Neuronal Biosensors Using Liposomal Delivery of Local Anesthetics

We have incorporated the anesthetics benzocaine, bupivacaine, and tetracaine into liposomes, generating dose−response curves. Liposomes are shown to...
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Anal. Chem. 1997, 69, 4120-4125

Neuronal Biosensors Using Liposomal Delivery of Local Anesthetics David R. Coon, Adeboye B. Ogunseitan, and Garry A. Rechnitz*

Hawaii Biosensor Laboratory, Department of Chemistry, University of Hawaii, Honolulu, Hawaii 96822

The use of liposomal drug incorporation significantly improves the sensitivity of neuronal sensors to poorly soluble drugs and extends the applicability of such sensors to previously undetectable compounds. Data presented support physiological models for liposomeneuron interactions and are consistent with the channelblocking actions of local anesthetics. We have incorporated the anesthetics benzocaine, bupivacaine, and tetracaine into liposomes, generating dose-response curves. Liposomes are shown to extend the range of the biosensor and improve its sensitivity. We have previously published a paper on biosensors using nerve tissue as the detection element in a biosensing system.1 Such systems are very sensitive to compounds that have their effect by interfering with the conduction ability of the nerve tissue. Neuronal biosensors using electrochemical or biomagnetic transducers have been developed for a wide range of analytes, including neural activity modulatory drugs such as toxins, agonists, and anesthetics.2,3 Neuroreceptors, either cloned and expressed in Xenopus oocytes or as intact ion channels of crustacean nerves, have been employed as molecular recognition elements. Thus far, the experimental arrangements have been limited to flowbased systems with constrained contact between the analyte and the nerve tissue. In consequence, only moderate sensitivity can be achieved, especially when the quantities of analyte dissolved are insufficient to cause a detectable change in the activity of the nerve tissue. In this paper, we describe the employment of liposomes to address these problems and demonstrate that improved sensitivities and good response to sparingly soluble analytes can be attained. We show that liposomes provide an effective and potentially economical means of delivering analytes to the neural sites where analytical dose responses are elicited. The changes in the conduction ability of the nerve tissue can be measured by very sensitive transducing devices such as biomagnetic “inductrodes” (sensors that can measure changes in magnetic fields) or potentiometric electrodes.4 The transducing device used in this study is composed of two loose-clamp capillary electrodes positioned at the membrane of an excised section of nerve tissue (Figure 1). The first electrode stimulates an action potential in the nerve tissue by providing a depolarizing current pulse to the nerve membrane. The second electrode, approximately 1 cm downstream from the first, detects the action (1) Leech, D.; Rechnitz, G. Anal. Chim. Acta 1993, 274, 25-35. (2) Wijesuriya, D.; Rechnitz, G. Anal. Chim. Acta 1992, 264, 189-196. (3) Rechnitz, G.; Coon, D.; Babb, C.; Ogunseitan, A.; Lee, A. Anal. Chim. Acta 1996, 337, 297-303. (4) Coon, D.; Babb, C.; Rechnitz, G. Anal. Chem. 1996, 68, 1671-1675.

