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Prajeesh et al: Development and Evaluation of Nanoparticle-Loaded Hydrogel of Co-trimoxazole 3131
Research Paper
Development and Evaluation of Nanoparticle-Loaded Hydrogel of Co-trimoxazole Prajeesh Kumar, Raj K. Narang, and Shivansh Swamy Nanomedicine Research Centre, Department of Pharmaceutics, Indo-Soviet Friendship College of Pharmacy, Moga, Punjab, India.
Received August 14, 2015; accepted October 16, 2015
ABSTRACT
In this study, development of nanoparticle loaded hydrogel of Cotrimoxazole that could simultaneously deliver trimethoprim and sulfamethoxazole at the site of wound to promote and accelerate wound healing is planned. Chitosan nanoparticles loaded with Co-trimoxazole were prepared by ionic gelation method. Chitosan nanoparticles were optimized and F5 formulation was selected 5:1 Chitosan TPP ratio for this Homogenization speed was kept at 5000 rpm and an average size of 209.8 ± 34.6 was obtained, PDI was found to be 0.26 ± 0.04, the zeta potential of the nanoparticles was found to be + 24.7 ± 3.12 and the entrapment efficiency of 89.7 ± 3.1 % was seen. Hydrogel was prepared and optimized on the basis of Conc. of Carbopol 940 1.2%, viscosity 14.953 ± 0.51, pH 5.9 ± 0.04, Swelling index 250 ± 4.71 and Spreadability 34 ± 3.5. Antimicrobial
study and Minimum Inhibitory Concentration (MIC) were performed and it was observed that the MIC of Co-trimoxazole was 2 μg/mL and the hydrogel formulation showed maximum zone of inhibition 3.7 ± 0.3 after 72 hours against plain drug and marketed formulation. The rate of wound contraction was calculated in percentage and at 15th day the control group had 72% ± 3.9%, Marketed formulation group had 36% ± 5.2% and the hydrogel formulation had reduced the wound size to mere 4% ± 1.3% only. Hydrogel loaded with nanoparticles of Co-trimoxazole that possessed optimum rheology and provided sustained drug release was successfully prepared. The developed hydrogel formulation was found to heal the wound 1.5 times faster than the marketed formulation thus providing us with a better alternative to other conventional wound dressings.
KEYWORDS: Hydrogel; Wound; Healing; Chitosan; Nanoparticles; Co-trimoxazole; Wound dressing.
Introduction
The first wound treatments were described 5 millennia ago. Since then, various principles of wound care have been passed on from generation to generation. At the present time, there are more than 5,000 wound care products. There are a number of different dressings and techniques available for managing wounds. Most modern dressings contain materials that are highly absorbent, such as alginates, foam, carboxymethyl-cellulose or carbopol (Thu et al., 2012, Hanna and Giacopelli, 1997). There are occlusive dressings and semi-occlusive dressings. There are growth factors, advanced honey-based dressings, and hypochlorous acid–based cleansers. Bioengineered tissue, negative pressure therapy, and hyperbaric oxygen therapy have changed the way we treat a lot of chronic wounds today (Blume et al., 2008). Nowadays, with new biopolymers and fabrication techniques, a wound dressing material is expected to have extraordinary properties which enhance the healing process of a wound. For an effective design of a functional wound dressing, characteristics of the wound type, wound healing time, physical, mechanical, and chemical properties of the dressing must be taken into consideration. Dressings
that create and maintain a moist environment, however, are now considered to provide the optimal conditions for wound healing (Field and Kerstein, 1994).
A wound is a type of injury in which skin is torn, cut, or punctured (an open wound), or where blunt force trauma causes a contusion (a closed wound) surface by physical, chemical, mechanical, and/or thermal damages. In pathology, it specifically refers to a sharp injury which damages the dermis of the skin. (Broughton 2nd et al., 2006) A more scientific definition of a wound is a disruption of normal anatomic structure and function of the skin. On the basis of wound healing processes, there are two types of wounds: Acute wounds (Li et al., 2007) and Chronic wounds (Moore et al., 2006).
Natural chitosan materials gained great interest in pharmaceutical sector because of its advantages like biodegradability, biocompatibility, non-toxicity, non-immunogenicity & low cost. Chitosan is a natural hydrophilic cationic polysaccharide derived by deacetylation of chitin compared to many other natural polymers, chitosan has a positive charge and is mucoadhesive (Ilium, 1998). It breaks down slowly to harmless products (amino sugars), which are completely
International Journal of Pharmaceutical Sciences and Nanotechnology
Volume 9Issue 1January – February 2016
MS ID: IJPSN-7-28-15-PRAJEESH
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absorbed by the human body. Chitosan has many advantages, such as its ability to control the release of active agents, it avoids the use of hazardous organic solvents while fabricating particles. chitosan has the special possibility of adhering to the mucosal surfaces within the body, a property leading to the attention to this polymer in mucosal drug delivery. The potential of chitosan for this specific application, has been further enforced by the demonstrated capacity of chitosan to open tight junctions between epithelial cells though well-organized epithelia (Zhang et al., 2010). Ionic gelation method has been used for the preparation of chitosan nanoparticles because it employees reversible physical cross-linking by electrostatic interaction, instead of chemical cross-linking, to avoid the possible toxicity of reagents and other undesirable effects. Tripolyphosphate (TPP) is a polyanion, which can interact with the cationic CS by electrostatic forces (Dash et al., 2011).
