Pluronic. Thus, these surfactants are effective regarding the PFCs but moderate else and not damaging the cell membranes. Accordingly the surfactants are tested for hemlysis, complement activation and inhibited proliferation of HeLa cells and Molt4 cells after an exposure time of 48 hours (table 111). No complement activation is observed for these surfactants. Only surfactant Ib causes hemolyeis at concentrations higher than 0.35 (w/v). The compounds are tested at concentrations from 0.078 to 10 I(W/V). Surfactants Ia to IIb cause even at low concentrations inhibited proliferation either of HeLa cells or Wolt4 cells. Therefore they are not biocapatible. Whereas in the case of IIIa to IVb there are better results. Surfactants IIIa and IVa are at last as biocoapatible as Pluronic F-68 which inhibits proliferation of HeLa cells only at concentrations higher than 1.25 S(w/v). Therefore further biological tests are in progress. It seems, that a branched prolongator promotes biocompatibility of a surfactant more than an unbranched one.
PERFLUOROCARBONS-SELECTION CRITERIA Second way to retard degradation is by means of the perfluorocarbons.As has been found, the main route for the elimination of fluorocarbons from the organism is transpiration with the exhaled air. The rate of elimination of various fluorocarbons varies within wide limits - from a few days to several years [a]. It is well known, the elinination rate from the body also depends on the chemical nature or structure of the PFC-molecules and their solubilities in media of low polarity. The solubility of PFCs is conveniently estimated from the critical temperature of solution in n-hexane (CSTB) determined from temperature-composition phase diagrams. The CTSH is a measure of the relative solubility of the PFCs in lipids and characterizes the rate of their passage through the alveolar membranes. According to this, PFCs with CTSBs lower than 28 OC are usually considered lipohilic or oleophilic and thoee with CTSBs above 42 OC lipophobic or oleophobic [71. Well, as can be seen in table IV, a well-defined inverse correlation exists between the rate of elimination and the stability of the emulsions. lripophobic PFCs form more stable emulsions than lipophilic PFCs. The disadvantage, however is, that lipophobic PFCs have a longer retention time in the body.
NEW THIRD COMPONENTS - THE CONCEPT OF IFACS A third way against Ostwald ripening was created by the Nottingham team Lowe, Sharma,
Bbllande and Davis.
809
PERFLUOROCARBON EMULSIONS
TAlU I PHYSICAL PROPERTIES OF PERFLUORIIATEO IFA-COIPOUIOS
(Meinert e t a l . )
0n F Y-(ff2)2-W n F 0
v
u
.0AF I-(ff2)3-I A F 0
u
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dimrp h olim -
U
n
A
u
v
19U
0 F I-(ff2),4F 0
dimrpholimPerflumodimrpholim-
a5
C14Fd202
A
n
v
u
0 F I-(Cr2)6-I F 0
PHYSICAL PROPERTIES OF F-OECALIN AN0 PERFLUORINATEO HSPO-AOOIrIVES
( S h e r m s er el..
Oxygen Transport
t o Tissue, Vol.
