Anal. Chem. 1903, 65,2254-2261
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Analytical SPLITT Fractionation in the Diffusion Mode Operating as a Dialysis-like System Devoid of Membrane. Application to Drug-Carrying Liposomes Shulamit Levin. and Galia Tawil Pharmaceutical Chemistry Department, School of Pharmacy, P.O.B. 12065, The Hebrew University of Jerusalem, Jerusalem 91120, Israel
This work reports a new technique for the rapid measurement of the fraction of free substrates in colloidal suspensions, specifically drugs in liposomes, usinganalytical SPLITT fractionation. The SPLITT fractionation channel mimics a dialysislike separation utilizing differential transport across a n ultrathin (-100 pm) lamina flowing through the cell. The fast-diffusing free drug molecules cross this thin lamina and dissipate over the channel, while the slowly diffusing colloids are confined to a narrow region next to their introduction side. The liquid stream in the cell is split into two substreams at the outlet. One of the substreams contains the colloids as well as the free drug, whereas the other substream contains only free drug molecules. The entire process is realized within less than 1 min, and a quantitative treatment of the signal of the substream containing the free drug facilitates the determination of the fraction of free drug in the suspension. This approach has been tested using a number of drugloaded liposomes. The results show that the distribution of drugs between the vesicles and the continuous aqueous phase in the suspension can be characterized by analytical SPLITT fractionation. Studies of several hydrophilic drugs entrapped i n liposomes showed that almost all the drug in the liposomal formulation was not entrapped,and in some cases, the amount varied with the conditions of the analysis due to drug burst from the liposomes. This occurrence can be observed using other techniques during which dilution is inherent to the procedure of analysis and can be used to screen formulations for resistance toward dilution. INTRODUCTION Split-flow thin (SPLITT) cells are ultrathin long unpacked flow channels, equipped with two inlets and two outlets, in which the flow is laminar. These cells were recently developed as tools for the continuous separation of suspended or solubilized components entering the cell at one inlet port.l-ll (1)Giddings, J. C. Sep. Sci. Technol. 1985,20,749-768. (2)Giddings, J. C. Sep. Sci. Technol. 1986,21,831-843. (3)Giddings, J. C. Sep. Sci. Technol. 1988,23,119-131. (4)Giddings, J. C. Sep. Sci. Technol. 1988,23,931-943. (5)Levin, S.;Myers, M. N.; Giddings, J. C. Sep. Sci. Technol. 1989, 24, 1245-1258. (6)Springston, S.R.; Myers, M. N.; Giddings, J. C. Anal. Chem. 1987, 59,344-350. (7)Fuh, C. B.; Myers, M. N.; Giddings, J. C. Anal. Chern. 1992,64, 3125-3132. 0003-2700/93/0365-2254804.00/0
The separation process was termed SPLITT fractionation (SF). The bimodal populations to be fractionated by SF are distributed laterally across different flow laminas by virtue of differential transport. Transport is generally driven by an externally applied field (electrical,5gravitational-), similar to the operation of fields in field-flow fractionation.12 However, lateral transport can be driven merely by diffusion, as a result of a concentration gradient."" The use of SPLITT fractionation for measurements of quantitative properties of sample components is termed analytical SPLITT fractionation (ASF). Examples of quantitative determinations include diffusion coefficients of proteins'O and the relative content of oversized particles above a cutoff diameter in particulate material, by gravitational sedimentation,' from which size distribution can be deduced. Preliminary exploratory studies of liposomes carrying hydrophilic drugs have indicated that ASF is capable of measuring the free drug released from the liposomes due to redistribution induced by dilution.11 These studies showed the way to a promising pharmaceutical application of ASF, the characterization of bimodal populated samples such as mixtures of colloids containing free small molecular weight substances. Such samples can be liposome~~~J3-15 and emulsions which are engineered to entrap drugs for controlledrelease purposes. The existence of free unbound drug outside the colloids opposes the concept of sustained or controlled release and should be minimized. Therefore, analysis of the fraction of free drug in colloidal drug formulations is essential a t every stage of their development. The object of this paper is the development and testing of the necessary methodologyfor the quantitative determination of the fraction of free substrates in colloidal samples. This concept is pursued here using a group of liposomes (phospholipid vesicles) containing various small molecular weight drugs. Principle of Operation. The operation of the SPLITT cell in the diffusion mode is illustrated in Figure 1,in which the side view of the channel is superimposed on the threedimensional scheme. A detailed description of the layer construction of a SPLITT channel has been previously
