DRUG DESIGN DEVELOPMENT AND DELIVERY JOURNAL (ISSN: 2631-3278)

Transdermal drug delivery offers an appealing alternative to injections and oral medications. However, applications of transdermal drug delivery are constrained to only a few drugs due to low skin permeability. The application of low‐frequency ultrasound enhances skin permeability, a phenomenon called low‐frequency sonophoresis. The skin consists of two important layers called the epidermis and dermis, which are on the fatty layer called hypodermis (subcutaneous tissue). The epidermis is the outermost layer of the skin. It consists mainly of cells called “keratinocytes”. This is caused by the evolution of cells formed in the lower layer and their accumulation on top of each other. At the top is the stratum corneum epidermis layer from almost completely dead cells. Stratum corneum acts as a primary barrier to drug delivery, transdermal drug delivery technique precedes to conventional drug delivery process. In this study, tramadol hydrogel is an opioid-like analgesic with much less adverse impact was carried out to rat skin. The tramadol hydrogel was applied on rat skin by using a novel developed sonophoretic device. There were 4 groups of Sprague Dawley male rats that were examined to evaluate analgesia. The first group was the control group, the second was the intraperitoneal (i. p. ) application group, the third was tramadol hydrogel (GT) without sonophoresis application and the last group was tramadol hydrogel with sonophoresis (GTS) application. It was shown that tramadol used with sonophoresis (GTS) increased analgesic effect threefold than tramadol hydrogel (GT) group 30 minutes later. Hot- plate Analgesia Meter was used and the efficacy was measured on 16 rats. Tramadol dosage was 28 mg per kilogram for each rat. The low-frequency sonophoresis device transducer was adjusted to 40 kilohertz (kHz) frequency for up to 60 minutes. Measurements were carried out at 0, 10, 20, 30, 40, and 60 minutes. There was a statistically significant difference between GT and GTS groups (P<0. 05, by Kruskal Wallis test). Moreover, the developed sonophoretic device application was successful and application low frequency 40 kHz was safe. Neither burn nor erythematous streaks were observed on rat skin by using low-frequency sonophoresis. 

Tramadol hydrogel; Hot plate analgesia test; Sonophoresis device; Transdermal drug delivery; Spraque Dawley rats; Low frequency

The skin is one of the most critical organs inside the frame and takes a huge role in the penetration of drug delivery structures [1]. Transdermal drug delivery systems have essential benefits as regards to conventional transport structures, accelerated affected patient comfort with much less pain, avoidance of degradation by means by the GI tract or enzymes and the hepatic first bypass metabolism, minimization of systemic toxicity, and clean manipulate of drug absorption, increases bioavailability can be used for long-term remedies of continual illness, continued protection of plasma drug levels, decreased negative drug effects, enhanced affected person compliance because oflessening in quantity and frequency of doses, less harm to tissues and powerful costly [2]. The skin talents as a barrier to inhibit drug delivery, stratum corneum layer is that the outermost layer of the skin, has a structure in which corneocytes are densely packed in a lipid matrix, forming the ‘bricks and mortar’ and that is the first crucial barrier [3]. The stratum corneum of the base layer is known as a granular layer is the formation of a mobile cell junction which blocks the drug from penetrating via the structure. The drug penetrates the stratum corneum initially, secondly passes wherethrough the deeper epidermis. As a consequence in the dermal layer has no drug accumulation [4].