IX)
They proposed that such unstable emulsions may be stabilized with respect to this process by the addition of small amounts of a third PFC-component, a so-called higher boiling point oil (HBW) [ E l . Our research program dealed with these interestiog findings and in the following, some remarks are given on the results. In table V some of the physico-chemical characteristics of F-decalin and the interfacial active compounds (IFACs) used by us and the BBWs, used by Lowe and co-worders, are given. The stabilizing effect of these IFACs and of HBWs on emulsions of F-decalin is illustrated in figure 2. Changes in stability parameter Dt/DO plotted against storage time for different additives are given in this figure. The stability parameter i s defined by considering the ratio of the measured diameter after storage to that immediately upon preparation. Accordingly the additives differ in their stabilizing effect. Before introducing our own theory, the two previous theories should be reflected, which a~8U.e Ostwald-Ripening to be the coarsening mechanism of PFC-enulsions. Thus Davis et a1 [I11 explain the effect of the third component as follovs: The third component bas a lower vapour pressure than the predominant oil. This added
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STABILITY PARMETER Dt /Do
10
30
20
4 0 STORAGE
TIME [ D A Y S ]
Relative stability of perfluordecalin emulsions stabilized with 2% (w/v) IFAC (based on F-diaorpholines). 0 Control:
A n=2;
n=3; W n=4; V n = 5 ;
+
n=6
CI
0
9
4
2.0
a W
c
w
f
a < a >
c d $ 1.0 L
in
0
20
60
40
(A) F-MCAUW (E) F - O E C A U N / P E R F L I . M B W P (C) F M C A L I N / P E R F L W m (0) F - U E C " / P E W P (E) F - D E C A L I N / P € R F L U L U H W T H E N E
STORAGE TIME (DAYS)
PICULB 1 Emulslfled p a r f l u o r o c h a ~ w l sas phySlOhglC8l oxygen-tta~¶POrt f l u x l a : assesment of a novel formulatron. In: OXYGEN TRANSPORT TO TISSUE. VOL. Eds. S i l v e r . 1.1%.& S i l v e r . A . , pp. 97-108, Plenun. London
Ix,
811
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PERFLUOROCARBON EMULSIONS
ia
0 1
'
IS
30
45
STORAGE TIME (DAYS)
HEINERT ET AL.
a12
IFAC
F-DIIIORPROLINO- O.lk(w/v) 9 2' ETHANE 64 11 PROPANE 58 0 BUTANE 79 20 PENTANE 83 17 REXANE 100 13
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sI
TRANSCONDENSATION RATE [i0-28& 0.54(~/~) 1' *2 49
61 75 84
96
l.Ol(w/v) 2'
11 2 14 32 16
35
a
45
2 3 3 7
49 59 79
2.04(~/~) 1' m2 23 17
--
46 50
1
a 2 1 1 2
material reduces slightly the vapour pressure of the perfluorocarbon bubbles, as defined by Raoult's law. The lower boiling PFC-molecules now move from the smaller bubbles to the larger ones (figure 1). Thus the third component is enriched in the small bubbles. The reduced vapour pressure according to Raoult's law balances the higher vapour pressure of the small bubbles (Kelvin effect). According to Lore et a1 1121. the stabilizing effect of higher boiling point oils (HBPOs) on PFC-emulsions is directly related to boiling point and hence molecular weight of the HBPO. Other groups [13, 14, 15, 161 observed that the cube of the mean radius of the particles increases linearly with time. They called the change of the cube of the mean radius with time transcondensation rate and defined the transcondensation rate as given in eq. 1, d (0)'
8 u V, C D 9 R T
@I-=
d t
(eq. 1 )
a 18 the man radllrs of the particles. o 1s the interfacial tenmol, V ls the mlar volme of subn~iare of &dIspm€d rhse. and C awl D a r e the mlubflity nd diffusim cnefficlent of the dlsFelse* S I I t e t ~ in the
&re
"=-
1
5.b "a
(eq. 2 )
"b
&re
and I am the t-tim ot the lmilndual crapolents and and 5 are the vollne fracticm of the OcDpnents UI the asperse phase.
mapenrum sdiu.
These groups have proved that the third component influences the transcondensation rate. Therefore the comon transcondensation rate is represented as given in eq. 2. In our theory we assume Ostwald-Ripening to be the mechanism of emulsion degradation. We demonstrated this for a control emulsion consisting of 20 8(w/v) F-decalin and 4 8 (w/v) Pluronic F-68. A plot of the cube of the average radius of PFC bubbles versus storage time for our emulsions stabilized by IFACa and of our control emulsion is shown in figure 3. It can be seen that the graphs consist of two different linear parts with the tranacondensation rates wl and w 2. Each linear part obeys equation 1. In PFCs, the carbon skeleton is completely surrounded by fluorine atoms and the heteroatoms are masked by perfluoroalkyl groups. Because of this PFCs are chemically inert. PFCs were regarded as ideal liquids being fully miscible with one another. The curves of interfacial tension in figure 4 show a steep decline at the beginning. These findings lead, however, to the conclusion that IFACs are enriched at the interface F-decalin/vater forming a retaining film at the interface. This film reduces Ostwald-Ripening by hindering the transmission of F-decalin from the bubble into water. The closer the retaining film is packed the better is the stabilizing effect of an IPAC. Table VI shows that the transcondensation rate w (as seen in the second part of the growth curve) decreases as increasing amounts o? IF C re added. The transcondensabut F-dimrpholinoethane tion rate w seem to have a limit at about 1.5*10-h will probabfy reach this value only at bigher concentrations.
