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Lipid nanoparticles are drug delivery systems that are able to increased bioavailability of poorly soluble drugs. They can be prepared with different lipid materials, especially natural lipids. Shea butter is a natural lipid obtained from the Butyrospermum parkii seed and rich in oleic and stearic acids. Nimesulide is a COX 2 selective anti-inflammatory that is poorly soluble in water. The purpose of this study was to develop and characterize shea butter lipid nanoparticles using a new technique and evaluate the in vivo activity of these nanoparticles. Lipid nanoparticles were prepared by melting shea butter and mixing with an aqueous phase using an ultra Turrax. The nanoparticles presented pH of 6.9 ± 0.1, mean particle size of 90 nm and a narrow polydispersity (0.21). Zeta potential was around -20mV and the encapsulation efficiency was 96.5%. Drug release was evaluated using dialysis bags and presented monoexponential profile with t1/2 of 4.78h (free drug t1/2 was only 2.32h). Antinociceptive activity was performed by the acetic acid model. Both nimesulide and nimesulide-loaded nanoparticles presented significant activity compared to the control. The in vivo anti-inflammatory activity was evaluated by paw edema and was statistically significant for the nanoparticles containing nimesulide compared to free nimesulide, blank nanoparticles and saline. In conclusion, the use of shea butter as encapsulating lipid was very successful and allowed nanoparticles to be prepared with a very simple technique. The nanoparticles presented significant pharmacological effects that were not seen for free drug administration.
Keywords: lipid nanoparticles, shea butter, nimesulide, anti-inflammatory, antinociceptive, natural lipid.
Lipid nanoparticles are usually prepared with triglycerides, glycerides, fatty acids and waxes1. Natural lipids have been proposed for the preparation of nanoparticles2,3. Mandawgade et al.2 used highly purified stearine fractions of fruit kernel fats from India. Colomé et al.3 described the use of cupuassu seed butter in the preparation of lipid nanoparticles by solvent evaporation and by high pressure homogenization. In both studies, lipid nanoparticles made of natural lipids were successfully prepared and characterized.
Shea butter is a natural solid lipid obtained from the Butyrospermum parkii (also known as Vitellaria paradoxa) seed. Shea butter main fatty acids are oleic acid and stearic acid4. Its use in cosmetics is already described and this butter presents cosmetic use as emollient and nourishing5. As far as we know, there are no papers describing the use of shea butter in nanoparticles, especially for oral use.
Lipid nanoparticles can be used orally to improve oral absorption of weakly soluble drugs6. Drugs like simvastatin and curcumin presented increase in oral bioavailability when encapsulated in lipid nanoparticles7,8. These nanoparticles were already evaluated in order to improve oral bioavailability of hydrophilic proteins, like salmon calcitonin9. Lipid nanoparticles have shown several advantages over conventional drug delivery systems as the increase in bioavailability, less variation among individuals and higher stability of the entrapped drug10-12. Compared to liposome, lipid nanoparticles revealed higher physico-chemical stability, leading to shelf-lives up to 1 or 2 years1.
Lipid nanoparticles can be prepared by different techniques, as high pressure homogenization, microemulsion, solvent evaporation and phase inversion3,13. High pressure homogenization present as advantages the absence of organic solvents; however special equipment is needed (high pressure homogenizer)13. Colomé et al.3 described the use of high shear force (ultra Turrax) for the production of lipid nanoparticles but using organic solvent in the production.
Nimesulide N-(4-nitro-2-phenoxyphenyl) methanesulfonamide is a first generation COX-2 selective nonsteroidal anti-inflammatory14. Nimesulide is a weak acid insoluble in water and soluble in acetone15. This drug inhibits selectively the COX -2, reduces the synthesis of chemistry mediators of inflammation, causing, potent anti-inflammatory and analgesic effects16,17.
Then, the purpose of this study was to develop and characterize shea butter nanoparticles containing nimesulide for oral application. The preparation method was developed to avoid the use of organic solvents and special equipments. The in vivo antinociceptive and anti-inflammatory activities were also evaluated for the obtained formulations.
2. MATERIALS AND METHODS
Shea butter was obtained by Alpha Química (Porto Alegre, Brazil), polysorbate 80, sorbitan monooleate and carrageenan were purchased from Sigma-Aldrich (São Paulo, Brazil), nimesulide was obtained from Henrifarma (São Paulo, Brazil). Acetonitrile was of HPLC grade and all other chemicals were of analytical grade and used as received.