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potential, which is then amplified, stored, and viewed. The viability of the nerve tissue is maintained as long as possible by using a flow cell designed in this lab. Local anesthetics have been employed in our laboratory to characterize the neuronal biosensor. They are convenient analytes because they have been well studied and their mechanism of action is relatively well understood. Local anesthetics function by binding inside the pore of the sodium ion channels distributed along the membrane of nerve axons. They cause a conformation change of the 260 kD R-subunit of the ion channel, preventing the depolarization of the membrane, and thus blocking generation of an action potential. The binding is reversible and dependent on the state of the channel and the nature of the drug. Until now, the neurosensor has been limited to drugs that can be adequately dissolved in the aqueous media necessary to maintain the nerve tissue. In order to extend the utility of neuronal biosensors, we use liposomes as drug delivery agents to the nerve tissue.5 Liposomes are phospholipid vesicles (60-600 nm) that can be loaded with channels or pharmaceuticals of interest. They are able to convey their contents, in aqueous media, until delivery by fusion with or endocytosis by cell membranes. They have been used in the fabrication of other biosensing systems.6 There are also reports in the literature of successful liposomal delivery of lidocaine, a local anesthetic, to nerve tissue in dogs.7 Liposomes can extend neural-based detection systems to include the analysis of drugs insufficiently soluble in aqueous media yet deemed to be of physiological significance. Not only can hydrophilic drugs be trapped in the aqueous lacuna of the vesicle, but hydrophobic drugs can be dissolved within the lipid bilayer (Figure 2). Since liposomes deliver their contents directly to the nerve membrane, they are able to deliver hydrophobic and hydrophilic drugs to the sensor. The benefits of liposomal delivery have been investigated using the local anesthetics benzocaine (ethyl p-aminobenzoate), bupivacaine (1-butyl-N-[2,6-dimethylphenyl]-2-piperidinecarboxamide), and tetracaine (4-[butylamino]benzoic acid 2-[dimethylamino]ethyl ester monohydrochloride). The limited aqueous solubility of benzocaine (1.69 mg/mL, pKa ) 2.51) is less than that of bupivacaine (2.4 mg/mL, pKa ) 8.1).8 These drugs are compared with the very soluble tetracaine hydrochloride which has previously been employed in our research.9,10 Previous attempts to detect aqueous solutions of benzocaine and bupiv(5) Gregoriadis, G. Liposomes as Drug Carriers: Recent Trends and Progress; John Wiley & Sons: New York, 1988. (6) Shiba, K.; Umezawa, Y. Anal. Chem. 1980, 52, 1610-1613. (7) Mashimo, T.; Uchida, I. Anesth. Analg. 1992, 74, 827-834. (8) Howard, P.; Meylan, W. Solubility Properties; CRC Press: NY, 1997. (9) Coon, D.; Babb, C.; Rechnitz, G. Anal. Chem. 1994, 66, 3193-3197. (10) Babb, C.; Coon, D.; Rechnitz, G. Anal. Chem. 1995, 67, 763-769. S0003-2700(97)00171-6 CCC: $14.00

© 1997 American Chemical Society

Figure 1. Experimental setup showing the flow cell and micromanipulator arrangement.

classed as an inactivated channel blocker because of its inability to successfully block batrachotoxin-modified channels. Bupivacaine, however, binds to these modified channels, successfully blocking them. It is, therefore, classed as an open channel blocker. Tetracaine is found to bind with both open and inactivated channels and is classed as intermediate between an inactivated and an open channel blocker.12 When benzocaine, bupivacaine, and tetracaine are incorporated into lipid vesicles, we are able to obtain dose-response curves for all three anesthetics. The use of liposomes has improved the sensitivity of the sensor and extended the range of possible analytes.

Figure 2. Depiction of a unilamellar liposome incorporating hydrophobic molecules within the lipid bilayer. The aqueous interior can also be used to transport hydrophilic drugs to nerve tissue sensors.

acaine were unsuccessful, presumably due to low levels of drug in aqueous solution. Current understanding of the action of local anesthetics on sodium ion channels relies heavily on the Hille modulated receptor hypothesis and its two principal premises, that only one binding site exists for all local anesthetics and that the affinity of the site for the drug depends on the state of the channel.11 The local anesthetics employed are distributed across the three broad categories that currently exist: open (depolarized), closed (resting), and inactivated (hyperpolarized or refractory) channel blockers. The categories describe the state of the channel for which any local anesthetic has the highest affinity. Batrachotoxin, a secretion of the South American poison frog, Phyllobates sp., binds selectively to sodium channels and leaves them in the open state. Benzocaine, a neutral local anesthetic, is (11) Hanck, D.; Mackielski, J.; Sheets, M. J. Gen. Phys. 1994, 103, 19-43.

