Doxy

ABSTRACT

Dye-like applications of antibiotics to silk produce infection-resistant materials for potential use in biomedical applications. Two antibiotics, doxycycline (doxy) and ciprofloxacin (cipro), are applied under a variety of conditions to silk and to silk that has previously been hydrolyzed at 40°C for 20, 40, and 60 minutes. FTIR spectroscopic analyses indicate that the drastically increased sorption of antibiotics by hydrolyzed silk is attributable to both chemical and conformational changes that occur with the hydrolysis. The high sorption of doxy by hydrolyzed silk does not necessarily yield a more infection-resistant material, as determined by a zone of inhibition test. Conversely, the same hydrolysis considerably increases both the sorption of cipro and the zone of inhibition of cipro-treated silk dyed at 65 and 85°C.

Silk is known for its long history of use in textile applications, but it has also been used for many years in biomedical applications [I]. The most popular silk from Bombyx mon consists of two main components: silk fibroin and sericin, the fiber and surrounding gum, respectively. About 85% of the highly repetitive units in silk fibroin are composed of glycine, alanine, and serine in a roughly 3:2:1 ratio [9, 10].

The main biomedical application of silk is as sutures for wound ligation in braided or multifilament forms. Over the past 100 years, silk has become the most commonly used natural suture, surpassing collagen [I]. Its highly ordered nature provides a combination of strength and toughness that is remarkable among natural fibers. In addition, modified silk fibroin has found various applications in the biological and biomedical fields as enzyme immobilizers, oxygen permeable membranes, and drug permeable films and matrices for mammalian cell culture [16].

Some biological responses of the human body to silk have raised questions about the biocompatibility of untreated (raw) silk [1], mainly due to the presence of sericin. Sericin removal followed by a treatment with exogenous materials such as wax or silicone minimizes adverse problems with biocompatibility and hypersensitivity to silk [1, 26]. Nevertheless, sericin is useful in producing cryopreservatives, anticoagulants, and biocompatible materials [26].

One of the biggest problems of implantation materials in vivo is bacterial infection caused by the implanted material [5, 6, 15, 17-19]. Such infections are combated by antibiotics, many of which have molecular masses and structural features that render them superficially similar to textile dyes [5, 25]. These similarities are close enough to be of practical use in developing materials with improved infection-resistant properties by using the textile dyeing technology. Thus, polyester and polyurethane materials have been "dyed" with fluoroquinolone antibiotics [5, 6, 25]. The interactions are of the right order such that the antibiotics are slowly and continuously released from the substrate, providing antibiotic activity that is maintained over periods of time far in excess of those obtained simply by "dipping" the material in an antibiotic solution [13]. Implantation devices and sutures are dipped or soaked in a solution of penicillin and heparin at the operating table immediately prior to insertion in the patient to prevent any infection or other complications such as thrombosis [5, 13]. Such attempts have not been completely effective because of the brief residence of antibiotics at the implantation site [13]. Therefore, sustained release of antibiotics becomes important for keeping the implantation site bacteriostatic for a longer time.

Unlike other common biomedical textiles such as polyester, silk contains various polar functional groups that might enhance antibiotic sorption [1, 21, 26]. The same functional groups might change the release characteristics at the same time, for better (greater interaction leading to a slower release and longer efficacy) or worse (the interactions might be great enough to prevent sufficient release for antibiotic activity to be sustained). The efficacy might also be modified by the application conditions, which could lead to antibiotics diffused throughout the fibers or confined more to the fiber surfaces. These considerations have prompted this study of the interaction of antibiotics with silk. In this work, we "dye" silk fabrics with two antibiotics, doxycycline (doxy) and ciprofloxacin (cipro).

Doxy is a semisynthetic tetracycline antibiotic [14], whereas cipro is a fluoroquinolone, a first generation DNA gyrase inhibitor [23]. These antibiotics are highly active against gram-negative and gram-positive bacteria, but are also the only antibiotics approved for treating anthrax (Bacillus anthracis) infection by the Centers for Disease Control and Prevention [3]. Figure 1 shows the chemical structures of doxy and cipro.