The conventional wound dressing materials are not suitable for acute and chronic wounds as far as rapid healing of a wound is concerned. Direct delivery of these agents to the wound site is desirable, particularly when systemic delivery could cause organ damage due to toxicological concerns. Also, new antimicrobial wound bandages with dry and cold fibrins for the release of tetracycline are used. These ingredients have a direct influence on the proliferation stage and activation of fibroblasts. Depending on a wound type and its healing, the most suitable wound dressing systems must be used. Because of unique properties of hydrogels to retain high amounts of water they help to produce more rapid healing by creating a moist environment that reduces the building up of necrotic tissue through apoptosis. Studies suggests that wounds should be kept in a moist environment, and hydrogel is a perfect medium to apply. The beneficial effects of a moist versus a dry wound environment include the prevention of tissue dehydration and cell death, accelerated angiogenesis, increased breakdown of dead tissue and fibrin (i.e., pericapillary fibrin cuffs) and potentiating the interaction of growth factors with their target cells (Field and Kerstein, 1994). Hydrogel provides a cooling barrier that permits water to evaporate from the surface and so produce a cooling effect. It is this cooling effect that helps to reduce the microcapillary circulation to the surface of the skin, so encouraging a reduction in erythema (redness), may lessen the building up of oedema and so reduce swelling to allow more even wound healing. This cooling effect will bring soothing comfort (Smith Jr et al., 1994). Scar quality is significantly superior in those wounds treated with a moist dressing (Atiyeh et al., 2003). The following properties are generally considered for all modern wound dressing materials.
Maintain the most suitable environment at the wound/ dressing interface
Absorb excess exudates without leakage to the surface of a dressing.
Provide thermal insulation, mechanical and bacterial protections.
Allow gaseous and fluid exchanges. Reduce wound odor and be easily removable
without trauma. Provide some debridement action (remove dead
tissue and foreign particles). Nontoxic, non-allergic, non-sensitizing (to both
patient and medical staff), sterile and non-scaring.
Hydrogels are a class of polymers that can absorb large amounts of water without dissolving. This is due to the physical or chemical cross linkages of hydrophilic polymer chains. The hydrophilic polymers contain 99% water and are highly flexible. Hydrogels mimic many of the properties of natural tissue so they are highly biocompatible. Hydrogels can be made porous or dense depending on usage requirements by altering their composition. Hydrogels can be prepared from monomers, pre-polymers, or existing hydrophilic polymers. These polymers are composed of oxygen, hydrogen, carbon and sometimes nitrogen bonds. Hydrogels can accommodate a large quantity of water and hence increase their drug loading capacities, impart tissue like properties, make them flexible. The reasons for hydrogels being suitable as wound dressing can be listed as follows.
(a) High Swelling (b) Highly biocompatible (due to high water content
and hence behave like natural tissue) and therefore immunological responses,
(c) Biodegradable, that makes these nanocarriers nontoxic,
(d) High drug loading capacity, (e) By tuning crosslinking densities drug release can
be regulated, (Ryu et al., 2010). (f) Can incorporate both hydrophilic and
hydrophobic drugs and charged solutes (Look et al., 2013).
In this study, development of nanoparticle loaded hydrogel of Cotrimoxazole that could simultaneously deliver trimethoprim and sulfamethoxazole at the site of wound to promote and accelerate wound healing is planned. The product was quality tested through a series of biopharmaceutical techniques.
Materials and Methods
Method of Preparation of Nanoparticles
Chitosan nanoparticles loaded with of SMX and TMP were prepared by ionic gelation method as reported by (Wadhwa et al., 2010) and (Bagre et al., 2013). Aqueous solution of TPP containing SMX and TMP was added drop wise to chitosan solution made in water acidified with 1% acetic acid. The chitosan solution was simultaneously homogenized at 6000rpm for sufficient time till the formation of nanoparticles.
Prajeesh et al: Development and Evaluation of Nanoparticle-Loaded Hydrogel of Co-trimoxazole 3133
Optimization of Chitosan Nanoparticles
Different process parameters were systematically investigated to determine their effects on the particle size, Polydispersity index (PDI) and entrapment efficiency of nanoparticles. The process parameters included speed of homogenization, concentration of chitosan and TPP.
Concentration of Chitosan
To optimize chitosan concentration different conc. of chitosan (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, and 0.7%) were used. Other parameters like conc. of TPP was fixed at (0.1%, 0.2%) at two different time points and homogenization speed was kept constant at 5000 rpm. Formulations were evaluated on the basis of the size and PDI of nanoparticles.