EXPERIMENTAL SECTION Procedure. Crayfish were caught in the Manoa stream on campus and kept in fresh water holding tanks until needed. Prior to an experiment, a crayfish was anesthetized by keeping it at -18 °C for 4-5 min. The ventral carapace was then removed, and a 1-2 cm section of the abdominal ganglion was excised. This was quickly transferred to a flow cell and pinned down with the aid of a dissecting microscope. The nerve was continually perfused with oxygenated buffered saline solution. Suction microelectrodes were attached onto the nerve with the aid of micromanipulators. One electrode was used to provide depolarizing current pulses to the nerve tissue, thus stimulating an action potential. The second electrode, placed downstream, potentiometrically detected the action potential. Analyte introduction was done by means of three-way switching valves connected to the inflow buffer line. Instrumentation. A Vanderbilt biomagnetic probe unit (Vanderbilt University) provided the gating output for the stimulus pulses.13 The pulse width is adjustable from 0.01 to 1.0 ms. This (12) Wang, G.; Mok, W.; Wang, S. Biophys. J. 1994, 67, 1851-1860. (13) Vanderbilt Biomagnetic Current Probe Model LSP-3 Users Manual; Vanderbilt University: Nashville, TN, 1990.

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signal was fed into a stimulus isolator (World Precision Instruments, Sarasota, FL). The isolator was used to adjust the current in the stimulating electrode to the threshold needed to generate an action potential in the nerve. It could provide a maximum of 10 mA. The signal from the detection electrode was fed into a differential preamplifier (Grass Instruments, Quincy, MA). The input signal was compared to that of a Pt wire immersed in the flow cell next to the nerve tissue. The output from the preamplifier was viewed on a digital oscilloscope (Model 54603B, Hewlett Packard). The image from the oscilloscope was captured and stored by an IBM-PC using Benchlink software (Hewlett Packard). Flow Cell. The flow cell dimensions are 3.17 cm × 1.27 cm × 1.91 cm. Two tubes protrude from opposite ends of the cell to allow introduction and removal of saline or analyte. Two Dynamax peristaltic pumps (Rainin, Woburn, MA) connected to either end of the cell were used to control inflow and outflow. The bottom of the cell is lined with Sylgard (Dow Corning, Midland, MI) to provide a soft surface for pinning the nerve to the cell floor. The volume of solution in the cell at any time was maintained at approximately 1 mL. Solutions and Reagents. Egg phosphatidylcholine dioleoyl, phosphatidyl-DL-glycerol, benzocaine, bupivacaine, and tetracaine hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). Phosphate-buffered saline (PBS) solution was made with 0.15 M NaCl and 0.01 M Na3PO4 and adjusted to pH 7.45 with 6 M NaOH. The nerve tissue was perfused with a modified van Harreveld (MVH) solution composed of 205 mM NaCl, 5.4 mM KCl, 13.5 mM, CaCl2, 2.6 mM MgCl2, and 10 mM Tris and was adjusted to pH 7.5 with maleic acid. All buffer salts were of analytical grade, purchased from Fisher Chemicals (Fair Lawn, NJ). Pure oxygen was bubbled through this solution for the duration of each experiment. Liposome Preparation. Cholesterol (40 mg) was placed in a 250 mL round-bottom flask. To this was added 5 mL of phosphatidylcholine (20 mg/mL), 1 mL of phosphatidyl-DL-glycerol (10 mg/mL), and 25 mL of chloroform. This solution was reduced with a Buchi rotary evaporator at 60 rpm, at 40 °C, under reduced pressure until a thin film was formed along the walls of the flask. The rotary evaporation was continued for an additional 15-20 min. The flask was then left at 25 °C for 30 min, and 20 mL of PBS was carefully added by pouring down the walls of the flask. The flask was then shaken to “swell” the liposomes and lifet them off the flask walls. Shaking by hand was done for 10 min before the flask was returned to the rotary evaporator. The flask was returned to the conditions described above, but this time at atmospheric pressure. This was continued for 20 min, after which the contents were transferred to a 20 mL centrifuge tube and allowed to stand at room temperature for 30 min. The tube was placed in an ice bath and sonicated with a titanium probe sonic dismembranator (Fisher Scientific, Model F50). The total sonication time was 20 min; however, this was carried out in 4 min pulses, between which was a 4 min rest period to avoid excessive heat buildup in the tube. The liposomes were left to stand for 1 h at room temperature and then centrifuged (Beckmann induction drive centrifuge, Model J2-21M) at 25 500 or 15 600 rpm for 45 min at 4 °C. The supernate was discarded, and the pellet was resuspended in 20 mL of PBS. Drug-Laden Liposomes. The procedure is the same as above, except that varying amounts of the local anesthetic of interest were dissolved in PBS, and this solution was used to swell 4122

