Natural Lipid Nanoparticles Containing Nimesulide Biology Essay

Lipid nanoparticles are drug bringing systems that are able to increased bioavailability of ill soluble drugs. They can be prepared with different lipid stuffs, particularly natural lipoids. Shea butter is a natural lipoid obtained from the Butyrospermum parkii seed and rich in oleic and stearic acids. Nimesulide is a COX 2 selective anti-inflammatory that is ill soluble in H2O. The intent of this survey was to develop and qualify shea butter lipid nanoparticles utilizing a new technique and measure the in vivo activity of these nanoparticles. Lipid nanoparticles were prepared by runing shea butter and blending with an aqueous stage utilizing an extremist Turrax. The nanoparticles presented pH of 6.9 ± 0.1, average atom size of 90 nanometers and a narrow polydispersity ( 0.21 ) . Zeta potency was around -20mV and the encapsulation efficiency was 96.5 % . Drug release was evaluated utilizing dialysis bags and presented monoexponential profile with t1/2 of 4.78h ( free drug t1/2 was merely 2.32h ) . Antinociceptive activity was performed by the acetic acid theoretical account. Both nimesulide and nimesulide-loaded nanoparticles presented important activity compared to the control. The in vivo anti-inflammatory activity was evaluated by paw hydrops and was statistically important for the nanoparticles incorporating nimesulide compared to liberate nimesulide, clean nanoparticles and saline. In decision, the usage of shea butter as encapsulating lipoid was really successful and allowed nanoparticles to be prepared with a really simple technique. The nanoparticles presented important pharmacological effects that were non seen for free drug disposal.

Keywords: lipid nanoparticles, shea butter, nimesulide, anti-inflammatory, antinociceptive, natural lipoid.

1. Introduction

Lipid nanoparticles are normally prepared with triglycerides, acylglycerols, fatty acids and waxes1. Natural lipoids have been proposed for the readying of nanoparticles2,3. Mandawgade et al.2 used extremely purified stearine fractions of fruit meats fats from India. Colom & A ; eacute ; et al.3 described the usage of cupuassu seed butter in the readying of lipid nanoparticles by solvent vaporization and by high force per unit area homogenisation. In both surveies, lipid nanoparticles made of natural lipoids were successfully prepared and characterized.

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Shea butter is a natural solid lipoid obtained from the Butyrospermum parkii ( besides known as Vitellaria paradoxa ) seed. Shea butter chief fatty acids are oleic acid and stearic acid4. Its usage in cosmetics is already described and this butter presents decorative usage as cream and nourishing5. Equally far as we know, there are no documents depicting the usage of shea butter in nanoparticles, particularly for unwritten usage.

Lipid nanoparticles can be used orally to better unwritten soaking up of decrepit soluble drugs6. Drugs like Zocor and curcumin presented addition in unwritten bioavailability when encapsulated in lipid nanoparticles7,8. These nanoparticles were already evaluated in order to better unwritten bioavailability of hydrophilic proteins, like salmon calcitonin9. Lipid nanoparticles have shown several advantages over conventional drug bringing systems as the addition in bioavailability, less fluctuation among persons and higher stableness of the entrapped drug10-12. Compared to liposome, lipid nanoparticles revealed higher physico-chemical stableness, taking to shelf-lives up to 1 or 2 years1.

Lipid nanoparticles can be prepared by different techniques, as high force per unit area homogenisation, microemulsion, solvent vaporization and stage inversion3,13. High force per unit area homogenisation nowadays as advantages the absence of organic dissolvers ; nevertheless particular equipment is needed ( high force per unit area homogenizer ) 13. Colom & A ; eacute ; et al.3 described the usage of high shear force ( extremist Turrax ) for the production of lipid nanoparticles but utilizing organic dissolver in the production.

Nimesulide N- ( 4-nitro-2-phenoxyphenyl ) methanesulfonamide is a first coevals COX-2 selective nonsteroidal anti-inflammatory14. Nimesulide is a weak acid indissoluble in H2O and soluble in acetone15. This drug inhibits selectively the COX -2, reduces the synthesis of chemical science go-betweens of redness, doing, powerful anti-inflammatory and analgetic effects16,17.

Then, the intent of this survey was to develop and qualify shea butter nanoparticles incorporating nimesulide for unwritten application. The readying method was developed to avoid the usage of organic dissolvers and particular equipments. The in vivo antinociceptive and anti-inflammatory activities were besides evaluated for the obtained preparations.


2.1. Materials

Shea butter was obtained by Alpha Qu & A ; iacute ; mica ( Porto Alegre, Brazil ) , polysorbate 80, sorbitan monooleate and carrageenin were purchased from Sigma-Aldrich ( S & A ; atilde ; o Paulo, Brazil ) , nimesulide was obtained from Henrifarma ( S & A ; atilde ; o Paulo, Brazil ) . Acetonitrile was of HPLC class and all other chemicals were of analytical class and used as received.

