Drug Deliv. and Transl. Res. DOI 10.1007/s13346-016-0296-9

ORIGINAL ARTICLE

Formulation and evaluation of proniosomes containing lornoxicam Jyotsana R. Madan 1 & Nitesh P. Ghuge 1 & Kamal Dua 2

# Controlled Release Society 2016

Abstract Proniosomes are the new generation provesicular drug delivery system of non-ionic surfactant, lecithin and cholesterol which upon reconstitution get converted into niosomes. The objective of current study was to develop stable and sustain transdermal delivery system for lornoxicam. Lornoxicam-loaded topically applied proniosomal gel was formulated, optimized, and evaluated with the aim to deliver drug transdermally. Lornoxicam-loaded proniosomal gels were prepared that contained Lutrol F68 and lecithin as surfactants, cholesterol as a stabilizer, and minimal amount of ethanol and trace water. The resultant lornoxicam-loaded proniosomal gel were assessed for stability and the proniosomes-derived niosomes were characterized for morphology, size, zeta potential, and entrapment efficiency, which revealed that they were suitable for skin application. The coacervation phase separation technique was used in formulation of lornoxicam proniosomal gel and the gel was further assessed for in vitro permeation of lornoxicam through the freshly excised rat skin and the cumulative permeation amount of lornoxicam from proniosome, all exhibited significant increase as compared to 1.0 % lornoxicam-loaded pure gel. The optimized F5 batch had shown maximum entrapment efficiency up to 66.98 %. It has shown sustained drug release for more than 24 h. The skin permeability of proniosomal gel was found to be 59.73 %. The SEM and zeta potential studies

* Jyotsana R. Madan [email protected]

1

Department of Pharmaceutics, Sinhgad Technical Education Society’s, Smt. Kashibai Navale College of Pharmacy, Pune, Maharashtra, India

2

School of Pharmacy and Biomedical Sciences, The University of Newcastle, Newcastle NSW 2308, Australia

showed formation of good and stable vesicles. Thus, proniosomes proved to have better potential for transdermal delivery of lornoxicam over conventional gel formulations. Keywords Proniosomes . Lornoxicam . Lecithin . Entrapment efficiency . Gel

Introduction Lornoxicam is a nonsteroidal anti-inflammatory drug belonging to an oxicam class having potent analgesic and antiinflammatory effects [1–4]. It is recommended widely for symptomatic treatment of pain and inflammation associated with rheumatoid arthritis, osteoarthritis and proven to have efficacy in the management of preoperative and postoperative pain associated with abdominal, orthopedic, gynecological and dental surgeries [5–8]. It requires repeated oral administration owing to its short duration of action and low elimination half-life. The major well-known side effects associated with all NSAIDs are gastric irritation and peptic ulcers [1, 3]. Lornoxicam has very low solubility in gastric acidic environment and remains unabsorbed for long periods in the stomach, aggravating the potential side effects [1]. One of the approaches to avoid its systemic side effects is to develop a topical drug delivery system of lornoxicam with a further advantage that it bypasses the hepatic first pass effect. Commercially, there is no formulation available for topical application of lornoxicam. The non-ionic surfactant vesicle strategy for improved transdermal drug delivery has been an area of research interest. Although surfactant vesicles overcome the skin permeation barrier and have good chemical stability, they possess certain physical incompatibility issues (leakage, sedimentation, aggregation, fusion, and loss of

Drug Deliv. and Transl. Res.

Lornoxicam (1 % w/w) was added in the vial and was covered with a lid to prevent loss of solvent and warmed in water bath at 65 ± 2 °C for 10 min or until the contents dissolved. Further, the mixture was allowed to cool at room temperature and aqueous phase (phosphate buffer pH 7.4) was dropped in to the vial which was kept again in water bath for 5 min or until the clear solution was formed. The solution was allowed to cool at room temperature until the formation of proniosomal gel.

vesicular integrity are the common) which affect the shelf life of the prepared dispersion [9–12]. The strategy to develop proniosomes as dry formulation which require reconstitution prior to use is a means of preserving physical stability and vesicles integrity. For the purpose of transdermal drug delivery proniosomes were formulated as gel suitable for topical application. Proniosomal gel takes skin water for reconstitution and forms niosomes, and the bilayer components (phospholipids and non-ionic surfactants) in proniosomes can act as penetration enhancers [5, 13]. Accordingly, in the current investigation, an attempt is made to develop lornoxicam transdermal proniosomal gel in order to supply sustained release of the drug to the affected tissues, improve patient compliance, and to avoid its potential gastrointestinal side effects.

Formulation of pure lornoxicam-loaded topical gel (PG) Carbopol-974p was soaked in approximately 20 ml of distilled water under mechanical stirring. Methyl paraben (0.2 %) and propyl paraben (0.02 %) were dissolved in ethanol and added to the carbopol dispersion under stirring. This dispersion was kept overnight for complete hydration of polymer. Lornoxicam (1 % w/w) was added with stirring to ensure uniform mixing of drug in dispersion. Triethanolamine was slowly added drop by drop to adjust the desired pH of the polymer. After complete dispersion, a fine gel is formed.