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the liposomes. After resuspension of the pellet, a 1 mL fraction was set aside for analysis of the incorporation efficiency. Determination of Liposome Content. There is a surfeit of analytical methods in the literature for the determination of liposome-entrapped material, and the methods employed here were adapted from standard procedures.14,15 A 50 µL aliquot from the 1 mL fractions saved from each liposomal preparation was placed in a microcentrifuge tube, to which was added an equal volume of a 10% solution of Triton X-100 (Sigma). This was placed in a 40 °C water bath for 30 min, after which the solutions were made up to 1 mL with 5 mM HCl. The resulting solution was filtered through a 0.2 mM Teflon membrane filter (Fisher). The solutions were then analyzed using HPLC. HPLC Experiments. HPLC grade 100% methanol with UV cutoff of 205 nm (Fisher) was used as solvent in standard preparations and as mobile phase. A 50:50 solution of acetonitrile (UV cutoff of 190 nm) and methanol was also employed as mobile phase to enhance peak resolution for some of the compounds. A 15 cm reverse-phase bonded C18 column (Supelco, Model 5-8935) with an inner diameter of 4 mm was used. The pump was a Shimadzu (Model LC 600) pump, and detection was done by a UV detector (Shimadzu, SPD-6A). Chromatographic peaks were integrated with a chromatointegrator (Hitachi, D2500). The injection port was fitted with a 20 µL sample loop (Rheodyne, Model 7125). A minimum of three runs of each sample were made. The concentrations of standard solutions of all three local anesthetics prepared were chosen to mirror the expected concentration ranges of the lipid incorporated drugs. Solutions of cholesterol and an aqueous solution of Triton X-100 were also run through the column. RESULTS AND DISCUSSION Controls. Liposomes without any drugs incorporated within them were run through the neurosensor system to test for an anesthetic effect on the nerve. No such effect was observed while the system was monitored for 30 min after the introduction of liposomes into the flow cell. A slight potentiation of the action potentials was noticed within 3-4 min of sample introduction. This effect, however, was transient, and action potentials subsequently returned to initial states. Drug-Laden Liposomes. Suspensions of benzocaine in buffer made by shaking 10-100 mg of crystalline benzocaine in 20 mL of PBS were used to swell the liposome vesicles. The liposomes were then introduced into the flow cell, and the action potential of the nerve tissue was monitored. The time to completely block all action potentials was chosen as the analytical signal. The time to block varied with the amount of benzocaine suspended in the preparation buffer. Figure 3 shows an action potential trace of a nerve before and after treatment with liposomeloaded benzocaine. To test the efficacy of benzocaine in aqueous solution, 1 g was shaken in 20 mL of PBS for 30 min, after which the suspension was gravity filtered. The filtrate was run over the nerve and monitored for 30 min. No block or other observable modulation of the signal was recorded. The compound action potentials are shown in Figure 4. Bupivacaine is a local anesthetic whose solubility is intermediate between those of the less soluble benzocaine and the more (14) Lalor, C.; Flynn, G.; Weiner, N. J. Pharm. Sci. 1994, 83, 1525-1528. (15) Lalor, C.; Flynn, G.; Weiner, N. J. Pharm. Sci. 1995, 84, 673-676.

Figure 3. Effect of loaded liposomes on the ability of the nerve to conduct action potentials. (A) The response of the tissue before the benzocaine-loaded liposomes were introduced to the cell. (B) Complete block of all action potentials 5 min after liposome introduction. One unit represents 200 mV or 1 ms on the potential and action potential duration axes, respectively.