2.2.1. Preparation of lipid nanoparticle suspensions
The nanoparticles suspensions were prepared using the quantities presented in Table I. The lipid phase was heated at 40°C until all components were melted. The aqueous phase was prepared by mixing all components under magnetic stirring. Then, the aqueous phase was poured over the lipid phase under agitation. The suspension was subjected to shaking in the ultra-Turrax T 125 IKA® at 24.000 rpm during 20 min and, finally, the lipid nanoparticles were obtained after cooling.
For comparison, a formulation without nimesulide was prepared under the same conditions. Thus, the formulations were named NP-N (Nanoparticles containing Nimesulide) and NP-B (Nanoparticles without Nimesulide).
2.2.2. Physico-chemical characterization of the lipid nanoparticles suspensions
The measurements of nanoparticles mean particle size and polydispersity index (PDI) were performed through dynamic light scattering using a Zetasizer® Nano-ZS Malvern model ZEN 3600 (Malvern Instruments, UK), after dilution of 500 times v/v in water. For the zeta potential measurements, the same equipment was used but diluting the samples with 10 mM NaCl.
The pH was measured using a Digimed®, model DM-22 potentiometer. The pH measurements were performed directly in the formulations in triplicate.
Drug loading was assessed by high pressure liquid chromatography (HPLC). For this, the NP-N was dissolved with acetonitrile, diluted in mobile phase and quantified by HPLC (Shimadzu, UV/VIS detector). Detection was set at 230 nm, the column used was Lichrospher 100 RP-18 (5 µm, 250 x 4 mm), the mobile phase of acetonotrile and water in a ratio of 55:45 (v/v), volume injection of 10 µl and the flow was 1.0 ml/min18. Encapsulation efficiency was assessed by ultrafiltration/ultracentrifugation, using Amicon Ultra centrifugal filters (Millipore, Ireland). NP-N (0.5 ml) was placed inside the filter, and centrifuged 5,000 rpm for 5 min. The filtered solution was analyzed in HLPC for the quantification of the free drug. The entrapped drug was calculated by the difference between the drug loading and the free drug.
2.2.3. In vitro release of nimesulide from nanoparticles
The release profiles were obtained by dialysis, using cellulose dialysis bags with cut of 10.000 Da. The bags were hydrated in water during 1h before the dialysis initiate. An aliquot of 5.0 ml of NP-N and NP-B was put inside the dialysis bag. The closed bags were put in beaker containing 50 ml release medium (phosphate buffer pH 7.4:polyethilenoglicol 400; 90:10). The beakers were kept at 37 °C and under magnetic stirring for 72 h. An aliquot of 3.0 ml was withdrawn in predetermined time intervals and quantified for nimesulide release by UV spectrophotometry at 283 nm. For comparison, nimesulide was dispersed in polysorbate 80 and placed inside the dialysis bag at the same concentration than NP-N. In all cases, skin conditions were maintained during the experiment.
The release profiles were evaluated by the application of mathematical models. The adjustment of the data to the monoexponential equation (Equation.1) and to the biexponential equation (Equation 2) was done using MicroMath Scientist® software19. The best adjustment was selected based on the correlation coefficient (r), Model Selection Criterion (MSC), and visual examination.
where C is the concentration in the release medium at time t, k is the release rate. C0 is the initial concentration. A is the amount released in the burst phase at rate α and B is the amount released in the sustained phase at rate β.
The animals (swiss mice and Wistar rats) used on the experiment were housed under conditions of optimum light (12/12 h light-dark cycle), temperature (22 ± 1°C) and they had free access to water and food. Rats and mice were kept in separate rooms.
All the animals were cared and manipulated according to Committee on Care and Use of Experimental Animal Resources from the Federal University of Santa Maria, Brazil.
2.2.5. Acetic acid-induced writhing test
Swiss male mice (n=8/group), weighting from 20 to 45g were divided in four groups: saline (control), nimesulide (nimesulide dissolved in saline) 10mg/kg, NP-N 10mg/kg and NP-B (same volume of NP-N).
The writhing test on rats was based on the method reported by Nogueira et al.20. The animals did not receive water or food for 12h before the experiment. The nociceptive effect was induced by an intraperitoneal application of 1.6% acetic acid at the dose of 0.1ml/10g. All formulations were orally administered immediately after the nociceptive agent. Then, the animals were kept in a controlled box during 20 min to observe of the number of writhing.
2.2.6. Paw rat edema test
This methodology was adapted to the described by Winter et al.21. The edema was induced at the rat's right hind paw by an intraplantar injection of the edematogenic agent (carrageenan) and compare with the contralateral paw.
Wistar male rats (n=8/group) weighting 180-360 g were divided in the same four groups described in 2.2.5. However, nimesulide dose was 5 mg/kg.