Experimental

An unbalanced plain weave silk fabric (fabric weight 61.3 g/m^sup 2^) containing sericin obtained from stocks within our department was used throughout the study. No sericin was removed prior to antibiotic treatment. Doxy and cipro were donated in pure form by Pfizer and Serological Products (Bayer-made), respectively, and used without further purification. Sodium hydroxide and glacial acetic acid were reagent grade, purchased from Aldrich Chemicals.

"Dyeing" of antibiotics on silk involved an Ahiba Polymat (Datacolor International) dyeing machine; 2% on weight of fabric (owf) of the antibiotic was applied at a liquor-to-fabric ratio of 20:1. Experimental parameters were dyeing temperature, time, and dyebath pH. The bath pHs were 2, 6.5, and 9 for doxy and 3, 5.5, and 10 for cipro: the lowest pH among them was the initial pH of the dyebath containing each antibiotic. Other pHs were controlled by adding 1% NaOH and 1% acetic acid and monitoring with a Corning pH meter 115. Dyeing temperatures and times were 25, 45, 65, 85, and 100°C for 1, 2, and 3.5 hours. After the process was run, the fabric was removed and the amount of antibiotic taken up by the silk was determined (see analyses below).

To investigate the effect of hydrolysis on sorption of antibiotics, the silk was treated in 1% NaOH for 20, 40, and 60 minutes at 40°C and a 20:1 liquor ratio in the Ahiba dyeing machine. After hydrolysis, the tensile strength of the treated fabric was measured with a Q-test (MTS Systems Corporation) CRE (constant rate of extension) instrument, according to ASTM D5035-95 (raveled strip) with a crosshead speed of 200 mm/minutes and gauge length of 76.2 mm.

To determine antibiotic sorption by silk, the concentration of residual antibiotics in the dyebath after dyeing was measured with a Gary 50 UV/VIS spectrophotometer (Varian Instruments, U.S.A.). The [lambda]^sub max^ values of doxy and cipro were determined as 274 and 276 nm, respectively. The relationships between absorbance and concentration were established at [lambda]^sub max^ for each antibiotic. There was no pH adjustment for doxy dyebaths after dyeing, since its absorbance at 274 nm and solubility were quite consistent at different pHs. However, cipro was insoluble at higher pHs, so cipro solutions for analysis were controlled to pH 3. The cipro solutions were also immersed in a water bath at 85°C for 15 minutes prior to appropriate dilution to ensure a complete dissolution of cipro for the absorbance reading. The data were analyzed in terms of this residual concentration. In addition, the amount of antibiotic taken up by the silk was determined as the "percent exhaustion," calculated as follows:

Exhaustion (%) = [(C^sub 0^ - C^sub r^)/C^sub 0^] × 100 ,

where C^sub 0^ is antibiotic concentration in blank solution, and C^sub r^ is the residual antibiotic concentration of the dyebath containing the substrate after dyeing.

The CIE lightness (L*) of doxy-dyed silk fabrics was evaluated with a Macbeth ColorEye system along with SLI-Form®/NG software (She Lyn Inc.). The infrared spectra of the untreated and hydrolyzed silk were obtained with a Sense FTIR spectroscope (SensIR Technologies) with an attached diamond ATR in the spectral region of 4000-700 cm^sup -1^ with 54 scans at 4 cm^sup -1^ resolution.

A zone of inhibition test determined the antimicrobial activity of the dyed materials. A stock solution of S. epidermidis was thawed at 37°C for 1 hour, then 1 µl of this stock was added to 10 ml of trypticase soy broth and incubated overnight at 37°C. From this solution, 10 µl was streaked onto trypticase soy agar plates. Untreated and antibiotic-treated silk segments were autoclaved, embedded into the streaked trypticase soy agar plates (n = 3 segments/time interval/treatment), and placed overnight in a 37°C incubator. Standard 5 µg antibiotic Sensi-Discs (n = 3) were also embedded at each time interval. The zone of inhibition of each piece was determined, taking the average of three individual diameter measurements. Meanwhile, pieces of the dyed materials were subjected to a "washing" to simulate blood flow conditions [5]. Samples were removed at intervals and the washing solution replaced each time. The zones of inhibition of these samples were determined, and thus zone size (mm) over time was determined for each parameter evaluated.