Figure 5. HPLC traces for a sample of benzocaine-containing liposomes lysed with Triton X-100 (1.03, 1.38, and 3.32 min elution times) (A), benzocaine standard (1.32 min elution time) (B), drugfree liposomes lysed with Triton X-100 (0.829 and 2.52 min) (C), and a 0.5% solution of Triton X-100 (0.808 min) (D).

Figure 4. Effect of a saturated aqueous benzocaine solution before (A) and after (B) passing over the nerve tissue for more than 20 min.

soluble tetracaine. Serial dilutions of saturated solutions of bupivacaine were used to swell liposomes. The liposomes were run through the neurosensor, and their effect on the action potential was monitored. The lowest concentration to achieve a full block of the action potential involved liposomes prepared from solutions 2.2 mM in bupivacaine. The response of the neurosensor to the drug-laden liposomes was concentration dependent, and the time to block decreased with increasing concentrations of bupivacaine. Tetracaine, the most soluble of the anesthetics employed, was made into solutions with concentrations ranging between 1.0 and 20 mg/mL. The yield of liposomes swelled with these solutions declined rapidly at tetracaine concentrations greater than 15 mg/ mL, and no liposomes were formed with the 20 mg/mL solution. A complete block of action potentials was achieved using liposomes prepared with 1.5-12 mg/mL tetracaine solutions. The time to achieve total block showed only a mild dose dependence. Drug Inclusion. The concentration of drugs incorporated within liposomes is related to but not equal to the concentration in the buffer used to swell the liposomes. To determine the concentration of drugs within liposomes and correctly establish the dose dependence of blocking times, liposomes were disrupted and their contents analyzed.

Benzocaine and tetracaine have high extinction coefficients (∼105) at or near the 274 nm wavelength of detection.16 The HPLC data from the standards provided retention times of the drugs as well as concentration-dependent peak areas. A linear regression analysis of these data was done, and the results were used in generating calibration curves. These curves were used to determine the amount of anesthetic within the liposomes. HPLC of the control (i.e., drug-free) liposomes and Triton X-100 was also done to assign peaks. Liposome solutions treated with the surfactant as described above were injected into the column, and HPLC data were collected. Figure 5 compares the chromatogram obtained from benzocaine standard solutions (A) with ones from a lysed benzocaine-loaded liposome (B), the control liposomes (C), and the Triton X-100 (D). Benzocaine peaks were easily resolved from the TX-100 peaks by the integrator, but this was not so for tetracaine or bupivacaine. An acetonitrile/methanol solution was used as mobile phase for bupivacaine separations. Bupivacaine eluted first with the mobile phase and was distinguishable from TX-100. The incorporation efficiency (IE), expressed as a percentage, is the ratio of encapsulated drug to the concentration of drug in the buffer solution. The IE was expected to increase with the concentration of the suspending solution and then decrease after the absorptive capacity was reached. An initial increase was observed, but the IE declined at drug concentrations higher than ∼4.0 mM. Tetracaine, being more soluble in aqueous buffer, was also expected to have higher incorporation efficiencies. Tetracaine showed the highest IE across the concentration ranges. The IEs (16) Caraballo, I.; Fernandez-Arevalo, M.; Holgado, M.; Vela, M.; Rabasco, A. J. Pharm. Sci. 1994, 83, 1147-1149.

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Figure 6. Distribution of the incorporation efficiencies of the local anesthetics into liposomes against the initial concentration of anesthetics in buffer solution. Table 1. Average Liposomal Incorporation Efficiencies compound

pKa

mean % inclusion

benzocaine bupivacaine tetracaine

8.2 8.1 8.5

10.9, 82.4a 9.64 11.7

a The incorporation efficiency of benzocaine in liposomes when the drug was dissolved in chloroform during the synthetic procedure rather than in aqueous buffer.