Tramadol is a centrally acting analgesic agent with µ-opioid agonist properties, blocks noradrenaline uptake [5]. Tramadol hydrogel is a similar molecule with the 4-phenyl-piperidine analog of codeine, which is also acting as an analgesic and painkiller. Its elimination half-life is about 6 hours [6]. Tramadol has a high solubility in the oral cavity. It is also known as Tramadol Hydrochloride prepared with Pluronics 127, it has both opioid and non-opioid properties [7]. It is primarily effective at the central nervous system (CNS). According to structure, Tramadol is similar to Codeine and Morphine. It is 6000 times fewer side effects than Morphine and is 10 times less powerful than Codeine [8]. It provides serotonin (5-HT) reuptake due to inhibition of ache distribution inside the spinal cord[9]. Sonophoresis facilitates to penetrate from stratum corneum absorption [10]. Fellinger and Schmidt published an article on sonophoresis in the early 1950s for the treatment of polyarthritis. It was applied hydrocortisone on the hand’s digital joints [11]. This technique provided us with better results considered hydrocortisone injections for bursitis treatment [12]. Sonophoresis can also be applied on a variety of drugs which have capability to assist the penetration as well. One of the most important applications of this method is for local anesthetics transdermal application [13]. Transdermal drug release has been examined in 2 subgroups as active and passive methods [14]. Passive methods are that the optimization of the drug method or delivery to enhance skin permeability [15]. However, these techniques have limits for the development of the skin permeability of bio-macromolecules [16]. On the contrary, the active strategies which comprise physical or mechanical techniques even for bio-macromolecules [17]. These techniques are micro needles, iontophoresis, chemical penetration enhancers, and sonophoresis. There are four main ultrasound parameters which are frequency, intensity, duty cycle, and application of time. Low-frequency sonophoresis has an important breakthrough in the formation and collapse of gaseous cavities(acoustic). Moreover, with the usage of acoustic spectroscopy, quantifying inertial cavitation has become handier [18]. It can produce intense micro streams, which is going up the bioavailability of the drugs [19]. Cavitation happens because of the nucleation of small gaseous cavities all through the negative stress cycles of ultrasound. As a result, cavitation provides the disordering of the lipid bilayers, and the formation of aqueous channels ensures to penetrate easily in the skin [20]. Levy et al. demonstrated that when convection and cavitation had been mixed with mannitol, inulin, they enhanced transport to skin [21]. Mitragotri et al. carried out a work of the synergistic effect of low-frequency ultrasound that is using 20 kHz with sodium lauryl sulfate. The addition of 1% sodium lauryl sulfate to the solution reduced the threshold to about 18 Joules / cm2 [22].

Biotechnology has its milestone on low-frequency sonophoresis which has been studied by scientists for the last 10 years. Sonophoresis provides electrical energy to turn into mechanical energy or vice versa. Low-frequency sonophoresis helps to increase diffusion and ultrasound waves cause convection. The most powerful feature of low-frequency sonophoresis is measuring frequency and drug delivery ratio which can be controlled by an ultrasonic transducer. It can help hospital staff members, deliver a drug in a controllable way.

Besides, low-frequency sonophoresis helps to delivery of low and high molecular (macromolecule like heparin and glucose) of drugs which includes hydrophilic drugs. Hence, it has an important technique for drug delivery systems.

Low-frequency sonophoresis presents advantages over other transdermal delivery methods. It can be tested by application time and ultrasound parameters [7, 22]. So it provides local delivery. The other advantage is that it can be controlled by varying frequency and intensity of ultrasound. In another advantage is that it can also be used with a drug-containing patch. It is the effective release of prescribed medications that cannot be easily achieved by conventional patches, since the dose may be discharged or released. For the solution to this problem, controlled therapeutic systems are preferred by physical means. Therefore, it provides to penetrate drug whenever it necessary from patients. It can monitor blood analyses as well as blood glucose for diabetes [23].

Low-frequency sonophoresis was used for therapeutic purposes with help of a piezoelectric disc, it is formed by the addition of a mechanism. As a result of rapid change in voltage with transducer motion. It consists of a high-frequency pressure wave(ultrasound). The active substance is provided from the ultrasound device. It is provided by a contacting agent which transmits energy to the skin. Ultrasound waves with mechanical changes in the skin resulting from the stratum corneum. Cavitation (cavity formation) occurs in keratinocytes and resulting in cavitation cell increased permeation, rapidly reversible cell damage. 

 Parameters
Ultrasound links to skin barriers. These barriers are stratum corneum thickness, high skin impedance, low skin hydration, low useable area for solid transportation, age, blood flow, follicles such as sweat and hair, trauma on skin, humidity and temperature, presence of chemicals and chronical usages of drugs.

There are four main ultrasound parameters which are frequency, intensity, duty cycle, and application of time. Lowfrequency sonophoresis has an extensive study on the dependence of permeability enhancement on frequency and intensity in the low-frequency which has been shown by Tezel et al [24].