Results and Discussion

EFFECT OF TEMPERATURE AND TIME ON DOXY SORPTION

The effects of dyeing temperature on doxy sorption of silk fabrics at three pH conditions are shown in Figure 2a. Sorption of doxy on silk was essentially consistent at different dyeing temperatures. The concentration of residual doxy was somewhat less at 100°C, but this was due to the decomposition of doxy at 100°C as shown by the loss of concentration in the blank bath (no silk). Note that at 100°C, the decreased concentration of residual doxy in the silk-containing bath was less than that in the blank bath. This suggests that the presence of silk tends to alleviate doxy decomposition at high temperatures.

Even at a dyeing temperature of 25°C, the % exhaustion of doxy on silk was over 40% at all three pHs (Table I). Such exhaustion values are lower than commercial textile dyes, but much higher than the exhaustion of antibiotics on other manufactured fibers such as polyester and polyurethane [5, 25]. Percent exhaustion did not increase with increased dyeing temperature. The high uptake of doxy on silk at a low temperature was primarily due to the presence of residual sericin on the silk surface. No sericin was removed from silk prior to dyeing, as described previously. Unlike fibroin, which is a highly ordered crystalline material with simple repeating units, sericin is much more amorphous and hydrophilic, containing considerable amounts of serine and other hydrophilic amino acids [1, 10, 21]. High temperatures generally increase the sorption rate of (dye) molecules to the substrate. In this case the increase could be minimized by concomitant sericin removal, which is facilitated at higher temperatures. Weight loss values of silk at 100°C were 1.2, 1.7, and 1.9 for 1 hour and 3.1, 3.4, and 6.2 for 3.5 hours at pH 2, 6.5, and 9, respectively, indicating removal of sericin by dyeing at high temperature and pH. Nonetheless, the % exhaustion after 3.5 hours increased gradually with increasing temperature. This suggests that the uptake by the fibroin is dominant. It also implies that the system is not at equilibrium, since dye uptake is exothermic, and equilibrium exhaustion is higher at lower temperatures.

Since both silk fibroin and doxy are amphoteric in nature with isoelectric points of zwitterions at around 5 [8, 21], we initially thought that doxy sorption on silk would largely depend on bath pH. However, doxy sorption varied little with pH, and the only effect we observed was a higher sorption and exhaustion of doxy at pH 2 (Figure 2a and Table I). At pH 2, we expected that that basic nitrogen atoms in silk and doxy molecules would be completely protonated, creating potential electronic repulsion. This result, however, suggested that other interactions such as hydrogen bonding play a greater role in doxy sorption. This postulation was also supported by consistent sorption of doxy in the presence of up to 20% salts (not shown). The minimal effect of electronic interaction was also shown in our previous study for sorption of amphoteric fluoroquinolone antibiotics on polyurethane film modified with carboxylic acid groups [25].

Pure doxy is in a yellowish powder form, and aqueous doxy solutions tended to darken on standing. The rate at which solutions darkened increased with simultaneous increases in temperature and pH. Solutions changed color rapidly at pH > ~7 and over longer times at lower pHs. UV/VIS spectroscopic analysis of solutions that had changed color revealed that the second absorption maximum peak (about 345 nm) did not change correspondingly, but over time its intensity decreased, showing that further changes were taking place in the solution (Figure 3). Nevertheless, absorbance at the first absorption maximum (274 nm) remained consistent despite all these variations in visible color, which we interpreted as indicating that the active doxy was stable through these processes.

The color of doxy solutions was reflected in the color of the silk samples after dyeing application, which ranged from light to dark brown. The color intensity depended on dyeing temperature and pH. To investigate the color change in more detail, we measured the CIE lightness (L*) of the doxy-dyed silk fabric normalized to that of pristine silk (L* = 82.1), as illustrated in Figure 2b. The lightness of silk fabric continuously decreased upon dyeing at temperatures above 45°C at all three pHs. The range of colors produced at different temperatures was readily apparent by simple visual analysis. Since doxy absorption at 275 nm was stable to the dyeing conditions, this suggests that the colored component that develops in solution is also substantive to silk.

To examine effect of dyeing time on doxy sorption, silk was dyed with doxy at 85°C. At this temperature, the absorbance of a blank bath at 274 nm was consistent for 3.5 hours, as illustrated in Figure 4a, again showing the stability of doxy to the application conditions. With silk present, the concentration of residual doxy in the dyeing bath continuously decreased, indicating a continually increasing sorption of doxy with time. As we mentioned previously, the continued increase in uptake suggests that equilibrium has not been reached, and that diffusion of doxy through the silk structure is comparatively slow. Again, as expected, the doxy-dyed silk darkened substantially with increased dyeing time. At pH 9, the decreased lightness (increased color change to brown) was maximal, representing the pH effect on the color change of the doxy molecule (Figure 4b).