Concentration of TPP
To optimize TPP different conc. of TPP (0.1% and 0.2%) were used. Other parameters like conc. of chitosan was fixed at (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, and 0.7%) at different time points while keeping homo-genization speed constant at 5000 rpm. Formulations were evaluated on the basis of the size and PDI of nanoparticles.
Homogenization Speed
For optimization of homogenization speed the nanoparticles were homogenized at varying rpm (5000, 6000, 7000, 8000 rpm) while other parameters like conc. of chitosan and TPP were kept fixed at 0.2% and 0.1% respectively. Formulations were evaluated on the basis of the size and PDI of nanoparticles.
Amount of Drug
TMP
The concentration of drug in formulation directly influences the entrapment efficiency. Amount of drug concentration was optimized by taking different amounts of drug i.e., 4, 6, 8, 10 and 12mg according to different drug/polymer ratios (0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1) with the polymer used in the preparation of nanoparticles (i.e., chitosan). Previously optimized formulations were repeated using different concentration of TMP. Different formulations for the drug were prepared with varying amount of drug i.e., 4, 6, 8, 10 and 12 mg. The formulations were evaluated on the basis of entrapment efficiency and the formulation with maximum efficiency was selected as the final optimized formulation.
Sulfamethoxazole
The concentration of drug in formulation directly influences the entrapment efficiency. Amount of drug concentration was optimized by taking different amounts of drug i.e., 4, 6, 8, 10 and 12 mg. according to different drug/polymer ratios (0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1) with the polymer used in the preparation of nanoparticles (i.e., chitosan). Previously optimized
formulations were repeated using different concentration of SMX. Different formulations for the drug were prepared with varying amount of drug i.e., 4, 6, 8, 10 and 12 mg. The formulations were evaluated on the basis of entrapment efficiency and the formulation with maximum efficiency was selected as the final optimized formulation.
First Derivative Spectra of Cotrimoxazole
Simultaneous detection is needed for detection of both the drugs and the spectra is obtained using the First derivative method to identify the individual drugs. The first-derivative scan offers a better separation and hence TMP can be determined at an absorbance with negligible contribution from SMX (called as Zero crossing point). Likewise SMX was determined at a wavelength when TMP has negligible contribution. The linear calibration graphs were obtained for both the drugs. This method involves the measurement of the absolute value of the derivative spectrum of the binary mixture at a wavelength corresponding to the zero crossing point of the derivative spectrum of the interfering component (Al-Saidi and Yonis).
Characterization of Optimized Nanoparticles
Optimized nanoparticles were characterized for various parameters like morphology, size and size distribution, entrapment efficiency.
Morphology
Scanning electron microscopy (SEM) was performed for to examine dimensional topography and distribution of exposed features. Aluminum stub was used as a sample holder. It is covered with double carbon tape, few drops of silver are placed at the sides for ensuring conductivity, a cover slip is then placed sample is placed in between and air dried. Gold coating is done in the presence of argon air. Now the stub is placed into machine and imaging is done after setting the desired magnification.
Particle Size and Distribution
Size and size distribution (PDI) of optimized formulation was determined by laser diffractometery using Beckman coulter Delsa TM Nano C Particle analyzer. For the measurement of size and polydispersity index (PDI) 2 mL of nanoparticles was placed into cuvetts of Beckman coulter and measurements were recorded.
Entrapment Efficiency
Entrapment efficiency (EE) was determined by measuring the concentration of free drug (unentrapped) (Fu et al., 2010). Nanoparticles were centrifuged at 15,000 rpm for 30 min. Then the supernatant was measured for the unentrapped drug in it.
Total drug – freedrugEE%
Total drug in nanoparticles
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Preparation of Nanoparticle Loaded Hydrogel
Fixed amount of Carbopol 940 was added to sufficient amount of distilled water to achieve a gel up to 2.5% conc. The solution was stirred till the polymer was completely dissolved and a viscous solution was obtained. Carbopol is an anionic polymer that needs neutralization to become gellified. Neutralizing agent such as NaOH was used to neutralize (Islam et al., 2004). To this viscous solution 3-4 ml of 50% NaOH was added that resulted in complete gelation of the solution and a gel mass was obtained. 2 mL of NaOH was again added to make the gel clear. (Phatak Atul and Chaudhari Praveen, 2012). Nanoparticle loaded gel was prepared by simply dispersing the centrifuged nanoparticles of both the drugs into the Carbopol gel with gentle stirring to attain uniform distribution of nanoparticles.
In-Vitro Evaluation of Nanoparticle Loaded Gel
Measurement of pH
The pH of the Carbopol gels was determined by digital pH meter (Model MK–VI, Kolkata, India). One gram of gel was dissolved in 25 mL of distilled water and the electrode was then dipped in to gel formulation for 30 min until constant reading obtained. And constant reading was noted. The measurements of pH of each formulation were replicated three times.