Emitted wave frequency is related to the size of the crystal. The attenuation of an acoustic wave is inversely proportional to its frequency. If the frequency increases, ultrasound penetrates less deeply under the skin. High frequencies range from 1-3 Mhz while low frequency ranges from 20 to 100 kHz. High frequencies were first surveyed as physical enhancers for transdermal delivery of drugs [25]. 

Many variables affect low-frequency sonophoresis. These are cavitation (pore induction), frequency, amplitude, intensity, and application of time.

Low-frequency sonophoresis has an important breakthrough in the formation and collapse of gaseous cavities(acoustic). Cavitation means the collapse and formation of gas bubbles in a liquid environment and the resulting collapse when exposed to a sound wave in such an environment. Cavitation is overwhelmingly meet with the coupling medium (the liquid consists in between the ultrasound transducer and the skin. The frequency and acoustic pressure amplitude are related to the maximum radius of the cavitation bubbles. During low-frequency sonophoresis, cavitation occurs within 15 micrometers of the stratum corneum and in order to overcome this, inert cavitation is created in the skin layers. Moreover, with the usage of acoustic spectroscopy, quantifying inertial cavitation has become more manageable [27]. It can produce violent micro streams, which goes up the bioavailability of the drugs. Cavitation happens because of the nucleation of small gaseous cavities during the negative pressure cycles of ultrasound. As a result, cavitation provides the disordering of the lipid bilayers, and the formation of aqueous channels provides to penetrates easily in the skin. 

Convection is a significant factor for low-frequency sonophoresis. Acoustic streaming (convective process) can augment the bioavailability of drugs such as Lidocaine.

Levy et al. demonstrated that when convection and cavitation were mixed with mannitol, inulin, they enhanced delivery to the skin. 

Attenuation of ultrasound waves leads to thermal increasement for low-frequency sonophoresis. Ultrasound waves cause heating of the medium. Thermal effects cause increase skin permeability. It provides to increases kinetic energy and diffusion of drugs, dilates points of entry of the skin, promotes drug absorption, and enhances the circulation of blood for in-vivo experiments. Duty cycle and ultrasound intensity are parameters that are directly related to thermal effects. Therefore, these parameters must be arranged for low-frequency sonophoresis application. 

Ultrasound application is not effective compared to the usage of low-frequency ultrasound combinations with other enhancers have been shown to be more efficient. Moreover, increasing transdermal transport, especially with the combination of ultrasound with other enhancers causes to decrease in the enhancers needed to help the drug flux. Therefore, a combination of ultrasound with other enhancers will increase the reliability by decreasing the strength of selected enhancers.

Mitragotri et al. carried out a work of the synergistic effect of low-frequency ultrasound that is using 20 kilohertz with sodium lauryl sulfate(SLS). It has been shown that the administration of sodium lauryl sulfate causes an approximately 3-fold increase in mannitol permeability and is only about 8 times greater than that of ultrasound for 90 minutes. It was also observed that the induced sulphate solution increased approximately 200-fold in the skin permeability of mannitol.

In particular, with the insufficiency of surfactants, the threshold ultrasound energy was about 141 Joules / cm2 to produce a detectable change in skin impedance. Mitragotri et al. have shown the addition of 1% sodium lauryl sulfate to the solution reduced the threshold to about 18 Joules / cm2 . The various results of this synergistic effect indicated that lowfrequency ultrasound indicated better spread and diffusion of the surfactant in the skin. 

The synergy between low-frequency ultrasound and iontophoresis is of great importance as it increases transdermal transport. This combination is particularly useful in the treatment of transdermal transport by Lee et al. By using heparin as a model drug, it has been shown to have a better and easier way to investigate the synergistic effect of ultrasound and iontophoresis on transdermal transport by Kost et al. Approximately 10 minutes prior to the administration of iontophoresis, the skin was once treated with 1% dodecyl pyridinium chloride solution. As a result, the increase in heparin flux of ultrasound and iontophoresis applications was recorded approximately 56-fold increased with these applications (Table 2).

Tramadol pharmacology
Tramadol is a centrally acting analgesic agent with µ-opioid agonist properties, which blocks noradrenaline uptake. Tramadol hydrogel is a similar molecule with the 4-phenyl-piperidine analogue of codeine, which is also acting as an analgesic and painkiller. It can be used by patients in the orthopedics spine clinic and may even be beneficial in patients with poor cardiopulmonary function, including patients with older people, obese and smokers, patients with liver or renal dysfunction, and nonsteroidal anti-inflammatory patients. It can be also used for post-operative pain relief. Its elimination half-life is about 6 hours.