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(8) Levin, S.; Giddings, J. C. J.Chem. Technol. BiotechnoL 1991,50,
A2-56
(9)Williams, P. S.;Levin, S.; Lenczycki, T.; Giddings, J. C. Znd. Eng. Chern. Res. 1992,31,2172-2181. (10)Fuh, C. B.; Levin, S.; Giddings, J. C. Anal. Biochern. 1993,208,
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(11)Levin, S.; Bar-Shua, T. In Liposome Technology, 2nd ed.; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1992;Vol. 11, Chapter 17,p 293. (12)Caldwell, K.D.Anal. Chern. 1988,60,959A. (13)Gabizon, A.;Peretz, T.; Sulkes, A.; Amselem, S.; Ben-Yoeef, R.; Ben-Baruch, N.; Catane, R.; Biran, S.; Barenholz, Y. Eur. Cancer Clin. Oncol. 1989,25, 1795-1803. (14)Gabizon, A. A.;Barenholz, Y. In Liposomes as Drug Carriers; Gregoriadis, G., Ed.; John Wiley & Sons: New York, 1988; p 365. (15)Goren, D.;Gabizon, A.; Barenholz, Y. Biochim. Biophys. Acta 1990,1029,285-294. 0 1993 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 85. NO. 17. SEPTEMBER 1. 1993 2255
L
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Figure 1. Schematic iliustrat!on of the principle of operation of the analyilcal SPLIT fractionation (ASF) cell. Samples are introduced through inlet substream a’, pure fluM canier flows in through inlet substream b’. and the adjustment of flow rates at substreams a and b affects the separatlon.
illustrated.6f6J1 There are two inlets and two outlets in the channel, drilled through the channel walls.” A thin (127pm) splitter serves as a physical partition between the two inlets and outlets to reduce mixing and turbulence as the two substreams converge into the total stream flowing through the channel. Generally, each substream in the cell has a different flow rate; v(a7 and V(b7 are the flow rates of inlet suhstreams a’ and b’, respectively, and v(a) and v(b) are the flow rates of outlet substreams a and h, respectively. It is, of course, necessarythat tbesumofthe inlet suhstream flow ratesequals the sum of the outlet suhstream flow rates: V(a’)
+ V(h’) = V(a) + v(b)
(1)
The sample-free carrier stream entering inlet h’, has a greater flow rate than the sample-containingstream entering at inlet a’. Consequently, as the two incoming substreams converge a t the splitter edge, an imaginary plane is created (termed as the inlet splitting plane or ISP), which divides the fluidelementsofthese twosuhstreams. This imaginaryplane bends upward toward the sample side wall to accommodate the greater stream flow of b’ (see Figure 1). As a result, the sample stream is compressedinto a narrow band against wall A. This hand forms the sample region and becomes the reservoir for the transverse separative diffusive transport.9 Another imaginary plane is created in the channel due to the substream construction, the outlet splitting plane (OSP, Figure 1). This plane divides the fluid elements that exit through outlet a, from those that exit through outlet b. A small molecular weight component that diffuses far enough can cross the outlet splitting plane by the time it reaches the outlet splitter and, thus, can emerge from both outlets a and b. The position of either the inlet or outlet splitting plane can be controlled hy varying the ratios of the flow rates of the two suhstreams at each end. In the diffusion mode, the inlet splitting plane is positioned above the outlet splitting plane, closer to wall A. Defined by the ISP and the OSP is a thin film of liquid, consisting of the fluid elements entering into inlet h’ and exiting through outlet a, termed the transport lamina or the transport region. As the sample components enter the cell through the substream a t a’ and pass beyond the physical inlet splitter, they are instantaneously compressed against wall A and then gradually dissipate into the transport lamina by lateral diffusion. The transport lamina can he compared to a thin dynamic membrane across which species must transverse to reach outletsuhstream b. Thisvirtualmembrane is typically -100 pm thick. Diffusionacrossthisregion iscorrespondinglyfast.