Viscosity Measurement
The rheological measurements were performed on the Brookfield Rheometer RS+. All measurements were carried out at room temperature 25 ± 10 °C. The rheological properties of the formulated gels were studied at three different shear rates (rpm) and the viscosity was measured in Pa.sec. 30 gm gel was placed in a beaker of volume 50 mL. The speed of the spindle was increased up to 400 s-1 and then decreased up to 0. From the data of shear rate, shear stress and viscosity different of viscosity and flow were plotted. Ostwald-deWaelepower-law model was used for the determination of consistency index (K) and flow index (n) according to the equation where shear stress is and D is the shear rate. The slope of the graph obtained between log versus log D gave the flow index (n) and the antilog of the y-intercept represented the consistency index (K). An n value less than 1 (n<1) means that the gel is pseudoplastic while n>1 means the gel follows shear thickening behavior. (Mandala and Bayas, 2004, Teipel and Forter-Barth, 2005, Vongvuthipornchai and Raghavan, 1987)
Degree of Swelling
The degree of swelling of developed gel was calculated by the following equation. The test was carried out in PBS buffer pH 5.5 at 37 °C.
Degree of Swelling (%) = F I
I
M – M×100
M
Where MF (final weight) is the weight of the swollen gel sample, MI is the initial mass of sample immersed in buffer medium.
Spreadability
Spreadability was determined by wooden block and glass slide apparatus. 0.1gm of gel was applied to the glass slide and about 20 gm of weight was applied to the pan and the time for upper slide to separate completely from the fixed slide was noted (Gupta et al., 2010). By applying the following formula Spreadability in Unit = g.cm/secwas calculated.
S M.L
T
Where, S is the spreadability of the gel M is the weight tide to upper slide L is the length of glass slide T is the time (in sec) taken by the upper slide to completely separate from lower one.
In vitro release Studies of Nanoparticles and Gel
In vitro drug release studies were performed by dialysis bag method using shaking incubator at rotation speed of 100 rpm. Saline Phosphate buffer (pH 5.5) was used as dissolution medium. Each dialysis bag (pore size: 12 KD, Sigma Chemical Co., USA) was filled with 1 mL nanoparticles. Volume and temperature of dissolution medium were 50 mL, and 37 oC ± 0.2 ºC respectively. At predetermined time interval samples (5 mL) were withdrawn, replaced with same volume of fresh media, filtered and assayed for drug content at 257 nm for SMX and 284 nm for TMP against blank by UV-Visible spectrophotometer.
Hence samples at each interval of time were collected and absorbance at both the λmax were observed and then substituted in the above equations to get the conc. of the in the samples. Each conc. value was converted to the amount of the drugs by simply multiplying by the volume of the medium. Hence % age drug in the medium at each interval of time was determined and this corresponded to the % age release of the drugs at those intervals of time.
Determination of Minimum Inhibitory Concentration
In this test, wafers containing antibiotics are placed on an agar plate where bacteria have been placed, and the plate is left to incubate. If an antibiotic stops the bacteria from growing or kills the bacteria, there will be an area around the wafer where the bacteria have not grown enough to be visible. This is called a zone of inhibition. An Antibiotic Disc Dispenser to dispense discs containing specific antibiotics is placed onto the plate containing the Staphylococcus aureus in Mueller- Hinton agar. Using a flame-sterilized forceps, each disc is pressed on to the agar to ensure that the disc is attached to the agar. Plates are incubated overnight at an incubation temperature of 37 °C (98.6 °F). The plates are then observed for inhibition starting at the lowest
Prajeesh et al: Development and Evaluation of Nanoparticle-Loaded Hydrogel of Co-trimoxazole 3135
concentration and this concentration is treated as the MIC(Tendencia, 2004).
Zone of Inhibition
The anti-bacterial activity of the optimized formulation was determined against Staphylococcus aureus, the micro-organism responsible for majority of bacterial skin infections to compare the therapeutic efficacy of the developed formulations with each other as well as with that of the plain drug. The bacterial strain (MTCC no-3160) was obtained from IMTECH Chandigarh. 100 μL of bacterial suspension was streaked over the plate containing Mueller-Hinton agar medium and was spread uniformly. 1×1 cm2 of Drug loaded nanoparticles incorporated into hydrogel were gently placed at the center of the solidified agar gel in different Petri dishes. The bacterial growth was compared among the different formulations and the plain drug (BAUER et al., 1959).
In-Vivo Studies
Animals used
Wistar rats (200-250 gm) were used as an animal model for the present study. Healthy Wistar rats (200-250 gm) of either sex were subjected to standard laboratory conditions (i.e., room temperature, 23 ± 2 °C; relative humidity, 55 ± 5 %; 12/12 hr. light/dark cycles) with free access to a commercial rodent diet and water. All experiments for a given treatment were performed using age-matched animals in an attempt to avoid variability between experimental groups.
In-vivo Evaluation of Drug Containing Nanoparticle Loaded Hydrogel
Wound healing test: The wound healing studies of the drug containing nanoparticle loaded gel were carried out to study the wound healing ability of the optimized gel formulation and to compare the efficacy of the gel with the marketed formulation for wound healing.