Tramadol has a high solubility in the oral cavity. It is also known as Tramadol Hydrochloride (Tr HC) and has opioid and nonopioid properties. It is primary effective on the central nervous system (CNS). This drug is similar to codeine and morphine as considered to structurally. However, it is 6000 times less active than morphine and is 10 times less effective than codeine. However, in 1995, it was rated as a treatment of acute pain with food and drug administration (FDA). Tramadol hydrochloride effects on low-affinity m-opioid and k-opioid receptors, and norepinephrine (NE), blocking monoamine receptor systems. It provides serotonin (5-HT) reuptake due to inhibition of pain distribution in the spinal cord. It has also a lower incidence of adverse effects. It waschosen for many patients for analgesia effect and many advantages.

Hot plate analgesia test 
The hot plate is one of the most widely preferred testings for analgesia test. Because it is helpful to determine the analgesic efficacy of experimental drugs in rodents. In this experiment, it was used the guidelines developed by Ankier S. I. (1974). A hot plate, May AHP 0603 is a brand name, has been adjusted to 54°C and the latency of the first reaction (licking of the paws or jumping response- a jump has been identified by all 4 paws leaving the heated surface) has been recorded. A cut-off period of 60 seconds has been considered to avoid any damage to the paws. Rats were placed onthe hot plate one by one and response latency was measured with a stopwatch(rats were used from YÜDETAM, Yeditepe University). Observations showed that the majority of animals reacted to the heat by licking their paws.  

Development of Transdermal Sonophoresis Device
Transdermal sonophoresis device was developed at Yeditepe University Biomedical Engineering Laboratories.

Lm555 oscillator, Texas Instrument, has been used for this experiment. In this experiment, the aim is to produce square wave pulses provided continuously by the 555 timer IC. On the other hand, the 555 timer IC has connected either in its monostable mode therefore it generates a back and front type switching action. The connection of the 555 timer IC in an unstable mode is a tricky part. When it was approved highly precise freeroaming waveform, very stable 555 oscillators has to use. Also, the RC circuit has to be connected to an oscillator which contains 2 resistors and capacitors. The 555 timer IC can be used which generates stabilized square wave output waveforms. Its duty cycle is between 50-100%.

The device has stopped working until the next trigger pulse. It initiates to act as an unstable multi-vibrator. It has great importance to continuously re-trigger the effect of the circuit. Pin 2 which is trigger input provides a triggering process connecting to 555 timer and threshold input to pin 6 acts as an unstable oscillator with together. Single timing resistor has a key act on this device because it has been split into two different resistors which are R1 and R2. Pin 7 which is discharge input has been linked to their junctions (As seen in Figure 3).

Tramadol Hydrogel Formulation
In this study, firstly 20 grams of Pluronic F 127 was weighed and dispersed into the 40 mL purified water. The dispersed polymer was put into the refrigerator overnight and was dissolved homogeneously as a hydrogel. Then hydrogel was incubated 2 days at 37 °C. The dispersed polymer was sterilized at room temperature. The solution was placed in an Eppendorf tube and then the Tramadol solution was incorporated into a hydrogel. Finally, a solution has sterilized in a cabinet with ultraviolet for 30 minutes.

Drug Loading 
The reason for mixing tramadol hydrogel with Pluronic F 127, Sigma Aldrich Chemical Co. , they are more or less permeable to body fluids, they do not avoid their transition to useful substances such as foods and oxygen. It has little friction with the surrounding tissues. It has also shown low adhesion to the mucous membrane and tissues. Epithelial cells in the stomach are protected from the acidic stomach acid thanks to the gel. Taking advantage of Blankenship’s studies, it was concluded that 28 mg/kg of tramadol hydrogel was the effective dose in rats, and since each of the rats had a weight of 250 grams, 7 mg/kg was administered to each in-vivo study [26].

