Electro-Spinning and Electro-Blowing of Hyaluronic Acid

Biomacromolecules 2004, 5, 1428-1436

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Electro-Spinning and Electro-Blowing of Hyaluronic Acid In Chul Um,† Dufei Fang,‡ Benjamin S. Hsiao,† Akio Okamoto,§ and Benjamin Chu*,† Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, Stonybrook Technology and Applied Research Inc., P.O. Box 1336, Stony Brook, New York 11790, and Denka Research Center, 3-5-1 Asahimachi, Machida, Tokyo 194-8560, Japan Received December 19, 2003; Revised Manuscript Received February 25, 2004

In this study, hyaluronic acid (HA) was electro-spun and electro-blown to prepare nonwoven nanofibrous membranes. Critical parameters for processing and corresponding effects on the membrane morphology were investigated using the methods of rheology and scanning electron microscopy (SEM). During electrospinning, the optimal HA concentration window for nanofibrous formation was determined within a narrow range of 1.3-1.5 w/v %, corresponding to a solution viscosity range of 3-30 Pa s at a shear rate of 1 s-1. SEM results indicated that, with increases in (1) the total concentration by blending of low molecular weight HA, (2) the evaporation rate by the addition of ethanol, and/or (3) the feeding rate of solution, the electrospinning performance for creating nanofibers was improved. However, the improvement was not sufficient to achieve a consistent production of high quality nonwoven nanofiber membranes. This problem was overcome by a new electro-blowing process using the combination of air flow and electro-spinning. Although air blowing at room temperature around the spinneret orifice did not exhibit a remarkable enhancement of nanofiber formation of HA, the performance was significantly improved with an increase in the air blowing rate. SEM results showed that the temperature of air-blowing was the most effective parameter in ensuring HA nanofiber formation. As the temperature of the blown air increased from 25 to 57 °C, the nanofiber formation became consistent and uniform. A high quality HA nonwoven membrane of nanofibers was successfully produced by blowing air at 57 °C with a 70 ft3/hr flow rate. Introduction Hyaluronic acid (HA) is a naturally occurring polysaccharide, commonly found in connective tissues in the body such as vitreous, umbilical cord, joint fluid, etc.1 It consists of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine (as shown in Figure 1). HA has been thought to act as a molecular filter, shock absorber, and support structure for collagen fibrils.2 Because of its unique rheological properties and complete biocompatibility, HA has been used quite extensively in many biomedical applications, including ophthalmology, drug delivery, dermatology, surgery, and medical implants.3,4 Recently, the electrospinning technique has attracted a great deal of attention as it is an effective means to produce nonwoven membranes of nanofibers. The electrospinning process was first demonstrated by Zeleny5 and patented by Formhals.6 Up to now, there are over 60 patents published based on the electrospinning process and more than 200 papers related to the technology of electro-spinning in the past 10 years. Overall, most of the electro-spinning research was focused on the development of nanofiber membranes as new materials for applications. However, an increasing number of papers have also dealt with many of the fun* To whom correspondence should be addressed. Phone: +1-631-6327928. Fax: +1-631-632-6518. E-mail: [email protected]. † State University of New York at Stony Brook. ‡ Stonybrook Technology and Applied Research Inc. § Denka Research Center.

Figure 1. Chemical structure of hyaluronic acid (HA).

damental physical parameters related to the jet formation, as a function of electrostatic field strength, fluid viscosity, solvent composition, and molecular weight of polymers in solution.7-16 The interconnected porous nanofiber networks in electrospun membranes, having a very high surface area-to-volume ratio, are particularly useful for biomedical applications, such as substrates for tissue regeneration, wound dressing articles, artificial blood vessels, and materials for the prevention of post-operative induced adhesions.17-31 Some selected recent examples are as follows. Li et al.27 reported that the nanofibrous structures in electro-spun membranes could positively promote the cell-matrix and cell-cell interactions, which exhibited the promising potential of electro-spun scaffolds for tissue engineering. Kenawy et al.28 demonstrated the possibility of using electro-spun membranes based on poly(lactic acid) (PLA), poly(ethylene-co-vinyl acetate) (EVA), and their blends as vehicles for drug delivery. Luu et al.29 reported that the electro-spun scaffolds containing