JIB,
813
PERFLUOROCARBON EMULSIONS
Y ImN 1 ml
Downloaded by [Universite Laval] at 09:03 21 April 2016
I
25.0
-.
20.8
-0.0
5.0
10.0
15.8
28.8
Pigire 1: Iitcrfacial teisioi of perfliorodecaliiliitcr vs coictitratioi of
2s.8
""""
pcrfliordiwr~loliioilk~ieo ($.-(CF~li-l~l
Decisive for the stabilizing effect of the IFAC is the time needed to form the retaining film. The shorter the time emulsions grow at transcondensation rate w1, the better is the stabilizing effect. Accordingly F-dimorpholinopropane and F-dimorpholinobutane are the most effective IFACs [171. SEHIFLUORINATED ALKANES Compounds of the type CmF CnH2 +1 (RpR ) [ l S l are also considered to be IFACs. They also stabilize PFC-emuiihlons h e a d y at low concentrations. Comparable to figure 3 the PFC-emulsions, stabilized with RFRH, grow with two different transcondensations rates (figure 5). But in contrary to the IFACs discusses befor, RFRHs are enriched in the interface in a different vay. Even at small concentrations RFRHs cover the PFC-bubbles completely with a monomolecular film. Accordingly the curves of the interfacial tension habe a deep decline at the beginning and reach a plateau at concentrations below 28 (figure 6 ) . Therefore RFRHS must be incorporated in the PFC/water interface in the way that R -chains are projecting into and the RH-Chains are jutting out of the PFC-bubbfes. Thus the PFC-bubbles act in their outer spheres like hydrocarbon-bubbles and the R -chains are used as anchor-groups for lipophilic surfactants. Thereby it is possihe to avoid the problems of fluorosurfactans. Therefore it can be concluded: In order to stabilize an emulsion against degradation by the effect of a third component. an IFAC-additive must be added, which even in small concentrations forms a strong retaining film and offers an acceptable half-life in the body.
MEINERT ET AL.
8
/
r
I
/1
10
9
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6
4
1
1
0
10
to
.
I
.
STORAGE
30
.
.
.
I
.
.
TIME [DAYS]
FIG 5: Cube of naan diameter of partieels as function of time for emulsions of perfluorodeeslin stabilized srith Pluronic i68 and 1 : ur 2 Z l w / r ) of various RpRHs. 1. perfluorodecalin; 2. C#l$sH17
1221; 3 . C6FI3CgHI7 1 1 2 ) ;
4. CgF17CzH5 1211; 5 . CgF17C2Hg ( 1 2 ) ; 6 . C ~ O F ~ ~ C12:? ); H ~ 7 . C ~ O F Z ~ C(i11 ~H~
NBT PERFLUORCARBONS For that reason we have prepared several derivatives of wrpholine, piperidine and pyrrolidine via electrochemical fluorination [I91
.