EFFECT OF TEMPERATURE ON CIPRO SORPTION

The thermal stability of cipro was better than that of doxy, as indicated by the consistent absorbance of a blank bath at [lambda]^sub max^ at 100°C and no color change (Figure 5). It also had a lower aqueous solubility than doxy, being soluble only at acidic pHs at room temperature [12, 23]. Among the three pHs we examined in this study, complete dissolution only occurred at pH 3 where it had maximum stability [23]. We therefore expected that pH would show more effect on the Sorption of cipro than of doxy.

As shown in Figure 5, cipro sorption at pH 3 did not vary significantly with dyeing temperature. Even at 100°C, the exhaustion of cipro was less than 20% at pH 3. However, at pH 5.5 and 10, concentrations of residual cipro were considerably lower at 45°C than those at higher temperatures. We believe the extremely low residual concentrations of cipro in the dyebath at 45°C and pH 5.5 and 10 were mainly due to insolubilization of cipro during dyeing. Cipro precipitated by the pH adjustment was physically trapped within the fabric structure, resulting in the low measured concentration of residual cipro in the bath.

Nevertheless, the effect of pH was clearly apparent in cipro applications at higher temperatures where cipro was soluble during dyeing: silk sorbed more cipro at pH 5.5 and 10 than at pH 3. The pK^sub a^ values of cipro were 6.09 for the carboxylic group and 8.74 for the nitrogen on the piperazinyl ring [12, 23]. Its isoelectric point was at pH 7.4, which was higher than that of silk [23]. Therefore, at pH 3, we expected that the carboxylic group of cipro was not ionized and basic nitrogen was completely protonated, resulting in the same positive charge for silk and cipro as for silk and doxy. The low sorption of cipro on silk at this pH demonstrated that an electronic repulsion played a major role. The disparity in electronic interaction of doxy and cipro with silk was probably due to the basicity difference between the amide nitrogen in doxy and the piperazinyl nitrogen in cipro. On the other hand, at pH 5.5 and 10, the ionic conversion of carboxylic groups might occur only partially, if at all, resulting in reduced electronic repulsion and, consequently, higher sorption of cipro onto silk than that at pH 3.

ANTIBIOTIC DYEING OF HYDROLYZED SILK

Silk hydrolyzed by 1% NaOH for 20, 40, and 60 minutes at 40°C lost 7.9, 8.0, and 8.6% by weight, respectively, indicating complete sericin removal even at the shortest time. The hydrolysis treatment was also expected to affect the fibroin structure. Retentions of mechanical properties of silk hydrolyzed at 20, 40, and 60 minutes were 83.8, 81.9, and 73.2% for strength, and 87.0, 84.2, and 63.4% for elongation, respectively.

Hydrolyzed silk showed dramatically increased doxy and cipro Sorption at all three temperatures examined (Figure 6). Dyeing was carried out at pH 2 for doxy and pH 3 for cipro where no pH control was necessary. Percent exhaustions of antibiotics on silk hydrolyzed for 60 minutes were between 86.3 and 90.1% for doxy and 78.2 and 82.4% for cipro, compared to the corresponding blank solution. These results clearly demonstrated that highly improved sorption and % exhaustion of antibiotics such as doxy and cipro was obtained by silk hydrolysis.

To study the structural changes that occurred in the silk as a result of the hydrolysis treatment, we conducted an FTIR study. Hydrolysis could conceivably cause chemical and/or conformational changes to the silk fibroin. Hydrolysis significant increased the heights of some representative FTIR absorption peaks, such as 3277 cm^sup -1^ (primary amine N-H stretching), 1621 cm^sup -1^ (amide I), 1513 cm^sup -1^ (amide II), 1442 cm^sup -1^ (CH^sub 2^ deformation), and 1227 cm^sup -1^ (amide III), as shown in Figure 7. We attributed such increases to increases in the concentrations of chemical species such as polar amino and carboxylic acids within the fibers, mostly due to scission of peptide bonds. The increase in peak intensity was most substantial in silk hydrolyzed for 20 minutes, which suggested that the first 20 minutes of hydrolysis had the greatest effect on chain cleavage of silk fibroin. Since the increase was in comparison with the pristine silk containing sericin, which was expected to be more polar, the increased chemical species in the hydrolyzed silk without sericin was quite substantial.