(a) Experimental design 9 rats (all weighing 150-200 gm) were randomized into three groups consisting of three animals in each group.
(b) Induction of wound Excision wound model was done using thiopental sodium as the anaesthetic was used to induce wounds in rats. Hair was removed by electric clipper. The skin of the dorsal thoracic region was excised to full thickness with the help of surgical blade to obtain a wound area of about 200 mm2. The animals were anesthetized with intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg). After anesthetizing the animals, the dorsal skin region of the animals was shaved to remove any hair present on the skin. Following this a 2 cm2 area and 0.5 mm full-thickness incision was made on the dorsal
skin using a sterilized surgical blade(Nayak et al., 2006).
(c) Rate of contraction The incised wounds were covered with hydrogel dressing and the changes in wound size over a period of 21 days were observed as the percentage of the original wound area. The three groups of animals included one control group in which no medication was applied on the induced wounds, the wounds of 2nd group of rats were covered with drug containing gels i.e., Co-trimoxazole containing nanoparticle loaded hydrogel. In order to compare the efficacy of the formulation with the marketed formulation, the wounds in one group of rats were covered with marketed formulation of Gentamicin (3rd group). The wounds of rats were observed at a regular interval of every 7 days in order to observe the progress of wound healing until the wounds were healed completely. Each group contained three wound induced rats.
Skin Irritation Test
Dermal irritation is defined as the production of “reversible damage of the skin following the application of a test substance”. It is generally assessed by the potential of a certain substance to cause erythema/ eschar and/or oedema after a single topical application on rat skin.
For this study the wounds of rats covered with gel formulations were observed for any kind of oedema or erythema on the skin during the period of healing as per the Draize skin irritation test and the results were calculated using the dermal irritation index (PDII) to classify them as non-irritant or irritant(Carbone, 2004).
TABLE 1
Formulation Evaluation Study protocol.
Group Treatment Route
1 Control Topical 2 Co-trimoxazole loaded Hydrogel Topical 5 Marketed Formulation Topical
Results and Discussion
Formulation Development and Optimization of Nanoparticles
Chitosan nanoparticles were prepared according to the procedure mentioned and further, it was optimized on the basis of various parameters affecting the required characteristics of the nanoparticles. These parameters include particle size and PDI of the nanoparticles. Process parameters included speed of homogenization, Concentration of chitosan, TPP.
Table 2 compiles the data of nanoparticles that were prepared by adding 10 mL of 0.1% Tri-poly phosphate drop wise to 10 mL of different conc. of chitosan (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, and 0.7%) with constant a homogenization at 5000 rpm. The formulation F12 with
3136 Int J Pharm Sci Nanotech Vol 9; Issue 1 January February 2016
a ratio of 5:1 was seen to exhibit desired size and polydispersity index.
TABLE 2
Optimization of Chitosan/TPP ratio using different parameters
Formulation CS: TPP
Speed (rpm)
Time (min)
Particle Size (nm)
PDI
F8 1:1 5000 10 174.4 ± 16.72 0.479 ± 0.06 F9 2:1 5000 10 231.4 ± 21.03 0.329 ± 0.04
F10 3:1 5000 10 278.2 ± 19.78 0.323 ± 0.05 F11 4:1 5000 10 245.5 ± 12.65 0.367 ± 0.04 F12 5:1 5000 10 232.2 ± 17.11 0.189 ± 0.03 F13 6:1 5000 10 844.3 ± 28.21 0.525 ± 0.06 F14 7:1 5000 10 1361.7 ± 32.24 0.597 ± 0.05
Homogenization Speed
Nanoparticles were prepared by adding 10 mL of 0.1% TPP drop wise to 10 mL of 0.5% chitosan with constant homogenization at different rpm (5000, 6000, 7000, 8000, 9000 rpm). The results are compiled in the Table 3.
TABLE 3
Optimization of homogenization speed.
Formulation CS: TPP
Speed (rpm)
Time (min)
Particle Size (nm) PDI
F15 5:1 5000 10 391.4 ± 32.12 0.302 ±0.07 F16 5:1 6000 10 181.9 ± 23.35 0.117 ±0.05 F17 5:1 7000 10 450.1 ± 26.64 0.357 ± 0.05F18 5:1 8000 10 578.3 ± 31.58 0.371 ± 0.08F19 5:1 9000 10 701.1 ± 34.92 0.411 ± 0.06
Optimization of Drug: Polymer Ratio
Drug polymer ratio was optimized on the basis of particle size, PDI, entrapment efficiency (EE) and drug loading. The optimized parameters like polymer conc., conc. of TPP and homogenization speed were fixed while the drug amount was changed till nanoparticles of optimum size and PDI with highest entrapment efficiency and drug loading were obtained.
(a) For Sulfamethoxazole
TABLE 4
Optimization of Drug/Polymer ratio of Sulfamethoxazole.