One subplantar injection of 0.1 ml of 1 % carrageenan solutions was used to induce the edema on the right hind paw of the rats. Then, the animals received orally the formulations. After 5 h, the animals were sacrificed and the edema was expressed by the difference in grams between the right and the left hid paws.
3. RESULTS AND DISCUSSION
3.1. Development and Physical-Chemical Characterization of Lipid Nanoparticles
The lipid nanoparticles were prepared by homogenization using high shear rates. This is a new technique that wasn't described before to obtain nanoparticles with narrow particle size distribution. The main advantage of this technique is that the production is easy, is not time consuming and isn't necessary organic solvents. This technique also reduced the need of specialized equipment as a high pressure homogenizer. The Table II presents the physico-chemical properties of NP-N and NP-B.
We can observe that NP-N [NP-B] presented mean particle diameter of 90.1± 0.2 [87.8 ± 0.4] nm and PDI of 0.21 ± 0.07 [0.26 ± 0.01]. There was no statistical difference between the considered samples. These results are in accordance to Bondi et al.22 that developed solid lipid nanoparticles containing nimesulide with different lipids. These particles presented a variation of 85 to 135nm of size and PDI ranging from 0.148 to 0.303. Besides, the PDI indicates a narrow size distribution and values less than 0.2 are considered normal and acceptable for colloidal suspensions23.
The zeta potential reflects the surface charge of the particles, which is influenced by changes in the interface with the dispersing medium due to the dissociation of functional groups on the particle surface or the adsorption of ionic species presented in aqueous dispersion on the particle surface24,25. For NP-N, zeta potential was -20.21 ± 1.88mV, and it was considered adequate for the stability of the nanostructures, due the repulsion electrostatic forces demonstrated by this high value24. Comparing the zeta potential of NP-N and NP-B, it can be observed that NP-N had a higher zeta potential, which suggest an increased stability of NP-N. Moreover, the results of zeta potential for NP-N are in accordance with other studies, like nanosuspensions containing diclofenac that obtained a zeta potential of -25mV26. Stable resveratrol-loaded nanocapsules presented zeta potential around -15mV27, indicating that the values obtained in the present study are satisfactory.
Several factors can influence the quantity of drug associated to nanostructured systems. Among these, the physico-chemical characteristics of the drug, pH, zeta potential and the quantity of drug added to the formulation were key factors to a successful encapsulation 24,25.
In this study, the drug loading was 87.7 ± 7.2 %. The encapsulation efficiency was 96.5%, indicating a very high encapsulation, mainly if compared with Bondi et al.22, that obtained nimesulide-loaded nanoparticles containing 17.8% of drug when using Compritol® as lipid. In this way, shea butter was a very adequate lipid to encapsulate nimesulide, showing high drug loading and encapsulation efficiency.
3.2. Drug Release Profiles of Nimesulide-loaded Nanoparticles
During the development of drug delivery systems, as nanoparticles, the extent and velocity of the release is very important to preview their in vivo behavior. One of the major limitations in evaluation the drug release profile is the difficulty to separate particles from the dissolved drug due to the small size of these carriers29. Indeed, a methodology that allows the quantification of low drug concentrations in the release medium is also necessary.
Both free nimesulide and NP-N showed maximum release of 12% in 72 h (Figure 1). Free nimesulide was statistically higher and faster, demonstrating the ability to control the drug delivery of the nanoparticles (ANOVA p <0.05).
These results are explained by a high degree of interaction between lipid and drug and differences in the drug deposition on the particle30. It is important to consider also the high hydrophobicity of nimesulide and the physical entrapment into the nanoparticles. These factors can improve the bioavailability and reduce the side effects22.
In the release study of nimesulide-loaded Compritol® nanoparticles22, 100% of drug was released in 14 h. However, the loading capacity only was 17.8%, which may have influenced the release of all drug amounts.
Studies comparing the slope of the release profiles nanoparticles prepared with different lipids found variations in the maximum released. Prednisolone release was 37.1 % for cholesterol and 83.8 % for Compritol® and the release occurred within 5 weeks30. The controlled release could be achieved by modifications of the chemical structure of the lipid. Moreover, surfactant concentration, production technique and temperature also affected the release profiles30.
Mathematical modeling was used to analyze the release profiles, where the monoexponential equation (which provides a kinetic constant that indicated the release rate) presented a better adjustment (r >0.99) (Table 3), similar to what was observed by Jäger et al.31 for the release indomethacin ethyl ester from nanocapsules.