10.1021/bm034539b CCC: $27.50 © 2004 American Chemical Society Published on Web 05/07/2004

Electro-Spinning and Electro-Blowing of HA

synthetic biodegradable polymers: PLA, poly(lactide-coglycolide) (PLGA), amphiphilic block copolymer of PLA and poly(ethylene glycol) (PEG), and DNA could be used for nonviral gene delivery. Jia et al.31 demonstrated that the electro-spun membranes can significantly improve the catalytic efficiency for immobilized enzymes which could be useful for enzymatic biotransformation. In view of these rapid developments, we anticipate that more work based on electrospun biomaterial membranes for varying biomedical applications will be demonstrated. As HA has already been widely used in varying biomedical applications, in both non-crosslinked and cross-linked forms,32-33 the production of nanofibrous HA membranes, especially in cross-linked form, non soluble to water, by electro-spinning is an attractive platform which may lead to many new applications. To the best of our knowledge, the electro-spinning HA solution (without cross-linking reaction) has never been accomplished. We believe that the major hurdle is the unusually high viscosity and the surface tension of HA solution, which significantly hinders the process of electro-spinning. In this study, we have systematically evaluated different means to modify the method of electrospinning in order to process higher viscosity solutions such as HA. Finally, we have implemented a unique air blowing feature to the conventional electro-spinning system to expand the capability of the electrospinning process and to overcome the shortcomings of the especially high viscosity in HA solutions. We termed this new technique “electro-blowing”. Effects of different processing parameters in both electrospinning and electro-blowing of HA solutions on the final membrane morphology are outlined and compared in this article. Experimental Section Preparation of HA Solution. High (Mw ) 3 500 000) (HA-C) and low (Mw ) 45 000) (HA-A) molecular weight hyaluronic acid sample was supplied by Denki Kagaku Kogyo Co. Ltd. (Denka, Japan). The HA sample was extracted from the culture broth of Streptococus equi and was purified.34 The sample preparation procedures were as follows. First, an acidic aqueous solution (pH ) 1.5) was obtained by adding HCl into distilled water. Then, homogeneous HA acidic aqueous solutions were prepared by dissolving the HA samples in the acidic aqueous solution through gentle stirring for electro-spinning and electroblowing processes. The concentration unit used was in w/v % (w in gram and v in milliliter). The acidic condition was further used for a subsequent cross-linking experiment, which will be described elsewhere. Electro-Blowing Process. The electro-blowing technology was developed based on the modification of a laboratorybuilt electro-spinning apparatus, which is shown in Figure 2. The electro-spinning apparatus setup consisted of the following components. The HA solution in the syringe was delivered by a programmable pump (Stonybrook Technology and Applied Research Inc., NY) to the exit hole of the electrode (spinneret with a hole diameter of 0.76 mm). The solution feeding rate ranged from 5 to 10 µL/min. A positive high-voltage supply (Glassman High Voltage Inc., NJ) was

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Figure 2. Scheme diagram for electro-spinning and electro-blowing setup used in this study.

utilized to supply the voltage over a range of 0-40 kV. The collecting plate was covered with aluminum foil, which was grounded to collect the electro-spun HA membranes. The electro-spinning and electro-blowing of HA typically required a high electric force. The highest electric field used in the present instrument was 40 kV, and the shortest distance between the two electrodes used was 9.5 cm, corresponding to an average applied field greater than 4 kV/cm. For the electro-blowing process, an air blowing system was attached to the electro-spinning apparatus so the modified apparatus had two simultaneously applied forces (an electrical force and an air-blowing shear force) to fabricate the nanofibers from a polymer fluid (Figure 2). The air blow system consisted of two components, a heater and a blower. The air generated by the blower was heated by passing it through the heating elements. The air blow features with various air temperatures and blowing rates were achieved by controlling the power output of the heater and the flow rate of the air. Figure 3 illustrates one design of the air stream guided by air ducts to pass around the spinneret so as to provide an additional pulling force (beside the electric force) to the polymer fluid droplet. The air stream could also be used to control the cooling rate of the fluid jet and the solvent evaporation rate. The air temperature profile could be varied quite substantially in the electro-blowing process, depending on the location where the temperature measurement was carried out. Three locations were used to determine the air temperature profile: the spot where the air came out of the air tube (A), the spot where the air came out of the gap around each spinneret (B), and the spot where the solution droplet came out of the spinneret (C). Among them, the temperature at spot C was most informative, as the solution temperature at the exit hole was almost the same as that of the air temperature at spot C. Consequently, spot C was chosen to monitor the air temperature. The electro-blowing process was performed under the same conditions (40 kV of electric field, 9.5 cm of distance between the electrodes) as the HA electro-spinning experiment, except that the HA solution feeding rate was set at 40 µL/min.