The peaks ascribed to conformational structures of fibroin were complex and largely overlapped in the normal FTIR spectra. Peak resolution can be improved by differentiating the zero-order absorbance spectrum with respect to the wavelength to produce the second-order derivative spectrum [11]. Thus, shoulder peaks were resolved into a number of sharp bands. As shown in Figure 8, the peaks at 1642 cm^sup -1^ (amide I), 1556 cm^sup -1^ (amide II), 1262 cm^sup -1^ (amide III), and 875 cm^sup -1^ (amide IV) increased significantly with hydrolysis time, representing an increase of [alpha]-helix conformation or random coil in the hydrolyzed silk. These FTIR data agreed well with antibiotic sorption, which showed the highest initial rate of uptake. Interestingly, however, the peaks attributed the [beta]-sheet conformation of the fibroin, such as 1709 and 1621 cm^sup -1^, also increased substantially, indicating that the remaining [beta]-sheet conformation became more distinct. It is well known that a distinct [beta]-sheet conformation can be obtained by heat and solvent treatment of silk fibroin [9, 20, 24]. An increase in the crystallinity of the [beta]-sheet conformation was further substantiated by the increase in crystallinity index with hydrolysis for 40 and 60 minutes, shown in Table II [4].

These results suggest that substantial increases in sorption of antibiotics in hydrolyzed silk are due to a combination of several factors, such as increased polar functional groups and amorphous regions. In addition, larger voids are more prevalent in the core area of silk fibroin, whereas the smaller voids are mainly located around or adjacent to its periphery [22]. An increase in the availability of larger voids with hydrolysis could enhance antibiotic sorption. Disruption of hydrogen bonding in silk fibroin during hydrolysis could also improve antibiotic sorption.

INFECTION RESISTANT PROPERTIES OF TREATED SILK

Figure 9a shows the zone of inhibition (mm) for silk fabric dyed with doxy for 3.5 hours at pH 6.5 at three different temperatures. Somewhat surprisingly, the silk fabrics dyed at lower temperatures (45 and 65°C) showed a more prolonged release of doxy. We expected that even with the same level of antibiotic on the fibers, the location of antibiotics within the fiber structure would vary with dyeing temperature. Thus, at 100°C doxy diffused more deeply within the fiber, and once leaching of the surface-bound doxy was complete, within four hours, little activity remained.

On the other hand, the zone of inhibition of the cipro-treated silk decreased quickly with washing time, and no activity remained beyond four hours for any application pH, as shown in Figure 9b. We therefore suspected that interactions of doxy and cipro with silk were quite different, occurring at different locations within the fibers or at a lower rate and/or extent of diffusion from the material under the conditions of the zone test.

The zones of inhibition of the treated hydrolyzed silk fabrics are shown in Figure 10. These zones were largely consistent at all treatment temperatures, probably because of high doxy sorption even on silk hydrolyzed for the shortest time. Some minor differences between samples occurred after four hours of washing, but at 24 hours, the zones of inhibition were the same regardless of treatment temperature or hydrolysis time. A comparison of the results in Figures 9a and 10 showed that the zone of inhibition of the doxy-treated hydrolyzed silk was less than that of the doxy-treated (at 45 and 65°C) unhydroIyzed silk at both zero and 24-hour wash times, respectively. Since doxy sorption by hydrolyzed silk was much greater, as previously shown in Figure 6, this result suggested that doxy sorbed by the hydrolyzed silk could be more strongly bound or located in a more interior area than that in the unhydrolyzed silk.