Materials and Methods Materials The drug (Lornoxicam) was obtained as gift sample from IPCA Laboratories Ltd., Mumbai, India. Lutrol F68 (Poloxamer 188) and Lipoid-80 H (Lecithin: soybean phosphatidylcholine) were obtained from BASF, Germany. Carbopol 974P NF was obtained from BASF, Mumbai, India. Other chemicals and reagents were of analytical grade.

Effect of bilayer composition on drug diffusion and drug entrapment To study the effect of variables on proniosome characteristics, different formulation batches were prepared. The amount of non-ionic surfactant Lutrol F68 and cholesterol were selected as two independent variables. Concentration of Lipoid-80 H was kept constant (Table 1). Entrapment efficiency (EE) and percent drug diffused were selected as dependent variables.

Methods Preparation of lornoxicam-loaded proniosomal gel

Characterization of lornoxicam proniosomal gel

Lornoxicam-loaded proniosomes were prepared according to the method (coacervation phase separation) reported by Vora et al. [14]. Briefly, in a wide-mouth vial, the non-ionic surfactant Lutrol F68, Lipoid-80 H, and cholesterol were mixed in the ratio 9:9:6 (w/w/w) with required amount of ethanol. Table 1 Components of lornoxicam proniosomal gel (LPG) formulation batches

Entrapment efficiency To 0.1 g of proniosomal gel, weighed in a glass tube, 10 ml of the aqueous phase (phosphate buffer pH 7.4) was added; the aqueous suspension was then

Formulation batches (codes)a

Lutrol F68 in mg

Cholesterol in mg

% entrapment efficiency (%EE) ± S.D.

% drug diffused

Vesicle size (nm)

Polydispersity index (PDI)

F1 F2 F3 F4 F5 F6 F7 F8 F9

80 80 80 90 90 90 100 100 100

80 60 40 80 60 40 80 60 40

49.302 ± 0.047 55.297 ± 0.058 48.252 ± 0.142 63.839 ± 0.133 66.989 ± 0.149 61.525 ± 0.163 58.124 ± 0.139 59.389 ± 0.093 53.300 ± 0.123

55.30 68.73 58.47 63.73 69.37 72.47 66.39 62.63 59.20

1088.58 375.10 434.46 1194.42 170.98 450.83 985.15 1176.71 150.01

0.375 0.823 0.812 0.530 0.702 0.357 0.228 0.378 0.453

a

All batches contain 90 mg of Lipoid-80 H and 8 mg of Lornoxicam

Drug Deliv. and Transl. Res.

sonicated in an ultrasonicator for 5 min. The formed niosomes containing lornoxicam were separated from unentrapped drug by centrifugation (REMI) at 9000 rpm for 45 min. The supernatant was recovered and assayed spectrophotometrically using UV spectrophotometer (Shimadzu), at 375 nm against phosphate buffer pH 7.4 solution. The entrapment efficiency of drug was calculated by the following equation: Entrapment efficiency ð%Þ ¼

Entrapped drug  100 Total drug added

Percentage Drug diffusion A Franz diffusion cell was used for diffusion studies. Dialysis membrane was mounted on the diffusion cell. A weighed amount of proniosomal gel was placed on one side of the dialysis membrane. The receptor compartment contained 10 ml of phosphate buffer saline pH 7.4 (PBS). The solution in the receptor compartment was continuously stirred at 100 rpm by means of Teflon-coated magnetic bead. Samples (1 ml) were withdrawn from the receptor compartment at 60-min intervals for a period of 24 h. The withdrawn sample was analyzed by UV spectrophotometer at 375 nm. Fresh phosphate buffer saline 7.4 (PBS) was added to replace the withdrawn sample volumes. Characterization of optimized lornoxicam proniosomal gel Particle size and polydispersity index Photon correlation spectroscopy (PCS) by using Nanophox (Sympatec, Germany) at room temperature was used to determine mean particle size and size distribution of drug-loaded proniosomal gel after hydration with PBS pH 7.4. The formed lornoxicam niosomal dispersion was added to filtered double distilled water to avoid multiscattering events. The width of the size distribution was indicated by the polydispersity index (PDI) using following formula: PID ¼

ðX 90 – X 10 Þ X 50

Scanning Electron Microscopy The morphology of the proniosomes after hydration with PBS pH 7.4 was determined by gold ion coating for 5 min followed by image capturing using a scanning electron microscope (Jeol JSM-6510, USA). Zeta potential analysis Charge on drug loaded vesicles surface was determined using Zeta potential analyzer (A Brookhaven instrument Corp). Analysis time was kept for 60 s and average zeta potential and charge on the proniosome preparation after hydration with PBS pH 7.4 was determined at 25 °C and 3 runs were carried out.