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Figure 3. Temperatures of blown air at different locations of the electro-blowing process.

Sample Characterization. The shear viscosity of the HA solution was measured by using a Rheometrics Mechanical Spectrometer (RMS-605E, Rheometrics Inc, NJ) to explore the proper viscosity range for nanofiber fabrication and to understand the effects of viscosity on the performance of electro-spinning and electro-blowing processes. The 50 mm plate-plate geometry was utilized for the measurements. The shear rate was controlled from 1 to 1000 s-1. The surface morphology of electro-spun and electro-blown HA membranes were investigated using scanning electron microscopy (SEM) (LEO1550, LEO, Germany) in order to examine the effects of processing parameters on the efficiency of nanofiber formation. The average fiber diameter was obtained from the SEM micrographs using a custom code image analysis program (the average diameter value was obtained from measurements of 50 different fiber segments in the SEM image of the HA membrane). Results and Discussion Electro-Spinning of HA Solution. Effects of HA Concentration. It has been demonstrated that polymer concentration (thus solution viscosity) plays perhaps one of the most important roles in electro-spinning.23,28,35 When the solution concentration is low, especially when it is near but below the overlap concentration (the overlap concentration of typical HA from synovial joints is about 0.135 w/v %36), the likelihood of intermolecular entanglement among the individual polymer molecular chains, although present, is relatively low. Under this condition, when the solvent is removed, a string-and-bead morphology is usually produced due to the fluctuations of concentration (in dilute solutions, the process turns into electro-spraying). On the other hand, if the polymer concentration is too high, the electric force may not be able to overcome the high viscosity of the solution, leading to a failure to form a polymer jet and consequently a failure to achieve the electro-spinning process. Therefore, it is logical to argue that the electro-spinning

Um et al.

process can only be operated within a concentration range, over which the polymer solution possesses sufficient molecular entanglements but a relatively weak resistance to form a polymer jet from the fluid droplet at the spinneret, i.e., the solution with a relatively weak surface tension. These requirements represent, to a certain extent, a contradiction, because a solution with a high degree of intermolecular entanglement is normally associated with high viscosity and high surface tension. Thus, electro-spinning is a process that can encompass only a limited range of solution viscosity and surface tension, especially when the applied electrical field strength is limited. In this study, the solution prepared from the 3.5 million molecular weight HA sample (HA-C) did not show a concentration range where the initial jet stream could consistently maintain its stability. This indicates that there is no suitable concentration window for the HA solution to meet all of the requirements of producing nanofibers by electro-spinning (we have tested the concentration range from 0.01 w/v % to 2 w/v %). Despite this discouraging finding, we observed that the variation of concentration could affect the fiber formation capability. This behavior is seen in Figure 4. Although no nanofiber structure was formed at 1.0 w/v %, electro-spinning started to produce nanofiber structure at concentrations higher than 1.3 w/v %. Although more fibers were formed at higher concentrations due to the increase in intermolecular entanglement, there was an upper concentration limit (1.5 w/v %) above which electro-spinning could not be carried out. This has been explained earlier. In the chosen electro-spinning conditions, the applied electric field was 40 kV, which was close to the practical limit for operation (higher electric potentials usually produce sparks, especially under high humidity conditions). At this potential (40 kV), the electrical field could not overcome the high viscosity and the surface tension of the HA solution at concentrations higher than 1.5 w/v %. Thus, the optimal concentration range for the production of nanofiber by electro-spinning of the chosen HA solution was from 1.3 to 1.5 w/v %, a very narrow window. The corresponding viscosity range was 3-30 Pa s (Figure 5). As the stability of the jet formation was poor even in the “optimal” concentration range (or the viscosity range), the electro-spinning results from this solution was very unsatisfactory. HA has an acidic group as well as the glucosamine segment, as shown in Figure 1. The presence of the weak acid makes the polymer a polyelectrolyte, leading to intermolecular associations between HA molecules and, consequently, unusually high viscosity for HA solution at even fairly low concentrations. The difficulty in preparing a highly concentrated HA solution is intimately related to the poor ability to electro-spin HA solutions. Another obstacle in achieving successful electro-spinning of HA solutions is the relatively high surface tension of HA aqueous solution. In the following sections, we tried to increase the HA concentration without substantially increasing the solution viscosity and to reduce the surface tension for fiber formation by electro-spinning within the present viscosity range. Polymer Blends by Addition of Low Molecular Weight HA. It is well-known that polymer chain entanglements above