Formu- lation
Drug: Polymer
Particle Size (nm)
PDI Zeta
pot. (mV)% EE
F32 0.2:1 253.1 ± 21.38 0.123 ± 0.04 +40.26 52.15 ± 1.4F33 0.3:1 271.1 ± 27.13 0.285 ± 0.04 +39.17 61.82 ± 1.8F34 0.4:1 280.5 ± 24.11 0.267 ± 0.02 +38.76 69.21 ± 2.1F35 0.5:1 296.1 ± 22.54 0.222 ± 0.04 +29.52 81.13 ± 2.4F36 1:1 315.4 ± 31.21 0.366 ± 0.05 +11.18 81.87 ± 2.7
(b) For Trimethoprim
TABLE 5
Optimization of Drug/Polymer ratio of TMP.
Formu-lation
Drug : Polymer Particle Size PDI
Zeta pot. (mV) % EE
F37 0.2:1 231.2 ± 19.32 0.134 ± 0.04 +39.16 56.63 ± 1.3F38 0.3:1 249.7 ±21.27 0.272 ±0.03 +37.05 64.14 ±2.2F39 0.4:1 251.1 ±16.39 0.228 ±0.04 +35.37 77.33 ±1.9F40 0.5:1 276.3 ±18.45 0.215 ±0.02 +30.92 88.42 ±3.5F41 1:1 348.2 ±22.34 0.426 ±0.06 +21.82 86.47 ±2.3
TABLE 6
Optimized parameters for final formulations (20 mL).
Optimized parameters F35 SMX F40 TMP Homogenization Speed (rpm) 6000 rpm 6000 rpmChitosan conc. (% w/v) 5 % 5 %TPP (% w/v) 1 % 1 %Drug Amount (mg) 10 10
Characterization of Optimized Nanoparticles
Morphology of Nanoparticles Scanning electron microscopy (TEM) Optimized drug loaded nanoparticles of both the
drugs were visualized under SEM for determining the shape and conformation of prepared Nanoparticles. SEM images of Cotrimoxazole-NP are shown in Fig. 1.
Fig. 1. SEM image of Co-trimoxazole loaded nanoparticles.
Particle Size and PDI
Size and size distribution of optimized Nanoparticles were determined by photon correlation spectroscopy method using Zetasizer (Bechman coulter).
Entrapment Efficiency
Entrapment efficiency of SMX and TMP loaded nanoparticles was estimated and repeated three times. All the formulations showed good reproducible results. Entrapment efficiency of SMX (F35) and TMP loaded nanoparticles (F40) was found to be 81.13 ± 2.4 % and 86.22 ± 2.5% respectively.
(a) Effect of amount of drug (SMX) on entrapment efficiency
Effect of amount of the drug on the entrapment efficiency is given in the following tabulated form in Table 4
0.2:1 0.3:1 0.4:1 0.5:1 0.6:10
20
40
60
80
100
0
20
40
60
80
100
Entrapment Efficiency Sulfamethoxazole/Chitosan Ratio
Sulfamethoxazole/Chitosan Ratio
%ag
e E
ntra
pmen
t E
ffic
ienc
y
Sulfamethoxazole/C
hitosan Ratio
Fig. 2. Effect of amount of Sulfamethoxazole on EE.
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of Nanoparticand Trimetdialysis bag
10 12 1
TrimethoprimSulfamethoxazol
TMP from chito
Drug Release
release from nal formulathat included zmodels. From
models it windividual drr Peppas currves of all
e from all cs wherein ion and diffus
in a sustainrent formulatid fig 8 for T
basis of varie 7.
3137
n at
ded
cles tho-g is
4
le
osan
the tion zero the
was rugs rve. the the the
sion ned ions
TMP
ious
3138
Fig. 8 Kine
Fig. 9. Kine
TABLE 7
Optimizatio
FormulatF1 F2 F3 F4
etic Drug Release
etic Drug Releas
on of hydrogel.
tion Conc. of g0.4 0.8 1.2 2.5
e Models for SM
se Models for TM
gel (w/v) p% 6.8 ±% 6.1 ±% 5.9 ±% 5.6 ±
X from nanopart
MP.
pH Visco± 0.12 2± 0.04 5± 0.04 14± 0.08 33
Int J
ticles
osity (Pa.sec).32 ± 0.36.16 ± 0.38
4.953 ± 0.53.41 ± .053
J Pharm Sci Na
Swelling Inde20 ± 0.28
100 ± 9.42250 ± 4.71450 ± 8.16
anotech Vol 9; I
ex % Sprea
216
Issue 1 Janua
adability (g.cm s89 ± 1.6 63 ± 7.03 34 ± 3.5 21 ± 2.9
ary February 2
sec-1)
2016
Prajeesh
ChapH oThe
6.8 to increaseacidic nthe adoptimiz
RheThe
was invretentioskin. Thviscositygel contwere 2.3Pa.sec, attributnetworkof the sof the ofig. 11 behavioincreasiThe slopversus shence cbehaviotime grdecreas
Fig. 10. S
Fig. 11. (F3).