for benzocaine, bupivacaine, and tetracaine against their concentrations in the buffers are shown in Figure 6. Two sets of liposomes containing benzocaine were prepared by dissolving the compound in chloroform and adding to the “drying down” stage. The IE for this procedure was much higher than that for the traditional suspension in aqueous buffer. The average IEs for all three local anesthetics are listed in Table 1. While the use of nonaqueous solvents significantly enhances the inclusion of the drugs into the liposomes, this method was not employed due to the deleterious effects of even small amounts of chloroform on nerve tissue. The time of the extra liposomal purification steps needed to remove the chloroform to an acceptable level made this method undesirable for fast-throughput, analytical sensors. Dose Response. The dose-related response of the sensor was evaluated by plotting the time to block against the calculated concentration of drug inside the liposomes. Plots for benzocaine, bupivacaine, and tetracaine are shown in Figure 7. They are all sodium ion channel blockers but function by slightly different mechanisms. Liposomes that would have maximal entrapment, be stable enough to transport drugs to their target, and deliver as quickly as possible (hence minimizing sensor response time) were desired. Some of the methods used to address each of these considerations include the use of a large surface area vessel for the drying down process, the addition of cholesterol to the lipids, and the incorporation of a high fraction of small unilamellar vesicles (SUVs). Cholesterol serves the same purpose in liposomes as in cell membranes, structural reinforcement. Sonication breaks up the multilamellar large vesicles (MLVs) into SUVs. The degree of entrapment by either MLVs or SUVs has been shown to be similar, even though the vesicles differ in size from 600 nm for MLVs to ∼40 nm for SUVs. Small unilamellar vesicles, however, penetrate the cell membrane easier than large vesicles, thus delivering their contents more quickly.17 4124 Analytical Chemistry, Vol. 69, No. 20, October 15, 1997

Figure 7. Dependence of time to block action potential with concentration of drug incorporated into liposomes. The stimulation strength varied from nerve to nerve but was within the range 0.023.85 mA.

The results show that drug-laden liposomes can be conveyed in an aqueous medium until they arrive at their target tissue and release their contents. The mechanism of this release has not been fully established but is believed to be one of four pathways. A liposome may first adsorb onto the surface of a cell and then slowly release its contents at the cell surface, or it may be endocytosed and then digested by lysozymes within the cell, thereby releasing its contents. The liposome may fuse with a cell, effectively becoming a part of the cell membrane and its contents, or it may simply exchange lipids with the cell. All four pathways result in drug delivery directly to the cell; hence, even low dosages show significant effects. Of the four mechanisms proposed, the first seems to be the least efficient, yet recent research has advanced evidence that supports it as the more probable one. Belgian researchers prepared liposomes incorporating either radioactive cholesterol or bupivacaine and injected these into rabbit cerebrospinal fluid.18 They found that cholesterol did not significantly label spinal nerves as would be expected for any of the three latter mechanisms. They also found a rapid uptake of radioactivity by spinal nerves exposed to radioactive liposomal bupivacaine, suggesting an exchange between liposome and nerve sheaths. However, it should be noted that this does not exclude the fourth mechanism, lipid exchange between liposome and cell membrane. Extensive research has been done on the use of liposomes as drug delivery agents. However, until recently, the focus has been on drugs with relatively high solubilities in aqueous media.14,19 Previous attempts to detect benzocaine and bupivacaine with the current neurosensor system were unsuccessful. This was believed to be due to the inability to dissolve appropriate levels of these compounds. The results show that inclusion of drugs into liposomes extends the viability of the neurosensor to the detection of neuroactive drugs that are weakly soluble in water. They also suggest that drug delivery is close to binding sites in concentrations high enough to effect a block of the ion channels. Early studies in the characterization of the neurosensor employed aqueous lidocaine and tetracaine as analytes.10 Since (17) Litzinger, D.; Buiting, A.; van-Rooijen, N.; Huang, L. Biochim. Biophys. Acta 1994, 1190, 99-107. (18) Boogaerts, J.; Lafont, N.; Carlino, S.; Noel, E.; Raynal, P.; Goffinet, G.; Legros, F. Br. J. Anaesth. 1995, 75, 319-325. (19) Weiner, N.; Niemiec, S.; Hu, Z.; Ramachandran, C.; Leib, L.; Egbaria, K. J. Drug. Target 1994, 2, 405-410.