Figure 4. Effects of HA concentration (w/v %) on the fiber formation of electro-spun HA: (a) 1.0 w/v %, (b) 1.3 w/v %, (c) 1.4 w/v %, and (d) 1.5 w/v %.

Figure 5. Shear viscosity of acidic HA aqueous solutions at various concentrations.

the overlap concentration promote the fiber formation during electro-spinning. However, the solution viscosity can rapidly increase at concentrations above the overlap concentration value. For associating polymers, such as HA, the solution viscosity often exceeds the operating viscosity range of the instrumentation for electro-spinning. Consequently, it is necessary to lower the solution viscosity but maintain a high level of polymer concentration. This is because at low polymer concentrations, an excess amount of solvent must be removed during the relatively short time period between the jet fluid stream leaving the spinneret and the fiber reaching the ground. With a relatively high amount of solvent, the electro-spinning process, even if it is operational, favors the bead formation due to the residual solvent and the resulting high surface tension. Thus, a highly concentrated

solution with a proper viscosity is essential for the successful fiber formation in the electro-spinning process. We find that the blending of polymers with lower molecular weight fractions can reduce the viscosity level without sacrificing the concentration value, thereby extending the operating range of HA with the existing instrumentation. In an effort to increase the total HA solution concentration, we mixed a fraction of low MW HA (HA-A) (Mw ) 45 000) to the high MW HA (HA-C) (Mw ) 3 500 000) polymer. Our objective is to use a minimum amount of high MW HA that favors the fiber formation and to have the total concentration of HA to be high enough so that sufficient solvent evaporation could be achieved within the short time period of electro-spinning. For pure HA-A, a fiber formation could not be achieved even with the highest electric potential (40 kV). Based on earlier experiments, the concentration range of 1.3-1.5 w/v % was found to be suitable for the fiber formation using the existing electro-spinning instrumentation. Thus, the concentration of 1.3 w/v % HA-C solution was used as the starting concentration, where the remaining 0.2 w/v % concentration of HA-C was substituted by 1 and 2 w/v % of HA-A in order to increase the total concentration of HA solution without substantially increasing the solution viscosity. As seen in Figure 6, the fiber content was increased with increasing HA-A concentration. In addition, the increase in the solution feed rate also facilitated fiber formation. Comparing the 1.5 w/v % HA-C solution with the 2 w/v % HA-A/1.3 w/v % HA-C mixed solution, a better morphology was achieved by the latter (solution at higher HA concentration) (Figure 6, parts B and C). Therefore, the addition of HA-A had a positive effect on the improvement of fiber formation in the electro-spinning of HA.

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Figure 6. Effect of adding low MW HA (HA-A) at two different flow rates (5 µL/min (A) and 10 µL/min (B)) to 1.3 w/v % high MW HA (HA-C) solution on the morphology of electro-spun membranes ((a) 0 w/v % HA-A (pure 1.3 w/v % HA-C), (b) 1 w/v % HA-A, and (c) 2 w/v % HA-A).

Effects of Ethanol Addition in Aqueous Based SolVent. A slight enhancement was obtained by mixing low MW HA (HA-A) with high MW HA (HA-C). Unfortunately, the result was still not satisfactory for viable production of nonwoven nanofiber membranes. The main problem was that the concentration of high MW HA could not be increased further (without the increase of solution viscosity) to a level that was suitable for nanofiber formation with our present electrospinning setup. Since the amount of high MW HA that could be substituted by lower MW HA was relatively small, i.e., on the order of 1-2%, the improvement by this approach was very limited. A further improvement was achieved by alternative means. As we could not decrease the solvent amount by increasing the solvent concentration, we could choose a solvent system that could evaporate at a faster rate. The polymer solution concentration should remain suf-