h et al: Develop
aracterization of hydrogel pH of the hy5.6 as the ced from 0.4 tonature of the ddition of 0.ed 1.2% gel (F
eological measrheological b
vestigated sinon time of the he effect of dify was also evtaining 0.4%, 32 ± 0.15; 5.16
respectively.ted to the inck as the concehear rate vs tptimized gel frespectively.
or of the gel wing shear ratepe (or flow indshear rate waconfirming a or of the gel wraph wherein ed with time a
Shear Stress ver
Log (Shear Stre
pment and Eval
of Hydrogel
ydrogel was fconcentrationo 1.2%. This wpolymer. Th
1N NaOH.TF3) was foundsurements behavior of tonce it descrigel formulatio
fferent concenvaluated. The
0.8%, 1.2% a6 ± 0.9; 14.95. This increacreased crosslentration of cathe shear streformulation ar. The plots wherein sheare with yield vdex, n) of the
as found to be pseudoplastic
was confirmed the viscosity
as shown in fig
sus Shear Rate o
ess) versus Log
luation of Nano
found to decren of the carbwas due to the pH was adj
The pH of td to be 5.9 ± 0
opical gel formibes spreadabon on the surf
ntrations of Caviscosities of
and 2.5% w/v ± 0.5 and 33ase in viscolinking of thearbopol increaess and their lre shown in fishow a pseur stress increvalue (non-Nelog plot of shless than 1 (n
c behavior. Thby the viscosiat constant sg. 12.
of Hydrogel (F3)
g (Shear Rate) o
oparticle-Loade
ease from bopol 940 he slightly djusted by the final .04.
mulations bility and face of the arbopol on f Carbopol
Carbopol .41 ± .053 osity was e polymer ased. Plots log values ig. 10 and udoplastic ases with
ewtonian). ear stress
n = 0.572), hixotropic ity versus
shear rate
).
of Hydrogel
Fr
ndespamcidwir
FH
opcwpdfhfo
ed Hydrogel of
Fig. 12. Viscositrate indicating a
In-Vitro D
In-vitro dnanoparticlesdialysis bag estimation spectrophotompH 5.5. This absolute valumixture at acrossing poininterfering codetermined atwere calculaintervals of trelease was ca
0 10
20
40
60
80
100
%ag
e C
umul
ativ
e D
rug
Rel
ease
Fig. 13. ComparHydrogel.
Minimum
The drug obtain the mipaper disks wconcentrationwere then previously cudisks were pforceps in lamhours. After for visible inobserved that
Co-trimoxazole
ty of the gel deca thixotropic beh
Drug Release f
drug releases of Co-trimox
is shown inof the dr
metry using Fmethod invo
ue of the deriva wavelengthnt of the domponent. Abt both wavele
ated in the the time as malculated as s
2 3 4 5 6
rative Cumulati
Inhibitory Co
was subjecteinimum inhibwere dipped in ranging from
placed on ultured with placed onto t
minar air flow incubation th
nhibition of tt at 2 μg/mL t
e
creases with timhavior of the gel.
from Hydroge
e profile of xazole in PBS
n Fig. 10. Thrugs was First derivativolves the meavative spectruh correspondiderivative spsorbances of ngths. Conc. osamples tak
mentioned in hown in Fig 1
6 7 8 9 10
SuTr
Time (hrs)
ive Release of S
oncentration
d to disk diffitory concentrnto drug solu
m 0.5 to 8μg/mMuller -HiltStaphylococc
the agar plaand incubate
he agar platehe microbial
the microbial
me at constant sh
l
gel containS (pH 5.5) ushe simultanedone by e method in P
asurement of um of the bining to the zpectrum of the sample wof both the dr
ken at differand percent
13.
0 11 12 13 14
ulfamethoxazolerimethoprim
SMX and TMP f
fusion methodration. Steriliution of differmL. These diton agar plcus aureus. Tate using steed at 37 °C fore were examin
growth. It wgrowth was s
3139
hear
ning sing eous UV
PBS the
nary zero the
were rugs rent tage
15
from
d to ized rent isks late The
erile r 24 ned was een
3140 Int J Pharm Sci Nanotech Vol 9; Issue 1 January February 2016
to be inhibited. Thus giving us the value of MIC for Co-trimoxazole drug as represented in table 8.
TABLE 8
MIC of Cotrimoxazole.
Concentration (μg/ml) Inhibition 0.5 - 1 -
1.5 - 2 + 3 + 4 + 5 + 6 + 7 + 8 +
Antibacterial Activity
The zones of inhibition of optimized formulations, plain drug and marketed formulation against Staphylococcus aureus were compared with each other as shown in table 9.
TABLE 9
Zones of inhibition (diameter) in Staphylococcus aureus (cm).