By measuring the fractions of a component emerging from both exits, the corresponding diffusion coefficient can be calculated.’O A different approach is suggested here, the quantitative treatment of the signal at outlet h, which represents the rapidly diffusing species only, as a result of a differential transport of the bimodal populated samples in a dialysis-like mode. The bimodal population is divided into theslowlydiffusingspecies (vesicles)that aretotally retrieved by outlet suhstream a and the rapidly diffusing species (unbound drugs) that are partially retrieved by both outlet suhstreams a and h. By monitoring the effluent a t outlet suhstream b, the concentration of the unbound drug can be deduced hy following the proper calibration procedure. The effectiveness of the SPLITT process is optimized by simply varying the flow rates of inlet and outlet substreams, varying the positions of the ISP and OSP, and controlling the thickness of the transport region. When the volumetric flow rate of the suhstream entering b’, v(b’),considerably exceeds that of the substream entering a’, v(a’), then the ISP is closer to wall A and the sample feed is more compressedagainst this wall; the degree of compression is dictated by the flow rate ratio V(a’)lV. Likewise the OSP (which marks the border between the flow streams that eventually emerge as substreams a and h) is controlled by the ratio of the outlet volumetric flow rates V(a)/V. The unique advantages of SPLITT cells derive from the simplelaminar flow and the narrow (submillimeter)geometry. The transport path is both short and homogeneous and extends across only a fraction of the thin dimension of the channel. Consequently, the high transport speed enables clean-cut separations and short analysis time. For typical cells havingathicknessof I81
Flgw 4. Fraction of free drug outside liposomes. &IC&. carrying drugs measured by ASF.
in MLV
Table IV. & / C h Obtained by Analytical SPLITT Fractionation (ASF) and by Ultracentrifugation (UC) of Three Liposomal Formulations at Different Total Concentrations. 0.1 mM UC ASF
0.5mM UC ASF
1mM
UC
3 mM
ASF UC ASF
theophylline 0.26 1.00 0.30 0.98 0.36 0.88 theobromine 0.93 0.74 0.89 0.70 0.94 caffeine 0.82 1.00 1.00 0.82 0.99 0.81 1.00 and compared to the total amount of the drug known to be present in the formulation. This comparison yields the proportion of the drug outside the colloids, C,JC,u. The value of C d C - can be between 0 and 1. When all the drug = is preferentially entrapped within the colloids, C,JC0. Whenno entrapment occurs, and thedrug is preferentially dissolvedin tbeaqueous continuousphasearound thecolloida, C,JC,, = 1. However,whenonlypartialentrapmentocms, 0 < C,JC,, < 1, and the distribution of the drug between thecolloidand thecontinuousaqueousphasecan beevaluated. I. HydrophilicandlipophilicDrugs. Aseries ofliposomes loaded withdrugswithvaryingsolubilityinwater were initially studied. Among the hydrophilic drugs were colchicine, caffeine, and 5-fluorouracil,and the lipophilicdrugs included colchiceine and tetrahydrocannabinol (Ag-THC). All the formulations contained approximately 1mM total concentration of the drug. The ratio of C..JC,w of the drug after fractionation by the SPLITT channel was evaluated from the peak emerging from outlet substream b, using calibration curves (see Experimental Section). Typical correlation coefficientsof the fit to a linear curve are shown in Table 111. The results for C,JCof these drugs are illustrated graphically in Figure 4. In most cases the C d C , , values, obtained for watersoluble drugs after fractionation in the cell, were close to 1, regardless of the values determined by phase separation; i.e., a very small fraction of the drug was entrapped. On the other hand. low C,JCM values were obtained for lipophilic drugs under most conditions. 11. Xanthines. A series of xanthin-, theophylline, caffeine, and theobromine, were studied to further examine the behavior of water-solubledrugs in liposomes. The ratio C d C, was measured by both phase separation and SPLITT fractionation at three different drugconcentrations. Caffeine and theophylline are readily soluble in water, whereas theobromine is moderately soluble in water. The results of C,JCM of the drug determined by SPLITT fractionation (SPLITT) are shown in Table IV. The values of C,JCof the drug after fractionation by SPLITT indicated that all three drugs were mostly outside the liposomes, regardless of