liposomes have been reported to effect slow release of encapsulated material, it was expected that the sensor response to tetracaine would be slower for liposomal tetracaine than for tetracaine in solution. Sensor response times as low as 2 min were reported for tetracaine in solution.9 The shortest time to block for liposomal tetracaine was about 7 min. At very low drug concentrations, however, the time to block for liposomal tetracaine showed the reverse of aqueous solutions, giving a slightly shorter time to block. This is most likely due to the direct deposition of drug to the cell membrane, thus concentrating its effect on the tissue as opposed to the behavior of tetracaine in the aqueous solution. This effect is lost when the aqueous drug concentrations increase. The proximal delivery of drug to nerve tissue led to expectations that sensor sensitivity to tetracaine would be significantly improved. The detection limit was, however, reduced by only a factor of 2, from the previous limit of 1.00 to 0.565 mM. This may be because a minimum amount of drug is needed to completely block compound action potentials in the nerve. Experiments to test the improvement of sensitivity by liposome entrapment have been conducted in vivo and in vitro by other researchers.20 Research employing tetracaine in liposomes delivered to volunteers showed an increase in activity with a lowering of drug dosage. Liposome-entrapped tetracaine produced stronger anesthesia with half the concentration required for other preparations.21 These results hold true for other local anesthetics such as benzocaine, dibucaine, and lidocaine. The concentration of a saturated solution of benzocaine in PBS was experimentally determined as 0.57 mg/mL. This is low compared to tetracaine, for which aqueous solutions of 20 mg/ mL are possible under standard conditions. Thus, it was expected that incorporation efficiencies would be lower for benzocaine. This was verified experimentally (Figure 6). However, the difference was smaller than expected. This may be due, in part, to the small volume of the liposomes (∼1%) of the total volume of the swelling solution and the ability of benzocaine to be dissolved within the lipid bilayer. Dissolution of benzocaine in chloroform greatly improves its incorporation (Table 1), in agreement with results (20) Touitou, E.; Junginger, H.; Weiner, N.; Nagai, T.; Mezei, M. J. Pharm. Sci. 1994, 83, 1189-1203. (21) Foldvari, M.; Gesztes, A.; Mezei, M. J. Microencapsul. 1990, 7, 479-489. (22) Boogaerts, J.; Lafont, N.; Donnay, M.; Luo, H.; Legros, F. Acta Anesth. Belg. 1995, 46, 19-24.

published by Weiner et al.19 This suggests that liposomes may be more efficient in delivery of compounds with a higher lipid solubility. The neurosensor, as it is currently configured, cannot directly discriminate between the depolarized, resting, or refractory states of the ion channels and their affinities for local anesthetics. Nevertheless, these state-dependent blocking properties may help explain the different sensitivities and response times of the sensor for the compounds employed. CONCLUSION The use of liposomes in the delivery of neuroactive compounds expands the range of drugs detectable by the neurosensor. Drugs that previously had been undetectable now show reasonable response times and dose dependence. The results also suggest that liposomal delivery improves the sensitivity of the sensor by lowering the concentration needed to effect a block. This is in line with the findings of other researchers in the field.21 The implications of this are significant, particularly in the field of biomedical research. Pharmaceutical research is time consuming and expensive. It is conceivable that potential drug agents are quickly dismissed and eliminated from further trials if initial results are unencouraging. This may be due, however, not to the agent itself but to the limitations of the delivery and testing system employed. Liposomal incorporation and delivery improves the sensitivity of neurosensors to the drug tetracaine, and further investigations involving other possible analytes may be promising. Liposome incorporation has already been found to be more effective for drug delivery without toxic side effects.22 Perhaps this method can improve the lifetime of neuronal biosensors by reducing the dosage and, thus, the toxicity of the agents delivered to the nerve. ACKNOWLEDGMENT The authors gratefully acknowledge the material support of the Larsen Research group and financial support from National Science Foundation Grant CHE-9216304. Received for review February 11, 1997. Accepted July 2, 1997.X AC970171V X

Abstract published in Advance ACS Abstracts, September 15, 1997.

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