ficiently high so that the polymer chains were above the overlap concentration level. Ethanol is known to have a lower boiling point (78 °C), a higher vapor pressure (7.87 kPa), and a lower surface tension (21.97 mN/m) than those of water (100 °C, 3.17 kPa, 71.99 mN/m, respectively). With the addition of ethanol to the HA aqueous solution, there should be an increase in the evaporation rate and a decrease in the surface tension, thereby improving the conditions for fiber formation in electro-spinning. Furthermore, as the solvent will be removed during the electro-spinning process, its temporary presence should not inhibit its use in various applications. Based on these considerations, ethanol was added into an acidic aqueous solution consisting of both high- and lowMW HA fractions in order to further enhance the electrospinning of HA.



Figure 7. Effect of the ethanol content on the morphology of HA nanofibers electro-spun from mixed HA acidic aqueous solutions (1.3 w/v % HA-C + 1 w/v % HA-A (A) and 1.3 w/v % HA-C + 2 w/v % HA-A (B)) in the presence of different ethanol content: (a) 0 v/v %, (b) 10 v/v %, and (c) 20 v/v %.

Figure 7 shows the effect of ethanol addition to mixed high/low MW HA solutions at two different ethanol contents on the performance of fiber formation in electro-spinning. For the first mixed high/low MW HA solution (1.3 w/v % HA-C + 1 w/v % HA-A), 20 v/v % ethanol was the maximum amount that could be added before the phase separation occurred, whereas for the second solution (1.3 w/v % HA-C + 2 w/v % HA-A), 10 v/v % ethanol was used as the maximum content. However, we found that, although the fiber content could be increased slightly by introducing ethanol, the jet remained unstable and the improvement was also not significant. The inconsistent fiber morphology is also seen in Figure 7. Thus, ethanol alone could not act as an effective enhancer for fiber formation in the HA electro-spinning process. Electro-Blowing of HA. Although faster evaporation was achieved by addition of ethanol, the enhancement was small due to the limited solubility of HA in ethanol. Nevertheless, the fast evaporation was still a good and viable strategy that could overcome some of the problems in electro-spinning of HA due to the unusually high viscosity of the HA solution. The evaporation of solvent can also be accelerated by

blowing air, especially at high temperatures. Therefore, we introduced an air blowing system to the conventional electrospinning apparatus, not only to enhance the solvent evaporation rate but also to introduce a large additional pulling force for the jet formation. We call this new system “electroblowing”. In the following sections, we performed electroblowing to prepare nanosized HA fibers and examined several pertinent parameters, including the air blowing rate and the air blowing temperature, on the electro-blowing performance of HA solution. Effect of Air Blowing Rate. Since the air flow around the jet stream is intimately related to the solvent evaporation rate, the effect of air blowing rate on the performance of electro-blowing of HA solution was examined. As shown in Figure 8, without air blowing, the electro-spinning of a 2.5 w/v % HA solution (HA-C) resulted in the formation bead-on-string structure with an extremely unstable jet stream. With an increase in the air blowing rate (e.g. 70 ft3/ hr), the capability of fiber formation was increased. At the blowing rate of 150 ft3/hr, although the process did not yield a consistent fiber production, a stable jet stream was obtained. These results clearly imply that air blowing is the viable and

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Figure 8. SEM micrographs of electro-blown HA with room temperature of air blown at different blow rates: (a) 0 ft3/hr, (b) 70 ft3/hr, and (c) 150 ft3/hr. The feeding rate and HA concentration were 40 µL/min and 2.5 w/v % (HA-C), respectively.

essential additional parameter that can facilitate the fiber formation capability, when combined with the electro-spinning process. However, the fact that fiber formation was not well established under the high air blowing rate indicated that the increase in the solvent evaporation rate remained insufficient by introducing air flow alone. One route to improve such a deficiency was to raise the temperature of the blown air. Effect of Air Blow Temperature. Different air temperatures were used to examine the effect of temperature on the electroblowing performance of HA solution. Results are illustrated in Figure 9. It was seen that, as the temperature of air was raised, the capability of fiber formation in electro-blowing was improved. For example, although the overall performance of fiber formation was improved at 39 °C, a small amount of fiber with an irregular size distribution was pro-