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, I993
the ratio measured by phase separation and their differences in water solubility. Similar results were reported using gel fdtration as well as dialysis measurements of free caffeine in liposomes, carrying mixtures of caffeine and salicylic acid.’a The ratio between the concentration of the drug outside the liposomes and the total concentration in the preparation was simultaneously determined hy phase separation. The phospholipid phase was separated from the continuous aqueous phase hy ultracentrifugation of the nonfractionated large multilamellar liposomal preparations. The results of C,JCare also presented in Table IV (under UC). The values of C,,&,w obtained by this procedure are not necessarily true. Results for the fraction of free drug in the liposomes tend to be lower because the drug molecules, loosely adsorbed on the phospholipid phase, are trapped with the phospholipid phase during its sedimentation. Nevertheless, the procedure can give a rough estimation of the minimum fraction of the free drug in the suspensions. The source of the high fraction of total concentration outside the liposomes could he the quantity of free drug initially present in the colloidal suspension due to poor entrapment. However, even if the initial entrapment was efficient, the hydrophilic drug molecules undergo redistribution due to dilution, which is inherent to the SPLITT fractionation process. The result is a burst of the drug out of the liposomes. Variations in the fraction of the free hydrophilic drug, depending on the conditionsof the analysis, may he observed due to redistribution of the drug and its leakage into the aqueous continuous phase outside the Liposomes. This problem is universal to colloidal drug formulations, and redistribution of entrapped hydrophilic drugs alsooccurs when othertechniques suchassizeexclusion chromatography and dynamic dialysis are used. The same problem also exists when static methods are used. For example, in aseparation by ultracentrifuge, the phcapholipid phase is sometimes resuspended,washed, and then separated again. The value of the fraction of the free drug determined this way depends on the washing procedure. Similarly, when a static dialysis is used, the fraction of the free drug may depend on the relative volumes of the reservoirs on both sides of the dialysis membranes. Optimal vsNonoptimalSuspensionof Liposomes. One of the goals in preparation of sustained-release colloidal formulations is to control the redistribution induced by dilution. Therefore, entrapment of hydrophilic drugs in liposomes and other colloidal carriers requires additional specificfeaturesin order to retain the drug within the colloids and prevent its instantaneous redistribution as a result of dilution. A liposomal suspension, which was designed for clinical use in humans and was described and characterized hy Barenholzand co-workers,’G16 was examined by analytical SPLI’M fractionation. The reported C,JCMof doxoruhicin in this formulation was &lo%, using size exclusion and ion exchange as described in ref 13. The clinical formulation was compared to a “homemade” simple formulation of doxorubicin, similar to those used in the preliminary studies of analytical SPLITT fractionation.” Results for C,JC,w of doxorubicin for both formulations are shown in Figure 51,II. The results confirm that in contrast to the homemade formulation (Figure 511) a high degree of entrapment of doxorubicin was observed in the clinical formulation (Figure SI). Although doxorubicin is hydrophilic, the clinical formulation was stable under the conditionsof the analysis, and it was not released from the liposomes. The homemade (18) Touitou, E.; Alhaique, F.;Dayan, N.;Lnry-SchafferF.Xanthine Limrnen: Charactsrization and Skin psrmeation Beha~+or. 2nd J e d e m Conferenee on Pharmaceutical Sciences and Clinical Pharmaeology J e d e m , Israel. May 24-29, 1992.