Hot Plate Analgesia Test  
Four groups were determined for measuring the analgesic effect on rats (in Figure2). The first was determined as a control group. 3 rats were placed with sonication at 40 kHz at 0, 10, 20, 30, 40, and 60 minutes. The second was the tramadol hydrogel group, which is name Contramal, its brand name is Abdi İbrahim. In this group, only tramadol hydrogel was put on rats directly on their backs that were shaved. The third was an intraperitoneal group. Each rat has taken 7 mg tramadol (28 mg/kg, each rat is 250 grams). The last was the sonication application group with tramadol hydrogel was applied on rats with a sonophoresis device. Tramadol hydrogel was supplied with a 40 kHz ultrasonic transducer; provided with a 15 volt via the power supply. In conclusion, each time jumping or paw licking response has been noted to understand analgesia effect (Table 1) [27].

Result 
Using low-frequency sonophoresis, acoustic cavitation is created on the skin, increasing the pore and permeability on the skin. As a result of the cavitation, acoustic microjets on the skin cause inhomogeneous pore formation. [28]. In-vivo experiments consisted oftramadol hydrogel and tramadol hydrogel with a lowfrequency sonophoresis application group. Tramadol hydrogel was improved drug penetration as it provides the destruction of the organic barrier [29]. A hydrogel that could stick finely to the epithelium can make increase the time retention of the system at the destination site [30]. Therefore, it has provided sufficient drug dose for the desired therapeutic healing effect; this is the maximum especially massive for transdermal controlled drug delivery [31]. Extensive efforts were made to expand bio-adhesive hydrogels to enable advanced drug delivery [32]. Ultrasoundbrought aboutthe disruption of ionic cross-links to setoff bursts of drug launch became observed [33]. Low frequency sonophoresis can instant disrupt the hydrogel structure [34]. Therefore, it’s far potentially advantageous based on its high Spatio temporal decision and deep penetration inside tissues [35]. It was observed no effect on latency in response to acute thermal pain in any of the rats that were given transdermal tramadol as hydrogel with sonication application at initial, 10, 20, and 30 minutes later. It was calculated that the transdermal as hydrogel with sonication application did not seem too effective until 40 minutes. Spraque Dawley rats that obtained 28 mg tramadol in keeping with kilogram body weight i. p. and transdermal packages with sonication had behavioral responses to tramadol that included minimized responsiveness to tactile stimuli and decreased cage interest no impact was discovered on latency. Acute thermal pain in any of the rats that have been given transdermal tramadol as hydrogel with sonication application at 10, 20 and 30 minutes (Table 2-6) [36].

In this study, it was found that when administering 40-60 minutes sonication was found effective. The maximum latency in response to acute thermal pain was observed after 60 minutes. The bioavailability of the transdermal hydrogel with sonication was increased almost two and three times (respectively after 40 and 60 minutes) more than transdermal hydrogel application. Hot plate latency test was used to compare latency in response to acute thermal pain after transdermal tramadol application with sonication (40kHz)[37]. This device investigated the possibility of developing transdermal tramadol with sonication application allowing the fast analgesic effect of tramadol in 40-60 minutes(in Figure1). Finally, it was observed 40 kHz was useful for this study, when the comparison with the high-frequency sonophoresis, low frequency provided larger bubbles (Figure 4-6) [38].

The utility of sonophoresis to the pores and skin will increase its permeability and let in penetrating of drug substances[39]. Transdermal delivery of hydrophilic substances like tramadol causes problems due to their lack of ability to integrate with the cellular membrane and penetrating through the stratum corneum [40]. In this study, it was observed that the efficacy of tramadol hydrogel was increased with sonophoresis as a trigger effect after 40 minutes. Tramadol drug penetration was tested with a hot plate analgesia test. 40 and 60minutes results were observed rat’s paw licking or jumping was tested Kruskal Wallis test for statistical analysis. At the end of the study, it was not observed no skin irritation with the lowfrequency sonophoresis technique. It was safe at 1. 5 W/cm2 energy density and 40 kHz frequency. In this way, low-frequency sonophoresis sooner or later causes greater modifications to the skin, growing its permeability greater than excessive frequency ultrasound, particularly for high-molecular-weight drugs [41]. In the simultaneous treatment, the drug and ultrasound had been performed at the same time and for this reason, pores and skin transmitting changed into better with the aid of the better diffusion as a result of structural modifications inside the skin and also because of convection ultrasound (Figure 7) [42].

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