As demonstrated in Table 3, the release rate of nimesulide from NP-N (k = 0.428 h-1) was statistical different from free nimesulide (k = 0.228h-1). The release profiles were constructed by plotting the concentration of nimesulide released a function of time (Figure 2).
3.3. Acetic Acid-induced Writhing Test
The acetic acid intraperitoneal administration induces pain by directly activation of non-selective communication channels located in the primary sensory pathways or, indirectly, by promoting the release of prostaglandins and other inflammatory mediators32. Therefore, this model is useful for assessment of analgesic or anti-inflammatory properties of drugs (as nimesulide), once the action of these drugs reduces the number the acetic acid-induced writhing.
Figure 2 shows the mean and standard error of acetic acid-induced writhing, after 20 min of acetic acid injection with concomitant treatment of the animals.. Animals treated with NP-N and nimesulide had mean writhing of 10.38 ± 2.82 and 9.63 ± 1.83, respectively. Results it is clear less that control group (33.13 ± 6.10) and NP-B (18.71 ± 4.93). The results showed that the group treated with NP-N and nimesulide significantly inhibited the writhing response induced by acetic acid (ANOVA p < 0.05 e Tukey < 0.05) when compared with the respective control groups.
Bochi et al.22 pre-treated mice, orally, with hydrophilic gel containing meloxicam nanocapsules (5 mg/kg) at different times before acid acetic application. The antinociceptive activity observed was significant in all cases. Santos33 pre-treated mice with meloxicam (2.8; 8.4; 28.4 µmol/kg) given intraperitoneally 30 min before acid acetic administration. They observed that the writhing inhibition was the dose dependent and significant in all cases. In addition, that the authors calculated that 50 % of writhing would be inhibited with 7.4 µmol/kg of meloxicam. Naveen and collaborators (2004) pre-treated mice with naproxen (5; 10; 20 mg/kg) and nitro-naproxen (6.83; 13.86; 27.73 mg/kg) orally, 30 min before noxious stimulus and both drugs significantly reduced the incidence of writhing.
3.4. Carrageenan-induced rat paw edema
Intraplantar carrageenan injection induces acute inflammatory process with marked edema formation resulting from the production of several inflammatory mediators34. The inflammatory response may be quantified by the increase in paw size (edema) and may be modulated by inhibitors of inflammatory cascade, such as nonsteroidal anti-inflammatory drugs33. Therefore, it is clear that anti-inflammatory efficacy of these drugs (as nimesulide) encapsulated or not in nanoparticles may be evaluated through the variation in the edematogenic process using animal models, like paw edema.
Figure 3 shows the differences in paw edema, after 5 h of induction of the edematogenic processes with concomitant treatment. In test rat paw edema, the group treated with NP-N or nimesulide (5 mg/kg) demonstrated mean of increase in paw weight of 0.65 g and 0.81 g, respectively. These values were considerably less than the control group (1.23 g) and NP-B groups (1.02 g). Due to a great variability among the individuals of the group treated with nimesulide, only the group treated with NP-N demonstrated significant inhibition of the edema (ANOVA p < 0.05 and Tukey p < 0.05). In this way, the encapsulation of nimesulide allowed a reduction in variability and a significant reduction in the edema, demonstrating the importance of the encapsulation to achieve a higher pharmacological activity.
Bernardi et al.35 evaluated the effects prophylactic and therapeutic use of indomethacin-loaded nanocapsules and free indomethacin (1mg/kg), administered intraperitoneally, 30 min before and 60 min after the injection of carrageenan. The anti-inflammatory activity of free indomethacin and was significant in comparison with control group.
Lenz36 administered topically (through of friction during 30 s), 50 mg of gel containing free nimesulide and gel containing nimesulide-loaded nanocapsules, 1h before the administration of the flogistic agent. The anti-inflammatory activities of the free drug free and the encapsulated drug were considered significant in comparison with group control.
It was possible to obtain lipid nanoparticles prepared by an innovative homogenization by high shear rates technique. Shea butter was a good lipid to encapsulate nimesulide with high drug loading.
The drug release profiles demonstrated a control in drug release from the nanoparticles. The profiles fit the monoexponential model. Regarding the in vivo activity, in the antinocipective model, both nimesulide and the nanoparticles containing nimesulide presented significant activity. However, in the paw edema model, only the encapsulated drug showed significant activity, demonstrating the importance of the nanoencapsulation in the pharmacological behavior of the drug.
In conclusion, the use of shea butter as encapsulating lipid was very successful and allowed nanoparticles to be prepared with a very simple technique. The nanoparticles presented significant pharmacological effects that were not seen for free drug administration.