As a part of our studies on PFC-oxygen-transport-eDulsions we have found that these perf luorinated compound6 exhibit favorable properties as IFAC-additives and as oxy-
gen-carrying agents in t e r m of both the emulsifiability, particle size stability and the phanadynamical properties. Within the same series of perf luoro-di-mrpbolinee or -di-piperidines by bridging the heterocycles with CF2-groups a continuous cbange in the wlecular weights and thereby in the pbysical properties is given (table VII - 1x1. Thus depending on the length of the CFZ-chain there only is a change of the distances of the polar centers but not in the positions of the heteroatoma each to the other. By the synthes; 7 of perf luoro-cyclohexylmorpholine, we have an excellent oxygencarrier, qualified for long-time stable emulsions (table X I .
a15
PERFLUOROCARBON EMULSIONS
60
Downloaded by [Universite Laval] at 09:03 21 April 2016
50
L L
FIG. 6: Interfacial tension of perfluorodecalin/uater vs concentration ot REX4 1. C ~ F ~ ~ C ~2. H CIOFz1C2AS; S ; 3 . CgF13CgH17
n F N-(ff,X-N
0
W
F 0
U
c 3 F N - (CFz),-N
F
n=0-6
FICUU 7: lerfliorocarbois apthesized ria BCF
816
MEINERT ET AL.
TllU VII Calculated Propertles Of Perfluorocarbons TSI
hr.
-CI
I 'C1
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-
"0
[
nW24hl
'
112
I dl
-
D1 m r p h o l 1nyl
0
20
4eo
125
21)
52
8.3
5
Dlmrpbol lnorrthana
1
31
510
144
15
50
2.9
I1 24
Dlmrpbol lnoethane
2
36
560
1e2
8
49
1.0
Dlmrpbol i n o p r o p n e
3
41
eio
178
4
47
0.3
55
Dl~rphollnohutnne
4
ae
660
194
3
4e
0. I
124
Dlmrphollnopentane
5
51
710
209
2
45
0.04
202
D l m r p b o l lrmhexane
e
56
780
224
1
43
0.01
043
Dlplperldyl
0
36
528
153
11
49
5.7
24
I
41
5713
170
6
48
2.0
55
Dlpl per Id1 no-tba
ne
Dlpl p r Id1 noetbnne
2
46
828
I06
3
46
0.7
124
Dlplerldlnopropane
7
51
678
202
2
45
0.2
282
Dlplperldlaobutane
4
56
?28
217
1
44
0.08
643
Dlplperldlnopentnne
5
51
778
231
1
43
0.03
i4ez
Dlplperldlnohornno
6
86
828
245
1
42
0.01
1327
TABLE VIII ECF of 0i.orpholino-derirmtivmi
mtmrting meteriml DrDduCtl
n n 0 F N - N FO
u u
n
n
OAN-CF2-N F 0
3
141
50
11
28
164
44
24
25
182
43
55
30
198
41
124
26
215
41
280
20
225
40
640
U
n
n
U
U
n
A
0 F N-ICF212- N F 0
0 FN-ICF,I,-N F 0
u
U
A
A
u
U
n
n
0 FN-ICF2Is-N FO
0 FN-ICF216-N FO U
U
celculetmd v m l u e i
TABLE IX ECF o f Oipipcridino-darirstirss starting matarial
CN-ltH2tn-N3
.sin
yimld
products
-N
s
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@-cF~-NTJ
-
02-aolub
bp
('t)
( 'I
n = 0
6
h a l f - l i r a elms* (days)
(rOl.X.37.C)
I
140-150
49
24
3
162-175
413
55
TllLK 1: ECF of rorpholinoc~clo~excie yield:
43 X
bp:
147.5
-
1413,5
12 Tarr. 37.C
p,:
oc
,
Product d l S t r i b u t l o n bp
("CI 141)
0 2 -solub. CSTH ( ~ o l . X . 3 7 ~ C I('C)
(151)'
47
excretion rate (days1
(50)-
11 (311'
13
(50)'
(413).
139
A
\
2 X
'
0 FN-C6F,3
(149)
U
* calculated ralues
for campariaon: bp
0 -solub.
(OC)
(v0l.X.37~C)
2
CSTH
I'C)
eacretion rate *
P,
(Tarr,37*C)
(days)
FOC
142
42
22
13
7
FTBA
177
41
so
2
I20
818
MEINERT ET AL.