2.2. Methods

2.2.1. Preparation of lipid nanoparticle suspensions

The nanoparticles suspensions were prepared utilizing the measures presented in Table I. The lipid stage was heated at 40 & A ; deg ; C until all constituents were melted. The aqueous stage was prepared by blending all constituents under magnetic stirring. Then, the aqueous stage was poured over the lipid stage under agitation. The suspension was subjected to agitating in the ultra-Turrax T 125 IKA® at 24.000 revolutions per minute during 20 min and, eventually, the lipid nanoparticles were obtained after chilling.

For comparing, a preparation without nimesulide was prepared under the same conditions. Therefore, the preparations were named NP-N ( Nanoparticles incorporating Nimesulide ) and NP-B ( Nanoparticles without Nimesulide ) .

2.2.2. Physico-chemical word picture of the lipid nanoparticles suspensions

The measurings of nanoparticles mean atom size and polydispersity index ( PDI ) were performed through dynamic light dispersing utilizing a Zetasizer® Nano-ZS Malvern theoretical account ZEN 3600 ( Malvern Instruments, UK ) , after dilution of 500 times v/v in H2O. For the zeta possible measurings, the same equipment was used but thining the samples with 10 millimeters NaCl.

The pH was measured utilizing a Digimed® , theoretical account DM-22 potentiometer. The pH measurings were performed straight in the preparations in triplicate.

Drug burden was assessed by high force per unit area liquid chromatography ( HPLC ) . For this, the NP-N was dissolved with acetonitrile, diluted in nomadic stage and quantified by HPLC ( Shimadzu, UV/VIS sensor ) . Detection was set at 230 nanometer, the column used was Lichrospher 100 RP-18 ( 5 µm, 250 ten 4 millimeter ) , the nomadic stage of acetonotrile and H2O 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, utilizing Amicon Ultra centrifugal filters ( Millipore, Ireland ) . NP-N ( 0.5 milliliter ) was placed inside the filter, and centrifuged 5,000 revolutions per minute 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 burden and the free drug.

2.2.3. In vitro release of nimesulide from nanoparticles

The release profiles were obtained by dialysis, utilizing cellulose dialysis bags with cut of 10.000 Da. The bags were hydrated in H2O during 1h before the dialysis novice. An aliquot of 5.0 milliliters of NP-N and NP-B was put inside the dialysis bag. The closed bags were put in beaker incorporating 50 ml release medium ( phosphate buffer pH 7.4: polyethilenoglicol 400 ; 90:10 ) . The beakers were kept at 37 & A ; deg ; C and under magnetic stirring for 72 h. An aliquot of 3.0 milliliter was withdrawn in preset clip intervals and quantified for nimesulide release by UV spectrophotometry at 283 nanometers. For comparing, nimesulide was dispersed in polysorbate 80 and placed inside the dialysis bag at the same concentration than NP-N. In all instances, skin conditions were maintained during the experiment.

The release profiles were evaluated by the application of mathematical theoretical accounts. The accommodation of the informations to the monoexponential equation ( Equation.1 ) and to the biexponential equation ( Equation 2 ) was done utilizing MicroMath Scientist® software19. The best accommodation was selected based on the correlativity coefficient ( R ) , Model Selection Criterion ( MSC ) , and ocular scrutiny.

( 1 )

( 2 )

where C is the concentration in the release medium at clip T, K is the release rate. C0 is the initial concentration. A is the sum released in the explosion stage at rate ? and B is the sum released in the sustained stage at rate ? .


The animate beings ( Swiss mice and Wistar rats ) used on the experiment were housed under conditions of optimal visible radiation ( 12/12 h light-dark rhythm ) , temperature ( 22 ± 1 & A ; deg ; C ) and they had free entree to H2O and nutrient. Rats and mice were kept in separate suites.

All the animate beings were cared and manipulated harmonizing 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 trial

Swiss male mice ( n=8/group ) , burdening 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 wrestling trial on rats was based on the method reported by Nogueira et al.20. The animate beings did non receive H2O or nutrient for 12h before the experiment. The nociceptive consequence was induced by an intraperitoneal application of 1.6 % acetic acid at the dosage of 0.1ml/10g. All preparations were orally administered instantly after the nociceptive agent. Then, the animate beings were kept in a controlled box during 20 min to detect of the figure of wrestling.

2.2.6. Paw rat hydrops trial

This methodological analysis was adapted to the described by Winter et al.21. The hydrops was induced at the rat ‘s right hind paw by an intraplantar injection of the edematogenic agent ( carrageenin ) and comparison with the contralateral paw.

Wistar male rats ( n=8/group ) burdening 180-360 g were divided in the same four groups described in 2.2.5. However, nimesulide dosage was 5 mg/kg.