Um et al.

duced because some droplets were still formed during the blowing process. As the temperature was increased to 47 °C, the jet stream became substantially stabilized, producing mainly the desired nano-fibrous structure with only a small fraction of bead formation. When the air temperature was raised to 57 °C, a consistent and stabilized jet steam was established, leading to a reproducible production of fine nanofibrous nonwoven membranes with fairly uniform diameters. The successful fabrication of HA nanofibers by electroblowing can be mainly attributed to the two new parameters introduced: the rate and the temperature of air blow. In essence, the introduction of air flow not only contributes to the stretching of the fluid jet stream, complementary to the applied electric field, but also to the evaporation of the solvent. With the increase in the air flow rate, both chain stretching capability and solvent evaporation rate are also increased. The temperature of the blown air also contributes to the solvent evaporation rate (e.g., the water vapor pressure of HA aqueous solution changed from 3.17 kPa at 25 °C to 17.32 kPa at 57 °C), but it plays an additionally unique role in facilitating the electro-blowing process. This can be rationalized as follows. When hot air is blown around the spinneret, the HA solution is also heated and reaches the temperature of blown air since the amount of solution at the tip of the spinneret is extremely small (about 4 µL). The temperature rise of the HA solution at the spinneret leads to a viscosity reduction. As seen in Figure 10, the solution viscosity is decreased by a factor of 3 (618 to 192 Pa s at 1 s-1) when the temperature is raised from 25 to 57 °C. The lower solution viscosity in combination with the electric force and the blown air leads to the formation of a stable and consistent jet stream. Thus, the new electro-blowing process permits the fabrication of nanofibers from associated HA solutions, whereas they cannot be efficiently processed by conventional electro-spinning methods alone. The fiber diameter was measured as a function of the air blow temperature in electro-blowing of HA solutions. At 25 and 39 °C, the fibers were not well developed and the diameters were irregular. However, as the air temperature was increased from 47 to 57 °C, the average fiber diameter was increased from 49 to 74 nm. The increase in the fiber diameter at higher temperatures could be due to the higher drying rate of the solution. This is because when the drying rate increases at high temperatures the concentration change of the polymer solution becomes faster, resulting in an increase in the solution viscosity of the jet stream and, consequently, an increase in the resulting fiber diameter. This observation is similar to the fact that a higher concentration in solution yields a larger diameter fiber in the electro-spinning process. On the basis of this observation, we conclude that the control of air blowing temperature is another important parameter that can be used to control the fiber diameter of electro-blown HA nanofibers. Conclusions In this study, we investigated several key parameters in the electro-spinning process and introduced a unique electro-



Figure 9. Effect of air blow temperature in the electro-blowing process of 2.5 w/v % HA-C solution: (a) 25 °C, (b) 39 °C, (c) 47 °C, and (d) 57 °C. The air blow rate was 70 ft3/hr.

spinning process. (1) The air blow process assisted the electric field to pull the HA solution into a jet stream at the spinneret. (2) A substantial decrease in the solution viscosity of the jet stream at the spinneret was achieved by elevating the air temperature. (3) The faster evaporation rate was accomplished by blowing air around the jet stream at high rate and high temperature. Acknowledgment. Financial support of this work was provided by a DoD-SBIR grant (DAAD16-03-C-0023) administered by the Stonybrook Technology and Applied Research, Inc. Dr. In Chul Um acknowledges the partial support by the Postdoctoral Fellowship Program of Korea Science and Engineering Foundation (KOSEF). References and Notes Figure 10. Shear viscosity of 2.5 w/v % HA-C solution at different temperatures.

blowing process to prepare nonwoven nanofiber membranes from associated and very viscous HA solutions. To improve the electro-spinning process, several new schemes were tested, including the control of HA concentration and viscosity by mixing of HA samples with different molecular weights, as well as by adding ethanol into HA aqueous solutions. These schemes, however, were not sufficiently effective to overcome the high viscosity of HA solution at relatively low concentrations. Only with the combination of air blowing and electro-spinning, we were able to prepare nanosized HA fibers with uniform diameters in tens of nanometers. This new process, termed electro-blowing, provided three advantages over the conventional electro-

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Electro-Spinning and Electro-Blowing of Hyaluronic Acid

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