On ECF of rorpholino-cyclohexene(1) as main products F-cyclohexylborpholine and F-nhexylwrpholine are formed.. In a side reaction splitting of the rorpholine ring takes place. The synthesis of new perfluorocarbona, acting both as oxygen carriers and as IFAC-additives, and of new classes of surfactants is our contribution to the preparation of PFC emulsions of second generation and to the understanding of their physicochemical properties. ACKNOWLEDGEMENTS
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We thank KaliChemie AG, Uannover, FRG and Hoechst AG, Gendorf, FRG for support of chemicals. All cytotoxic testing8 were performed by H. Northoff and L. Haidmann, DRKBlutspendezentrale Ulm, 7900 Ulm, FRO. This work has been carried out with financial support of the Bundesministerium fllr Forschung and Technologie, Federal Republic of Germany. REFERENCES [I] K.C. Lowe, Blood Substitutes, Ellis Horwood, Chichester, 1988 121 The Green Cross Corporation, FC-43 Emulsion, Technical Information Ser. No. 3, Osaka, 1976 [31 W.I. Uiguchi and J. Nisra, J. Pharm. Sci. 51, 459 (1962) [41 A.S. Kabal'nov, Yu.D. Aprosin, O.B. Pavlova-Verevkina, A.V. Pertsov and E.D. Shchukin, Kolloidnyi Zhurnal, 48, 27 (1986) 151 E. Dellacherie, P. Labrude, C. Vigneron and J.G. Riess, CRC Crit. Rev. in Therapeutic Drug Carrier Systems, Vol. 3, Issue I, 41 (1987) [61 J.G. Riess, Artificial Organs, 8, 44 (1984) [71 G.Ya. Rozenberg and K.N. Nakarov, Zhurnal Vses. Khim. Ob-va ia. D.I. Nendeleeva, 30, 27 (1985) [8] S.K. Sharma, A.D. Bollands, S.S. Davis and K.C. Lore, Oxygen Transport to Tissue, IX, I.A. Silver and A . Silver, 1987 p. 97ff; S.K. Sharaa, K.C. Lowe and S.S. Davis, Biomat., Art, Cells, Art, Org., 16 (1988) 447 [9] K. Yamanouchi, N. Tanaka, Y. Tsuda, K. Yokoyaba, S. Awazu and Y . Kobayashi, Chem. Pharm. bull, 33, 1221 (1985) 1101 U. Neinert and J. Blader, in Instrumentalized Analytical Chemistry and Computer Technology (W. Gllnther, J.P. Natthes and H . 4 . Perkampus, eds.) GIT Verlag, DarMtadt, 1990 [111 S.S. Davis, H.P. Round and T.S. Purewal, J. Coll Interface Sci., 80. 508 (1981) [12] S.K. S h a m , A.D. Bollands, S.S. Davis and K.C. Lowe, in Oxygen Transport to Tissue, IX (I.A. Silver ans A. Silver, eds.), 1987 [131 A.S. Kabal'nov. A.V. Pertsov, Yu.D. Aprosin and E.D. Shchukin, Kolloidnyi Zhurnal, 41, 1048 (1985) [141 A.S. Kabal'nov, Yu.D. Nakarov, L.L. Gervits, A.V. Pertsov and E.D. Shchukin, Kolloidnyi Zhurnal, 48, 393 (1986) [151 0.6. Pavlova-Verevkina, Yu.D. Aprosin, L.V. Novopashina and N.I. Afonin, Kolloidnyi Zhurnal, 49, 178 (1987) [161 A.V, PertBOV, A.S. Kabal'nov and E.D. Shchukin, Kolloidnyi Zhurnal, 46, 1172 (1984) [171 U. Neinert and J. Nader. J. Fluorine Chem., 51, 335 (1991) [la] U.P. Turberg and J.E. Brady, J. Am. Chem. SOC., 110. 7797 (1988) [191 8. Neinert. R. Fackler. J. Nader, P.Reuter and W. Rbhlke, J. Fluorine Chem., 51, 53 (1991)