One subplantar injection of 0.1 milliliter of 1 % carrageenan solutions was used to bring on the hydrops on the right hind paw of the rats. Then, the animate beings received orally the preparations. After 5 H, the animate beings were sacrificed and the hydrops was expressed by the difference in gms between the right and the left hid paws.


3.1. Development and Physical-Chemical Characterization of Lipid Nanoparticles

The lipid nanoparticles were prepared by homogenisation utilizing high shear rates. This is a new technique that was n’t described before to obtain nanoparticles with narrow atom size distribution. The chief advantage of this technique is that the production is easy, is non clip consuming and is n’t necessary organic dissolvers. This technique besides reduced the demand of specialised equipment as a high force per unit area homogenizer. The Table II presents the physico-chemical belongingss of NP-N and NP-B.

We can detect that NP-N [ NP-B ] presented average atom diameter of 90.1± 0.2 [ 87.8 ± 0.4 ] nanometer and PDI of 0.21 ± 0.07 [ 0.26 ± 0.01 ] . There was no statistical difference between the considered samples. These consequences are in conformity to Bondi et al.22 that developed solid lipid nanoparticles incorporating nimesulide with different lipoids. These atoms presented a fluctuation of 85 to 135nm of size and PDI runing 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 potency reflects the surface charge of the atoms, which is influenced by alterations in the interface with the scattering medium due to the dissociation of functional groups on the atom surface or the surface assimilation of ionic species presented in aqueous scattering on the atom surface24,25. For NP-N, zeta potency was -20.21 ± 1.88mV, and it was considered adequate for the stableness of the nanostructures, due the repulsive force electrostatic forces demonstrated by this high value24. Comparing the zeta potency of NP-N and NP-B, it can be observed that NP-N had a higher zeta potency, which suggest an increased stableness of NP-N. Furthermore, the consequences of zeta potency for NP-N are in conformity with other surveies, like nanosuspensions incorporating diclofenac that obtained a zeta potency of -25mV26. Stable resveratrol-loaded nanocapsules presented zeta possible around -15mV27, bespeaking that the values obtained in the present survey are satisfactory.

Several factors can act upon the measure of drug associated to nanostructured systems. Among these, the physico-chemical features of the drug, pH, zeta possible and the measure of drug added to the preparation were cardinal factors to a successful encapsulation 24,25.

In this survey, the drug burden was 87.7 ± 7.2 % . The encapsulation efficiency was 96.5 % , bespeaking a really high encapsulation, chiefly if compared with Bondi et al.22, that obtained nimesulide-loaded nanoparticles incorporating 17.8 % of drug when utilizing Compritol® as lipid. In this manner, shea butter was a really equal lipoid to encapsulate nimesulide, demoing high drug burden and encapsulation efficiency.

3.2. Drug Release Profiles of Nimesulide-loaded Nanoparticles

During the development of drug bringing systems, as nanoparticles, the extent and speed of the release is really of import to preview their in vivo behaviour. One of the major restrictions in rating the drug release profile is the trouble to divide atoms from the dissolved drug due to the little size of these carriers29. Indeed, a methodological analysis that allows the quantification of low drug concentrations in the release medium is besides necessary.

Both free nimesulide and NP-N showed maximal release of 12 % in 72 H ( Figure 1 ) . Free nimesulide was statistically higher and faster, showing the ability to command the drug bringing of the nanoparticles ( ANOVA P & A ; lt ; 0.05 ) .

These consequences are explained by a high grade of interaction between lipid and drug and differences in the drug deposition on the particle30. It is of import to see besides the high hydrophobicity of nimesulide and the physical entrapment into the nanoparticles. These factors can better the bioavailability and cut down the side effects22.

In the release survey of nimesulide-loaded Compritol® nanoparticles22, 100 % of drug was released in 14 h. However, the burden capacity merely was 17.8 % , which may hold influenced the release of all drug sums.

Surveies comparing the incline of the release profiles nanoparticles prepared with different lipoids found fluctuations in the upper limit released. Prednisolone release was 37.1 % for cholesterin and 83.8 % for Compritol® and the release occurred within 5 weeks30. The controlled release could be achieved by alterations of the chemical construction of the lipoid. Furthermore, surfactant concentration, production technique and temperature besides affected the release profiles30.

Mathematical mold was used to analyse the release profiles, where the monoexponential equation ( which provides a kinetic invariable that indicated the release rate ) presented a better accommodation ( R & A ; gt ; 0.99 ) ( Table 3 ) , similar to what was observed by J & A ; auml ; ger et al.31 for the release Indocin 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 ( thousand = 0.228h-1 ) . The release profiles were constructed by plotting the concentration of nimesulide released a map of clip ( Figure 2 ) .