On the other hand, zones of inhibition of cipro-treated, hydrolyzed silk were quite different from that of the corresponding doxy-treated material, as illustrated in Figure 11. Hydrolyzed silk treated with cipro at 45°C showed no antibacterial activity at 24 hours (like the cipro-treated unhydrolyzed silk, Figure 9), whereas substantial zones of inhibition were apparent for those treated at 65 and 85°C. The only exception was in the cipro-treated silk hydrolyzed for 40 minutes. We can provide no explanation for the different zone of inhibition at 24 hours with the silk hydrolyzed for 40 minutes. Nevertheless, a highly infection-resistant silk with sustained release could also be obtained by applying cipro with careful control of hydrolysis and treatment conditions. Note that the zone of inhibition was generally greater in cipro-treated silk than doxy-treated silk, suggesting the superior antibacterial activity of cipro compared to doxy against the bacterium (S. epidermidi) used in this study.

Silk is known to lose the majority of its tensile strength within 1 year in vivo and, consequently, is regarded as a long-term absorbable suture [I]. Continuous leaching of antibiotics could be possible from silk fabrics treated with antibiotics, but showing no antibacterial activity after certain wash times, during their proteolytic degradation in vivo. Some examples include doxy-treated silk at a high temperature and cipro-treated silk hydrolyzed for 40 minutes. Therefore, antibiotic-treated silks showing a zone of inhibition at 24 to 48 hours could find end-use as a short-term infection-resistant materials, whereas the same material that might release antibiotics during in vivo degradation could be useful as a long-term infection-resistant material. Further study is needed to confirm antibiotic release from biodegrading silk in vivo. Note that the zone of inhibition test was only for wash times of up to 24 or 48 hours, and more extensive testing might find antibiotic activity maintained for much longer periods.

Conclusions

To produce infection-resistant biomaterials, silk fabric is "dyed" with two common antibiotics, doxy and cipro, at a range of dyeing temperatures, times, and pHs. Sorption of doxy on silk is steady at around 40% at different dyeing temperatures, because of the presence of the highly amorphous and hydrophilic sericin. On the other hand, dyeing time affects doxy sorption, suggesting the necessity of long dyeing times such as 3.5 hours to achieve high sorption of doxy on silk. The pH has little effect on uptake despite the amphoteric nature of silk and doxy, indicating the importance of other interactions such as hydrogen bonding in doxy sorption.

Cipro is more stable under dyeing conditions. At pH 5.5 and 10 at low temperatures, precipitated cipro is physically trapped in the fibers, resulting in exceptionally low residual concentrations in the bath. At pH 3, electronic repulsion results in a consistently low (about 20%) uptake at all dyeing temperatures. Unlike doxy, cipro is sorbed to a greater extent by the silk at pH 5.5 and 10. Such a disparity between doxy and cipro is ascribed to the different basicities of nitrogen within the two antibiotics. Weaker electronic repulsion could cause higher sorption of cipro at pH 5.5 and 10.

Release of doxy estimated by the zone of inhibition is much slower with silk fabrics dyed at lower temperatures such as 45 and 65°C than with those dyed at 10O°C. On the other hand, cipro application on unhydrolyzed silk does not provide sustained antibacterial properties.

Silk hydrolysis dramatically increases the sorption of doxy and cipro. High sorption and exhaustion of both antibiotics can easily be obtained by hydrolyzing silk for 20 minutes at 4O°C with about 20% strength loss. The FTIR spectrum and its second derivative confirm the scission of peptide bonds and conformational changes in silk fibroin during hydrolysis reaction. However, a zone of inhibition test on antibiotic-treated, hydrolyzed silk indicates that increased doxy sorption by hydrolyzed silk does not result in a greater zone of inhibition of antibiotics at up to 24 hours of wash time. Conversely, silk hydrolysis for 20 or 60 minutes considerably increases the zone of inhibition of cipro-treated material at 65 and 85°C. Therefore, in terms of infection-resistant properties, a hydrolysis treatment for doxy-treated silk is not essential, but it does produce silk with sustained cipro release.

ACKNOWLEDGEMENT

This research was funded by the U.S. Army (DAAD1602P0720).

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Manuscript received April 29, 2003; accepted june 20, 2003.

HYUNG-MIN CHOI,1

School of Textiles, Soongsil University, Seoul, South Korea

MARTIN BIDE

Department of Textiles, University of Rhode Island, Kingston, Rhode Island, U.S.A.

MATTHEW PHANEUF, WILLIAM QUIST, AND FRANK LOGERFO

Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, U.S.A.

1 To whom all the correspondence should be addressed: tel: +822-820-0626, fax: +822-817-8346, email: hchoi@ssu.ac.kr

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