Formulations Time intervals
After 12 hours
After 24 hours
After 48 hours
After 72 hours
Plain drug 1.8 ± 0.1 2.5 ± 0.3 2.7 ± 0.2 2.2 ± 0.2 Hydrogel 2.1 ± 0.4 3.3 ± 0.4 3.6 ± 0.2 3.7 ± 0.3 Marketed 1.4 ± 0.2 2.1 ± 0.3 2.4 ± 0.2 2.1 ± 0.2
In Vivo Studies In vivo studies for the developed hydrogel
formulation were carried out on Wistar rats (150-200 mg). The wound healing studies of the drug containing nanoparticle loaded gel were carried out to study the wound healing ability of the optimized gel formulation and to compare the efficacy of the gel with the marketed formulation for wound healing.
Physical Estimation of Wound Healing
Changes in wound area during the progress of wound healing
The physical estimation of wound healing can be clearly observed in figure 12, 13 and 14. These figures depict the process of healing of all the 3 groups of rats. The wounds of rats were observed on every 5th day for the progress of healing and the decrease in the size of the wound. The area of the wound decreased considerably with the passage of time. Highest rate of reduction in area of wound was observed in case of Cotrimoxazole loaded hydrogel followed by marketed formulation and control. The percentage size of wounds for all the three groups during the entire period of healing at regular intervals is reported in graph in fig 14.
0 5 10 150
20
40
60
80
100
CONTROLHYDROGEL MARKETED
Days
Per
cen
tag
eS
ize
Fig. 14. Effect of Hydrogel, Marketed formulation on mechanically induced wounds on percentage wound size.
Fig. 15. Physical examination of wound area at of control group (in days).
Fig. 16. Physical examination of wound area at of Co-trimoxazole loaded hydrogel.
Prajeesh et al: Development and Evaluation of Nanoparticle-Loaded Hydrogel of Co-trimoxazole 3141
Fig. 17. Physical examination of wound area at of marketed formulation.
Skin Irritation Test
All the types of applied gels showed no kind of reaction on the skin. There was no sign of any erythema/ eschar and/or oedema on rat skin which indicated the compatibility of the gel with the skin. As per the Draize skin irritation test -the dermal irritation index (PDII) was found to be 0 for erythema and 0 for oedema. The formulation was categorized as a nonirritant on the basis of 0.0 PDII.
Discussion
Hydrogel loaded with nanoparticles of Co-trimoxazole that possessed optimum rheology and provided sustained drug release was successfully prepared.Chitosan nanoparticles loaded with Co-trimoxazolewere prepared by ionic gelation method. Aqueous solution of TPP containing the drug was added drop wise to chitosan solution made in water acidified with 1% acetic acid. The chitosan solution was simultaneously homogenized at 6000 rpm for sufficient time till the formation of nanoparticles Hydrogel preparation was done using fixed amount of carbopol 940 was added to sufficient amount of distilled water to achieve a gel of 1.2 conc. The solution was stirred till the polymer was completely dissolved and a viscous solution was obtained, to this viscous solution 3-4 mL 0.1N NaOH of was added that resulted in complete gelation of the solution and a gel mass was obtained. Nanoparticle loaded gel was prepared by simply dispersing the centrifuged nanoparticles of the drug into the Carbopol gel with gentle stirring to attain uniform distribution of nanoparticles. The skin of the dorsal thoracic region was excised to full thickness with the help of surgical blade to obtain a und area of about 200mm2. The incised wounds were covered with hydrogel dressing and the changes in wound size over a period of 15 days were observed as the percentage of the original wound area.
Chitosan nanoparticles were optimized and F5 formulation was selected 5:1 Chitosan TPP ratio for this Homogenization speed was kept at 5000 rpm and an average size of 209.8 ±34.6 was obtained, PDI was found to be 0.26 ± 0.04, the zeta potential of the nanoparticles was found to be + 24.7 ± 3.12 and the entrapment efficiency of 89.7 ± 3.1 % was seen. Hydrogel was prepared and optimized on the basis of Conc. of Carbopol 940 1.2%, viscosity 14.953 ± 0.51, pH 5.9 ± 0.04, Swelling index 250 ± 4.71 and Spreadability 34 ±
3.5.Plots of the shear rate vs the shear stress and their log values of the optimized gel formulation were arranged. The plots show a pseudoplastic behaviour of the gel wherein shear stress increases with increasing shear rate with yield value (non-Newtonian). The slope (or flow index, n) of the log plot of shear stress versus shear rate was found to be less than 1, hence confirming a pseudoplastic behavior. Wound contraction rate studies were done and results show better accelerated healing than marketed preparations. The rate of contraction was calculated in percentage and at 15st day the control group had 72% ± 3.9%, Marketed formulation group had 36 % ± 5.2% and the hydrogel formulation had reduced the wound size to mere 4% ± 1.3% only.
The developed hydrogel formulation was found to heal the wound faster than the marketed formulation. Skin irritation studies also showed no sign of erythema/eschar and/or oedema on rat skin which indicated the compatibility of the gel with the skin.
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Address correspondence to: Prajeesh Kumar, Indo-Soviet Friendship College of Pharmacy, Ferozepur Road, Ghal Kalan Moga 142001, Punjab, India. Mob: +91-9815915035; E-mail: [email protected]