3.3. Acetic Acid-induced Writhing Trial

The acetic acid intraperitoneal disposal induces pain by straight activation of non-selective communicating channels located in the primary centripetal tracts or, indirectly, by advancing the release of prostaglandins and other inflammatory mediators32. Therefore, this theoretical account is utile for appraisal of analgetic or anti-inflammatory belongingss of drugs ( as nimesulide ) , one time the action of these drugs reduces the figure the acetic acid-induced writhing.

Figure 2 shows the mean and standard mistake of acetic acid-induced writhing, after 20 min of acetic acid injection with attendant intervention of the animals.. Animals treated with NP-N and nimesulide had average writhing of 10.38 ± 2.82 and 9.63 ± 1.83, severally. Result it is clear less that control group ( 33.13 ± 6.10 ) and NP-B ( 18.71 ± 4.93 ) . The consequences showed that the group treated with NP-N and nimesulide significantly inhibited the wrestling response induced by acetic acid ( ANOVA P & A ; lt ; 0.05 vitamin E Tukey & A ; lt ; 0.05 ) when compared with the several control groups.

Bochi et al.22 pre-treated mice, orally, with hydrophilic gel incorporating meloxicam nanocapsules ( 5 mg/kg ) at different times before acerb acetic application. The antinociceptive activity observed was important in all instances. Santos33 pre-treated mice with meloxicam ( 2.8 ; 8.4 ; 28.4 µmol/kg ) given intraperitoneally 30 min before acerb acetic disposal. They observed that the wrestling suppression was the dose dependant and important in all instances. In add-on, that the writers calculated that 50 % of wrestling would be inhibited with 7.4 µmol/kg of meloxicam. Naveen and confederates ( 2004 ) pre-treated mice with Naprosyns ( 5 ; 10 ; 20 mg/kg ) and nitro-naproxen ( 6.83 ; 13.86 ; 27.73 mg/kg ) orally, 30 min before noxious stimulation and both drugs significantly reduced the incidence of writhing.

3.4. Carrageenan-induced rat paw hydrops

Intraplantar carrageenin injection induces acute inflammatory procedure with pronounced hydrops formation ensuing from the production of several inflammatory mediators34. The inflammatory response may be quantified by the addition in paw size ( hydrops ) and may be modulated by inhibitors of inflammatory cascade, such as nonsteroidal anti-inflammatory drugs33. Therefore, it is clear that anti-inflammatory efficaciousness of these drugs ( as nimesulide ) encapsulated or non in nanoparticles may be evaluated through the fluctuation in the edematogenic procedure utilizing carnal theoretical accounts, like paw hydrops.

Figure 3 shows the differences in paw hydrops, after 5 H of initiation of the edematogenic procedures with attendant intervention. In trial rat paw hydrops, the group treated with NP-N or nimesulide ( 5 mg/kg ) demonstrated mean of addition in paw weight of 0.65 g and 0.81 g, severally. These values were well less than the control group ( 1.23 g ) and NP-B groups ( 1.02 g ) . Due to a great variableness among the persons of the group treated with nimesulide, merely the group treated with NP-N demonstrated important suppression of the hydrops ( ANOVA P & A ; lt ; 0.05 and Tukey p & A ; lt ; 0.05 ) . In this manner, the encapsulation of nimesulide allowed a decrease in variableness and a important decrease in the hydrops, showing the importance of the encapsulation to accomplish a higher pharmacological activity.

Bernardi et al.35 evaluated the effects contraceptive and curative usage of indomethacin-loaded nanocapsules and free Indocin ( 1mg/kg ) , administered intraperitoneally, 30 min before and 60 min after the injection of carrageenin. The anti-inflammatory activity of free Indocin and was important in comparing with control group.

Lenz36 administered locally ( through of clash during 30 s ) , 50 milligram of gel incorporating free nimesulide and gel incorporating nimesulide-loaded nanocapsules, 1h before the disposal of the flogistic agent. The anti-inflammatory activities of the free drug free and the encapsulated drug were considered important in comparing with group control.


It was possible to obtain lipid nanoparticles prepared by an advanced homogenisation by high shear rates technique. Shea butter was a good lipoid to encapsulate nimesulide with high drug burden.

The drug release profiles demonstrated a control in drug release from the nanoparticles. The profiles fit the monoexponential theoretical account. Sing the in vivo activity, in the antinocipective theoretical account, both nimesulide and the nanoparticles incorporating nimesulide presented important activity. However, in the paw hydrops theoretical account, merely the encapsulated drug showed important activity, showing the importance of the nanoencapsulation in the pharmacological behaviour of the drug.

In decision, the usage of shea butter as encapsulating lipoid was really successful and allowed nanoparticles to be prepared with a really simple technique. The nanoparticles presented important pharmacological effects that were non seen for free drug disposal.