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Flgure 5. Fractlon of free drug outside llpowme~&IC, In two doxwublclnfMmulatlom: (I)opUmkedfwmu$tlonofdoxaubklnaimed for clinicaluse In humans; (11) homemade formulations of doxoNbicln. Injection volume. 20 fiL: l4a)lV= 0.7. Conditlons of l4a‘)lVsnd V are ihe same as in Flgue 2.
lipwomes displayed some degree of doxorubicin attachment, probably due to a degree of electrostatic interaction.” The SPLITT cell cannot indicate whether the drug is trapped inside theliposomeor bound tothesurfaceofthe phwpholipid bilayer. Various destabilizers were added to the carrier fluid in an attempttoaffectthe fractionoffreedrugin thestableclinical formulation. Relatively high concentrations of surfactants (up to 0.1% w/v) or 2-propanol (up to 35% v/v) failed to change the CdC-, which remained approximately0.1.The optimizedformulationwasprovento be intact in the SPLITT fractionation cell. In contrast, the homemade suspension wasnotstabletothedilution induced bythe SPLITTchannel, and doxorubicin readily leaked from the liposomes under various conditions. This difference in behavior suggests that the SPLITT cell, operated in the diffusion mode, is also feasible for a rapid preliminary screening of drug-loaded liposomes for stability toward dilution.
CONCLUSION Analytical S P L I l T fractionation is capable of measuring the C,JC,w of colloidal formulations loaded with drugs, aimed for sustained-release usage. This ratio yields the distribution of the drug between the phospholipid vesicles and the continuous aqueous solution in which they are suspended. The ASF process could be readily automated using an autoinjector, providing in many cases the capability of making one or more determinations per minute on an ongoing basis. Another promising area where SF might be useful at the analytical levelis in the determinationofdiffusion
ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993
coefficients of colloids, by measuring the relative content of the two outlet substreams, from which their average size can be estimated. In principle, the system can be used with a variety of mobile fluid compositions flowrates,injection volumes,and detection modes. A great deal of work needs to be done in order to establish the technique and to validate its operation and the lack of artifacts from the analytical procedure. Effects such as shear stress, degree of dilution, and geometryof the channel still need to be investigatedto make sure they do not introduce artifacts. This is a new technology that operates in a dialysislike mode, devoid of membrane, which may greatly simplify the characterization of complex and delicate colloidal SUBpensions.
ACKNOWLEDGMENT This work has been supported by the "Fund for Applied Research" of the Hebrew University from the Wolfson Foundation. We are grateful for the generous donation of the SPLITT channel by Prof. Giddings of the FFFRC, The University of Utah. We express our special gratitude to Dr. S. Williamsfor letting us use the Fortran programs from which the transport parameters were calculated. We appreciate the contribution of Dr. DovTamarkin and Ms. Dana Shwartz from Clilco Inc., Jerusalem, Israel, in the preparation of homemade drug-carrying liposomes. We also thank Prof. Y. Barenholz for his useful discussions regarding liposomes, as well as for his contribution of doxorubicin, and the clinical doxorubicin-loadedliposomalformulation. We acknowledge the help of the following colleagues: Dr. Israel Ringel, the PharmacologyDepartment; Prof. R. Mechoulam,the Natural Products Department, and Prof. S. Benita, the Pharmacy
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Department, for their donation of drugs to this study.
GLOSSARY outlet substream carrying the colloids and the drugs inlet substream carrying the sample outlet substream carrying just the free drugs inlet substream carrying pure fluid carrier channel breadth initial sample concentration fraction of free drug unbound to the colloids diffusion coefficent retrieval factor at outlet substream a retrieval factor at outlet substream b channel length channel void time dimensionless transport parameter channel void volume flow rate of outlet substream a flow rate of inlet substream a' flow rate of outlet substream b flow rate of outlet substream b' total flow rate channel thickness sample region thickness coordinate across the channel thickness coordinate along the channel axis
RECEIVED for review February 17, 1993. Accepted June 1, 1993.