Differential Scanning Calorimetry Thermal characteristics of the lornoxicam proniosomes after hydration with PBS pH 7.4 were evaluated using differential scanning calorimetry (Perkin Elmer 4000, USA) instrument. The analysis was performed on 1 mg proniosomal gel samples sealed in standard aluminum pans. Thermogram of lornoxicam proniosomes and bulk Lornoxicam was obtained at a scanning rate of 10 °C/min in a temperature range of 30 to 300 °C. X-ray diffraction Lornoxicam proniosomes after hydration with PBS were evaluated for solid-state characteristics by Xray diffraction technique. Bulk lornoxicam and drug-loaded proniosomal dispersion was scanned at a scanning speed of 2°/min using a Phillips X-ray diffractometer equipped with an X-ray generator operating at a 40 kV voltage and 20 mA current. Ex vivo skin permeation studies Ex vivo skin permeation studies were performed using modified Franz diffusion cell. It was carried out using abdominal skin of rat (male albino Wistar rat weighing 250 ± 20 g). The skin was clamped in such a way that the dermal side would be in contact with receptor medium The receptor chamber with cross-sectional area of 4.32 cm2 was filled with receptor medium (PBS pH 7.4). The gel was applied uniformly on dorsal side of rat skin and donor compartment was placed and temperature was maintained 37 °C ± 0.5 °C at 100 rpm. At set interval of 18 h, 1 ml of the sample was removed and analyzed for percent of drug permeated into the receptor phase from the formulations by UV spectrophotometer at 375 nm.

Results and discussion Results Preparation of lornoxicam-loaded proniosomal gel Lutrol F68 and cholesterol were mixed with alcohol and aqueous phase to form the lornoxicam-loaded semisolid proniosomal gel. The proniosomes can spontaneously convert to a niosomal dispersion upon dilution with excess aqueous phase under gentle shaking. The mechanism behind the formation of proniosomal gel may be as following: lamellar liquid crystal may be formed from non-ionic surfactant (Lutrol F68) and the lecithin (Lipoid-80 H) at kraft temperature in the presence of little quantity of alcohol, and this crystalline phase will convert to niosomal dispersion with excess water [15]. The proniosome gel takes water from skin for immediate hydration so that it may avoid the stability problems associated with aqueous niosome dispersions such as fusion, aggregation, and leakage.

Drug Deliv. and Transl. Res.

Effect of bilayer composition on drug diffusion and drug entrapment Surfactant is an important component in the formation of niosomal vesicles and the variation in the concentration may affect the entrapment efficiency. The data shows that the variation in the concentration of surfactant (Lutrol F68) from 80 to 90 mg showed a significant increase in the entrapment efficiency, whereas the further increase in concentration from 90 to 100 mg (F9) decreased the entrapment efficiency to 53.3 % (initial increase in the concentration of surfactant may increase the number of niosomes formed; therefore, the volume of hydrophobic domain increases and hence increase in entrapment efficiency). However, the further increase in concentration showed decrease in entrapment efficiency, possibly due to formation of mixed micelles along with the niosomal vesicles with high concentration of surfactants, which may lead to lowering of entrapment efficiency. It is reported that the size of micelles is <10 nm, thus fewer amounts may be entrapped inside the vesicles. It may be due to this reason that the higher surfactant concentrations form vesicles with low entrapment efficiency. Thus, it has been concluded that 90 mg surfactant is the optimum quantity for proniosomal gel. The concentration of cholesterol plays an important role in the entrapment of drug in the vesicles. The variation in the concentration of cholesterol significantly affects the entrapment efficiency. The observed entrapment efficiency was increased significantly when cholesterol amount was increased from 40 mg (F6, %EE 61.52) to 60 mg (F5, %EE 66.98), but further increase in the cholesterol to 80 mg decreased the entrapment efficiency (F4, %EE 63.83). The increase in entrapment efficiency shows that the cholesterol, which acts as the vesicular cement in the molecular cavities of surfactant bilayer, and abolishes the gel to sol transition, thereby forms less leaky vesicles. Therefore, the increase in the rigidity decreases the permeability of the entrapped drug and hence improves the entrapment efficiency. However, when cholesterol amount was increased further from 60 to 80 mg, the opposite result occurred. The reason behind decreased entrapment efficiency may be due to the reason that a cholesterol molecule will compete with drug for the space within the bilayer, remove the drug from the bilayer and in addition to this will disrupt the vesicular membrane structure. The % of diffusion of lornoxicam through dialysis membrane was determined (Table 1). The increased diffusion due to increase in surfactant concentration (Batch F5) may be due to the non-ionic surfactant present in it, which modifies the structural composition of stratum corneum Table 3 Characterization of lornoxicam-loaded proniosomal gel (Batch F5). Data represent mean ± SD (n = 3)

Table 2 Comparative In-vitro drug diffusion study through dialysis membrane

Time (h)

F5 batch (% diffused)

PG (% diffused)

0 1

0 5.17

0 23.15

2 3

14.22 19.12

49.96 68.32

4

23.41

82.73

5 6

28.23 34.72

89.65 98.76

7 8

38.82 43.21

9

46.46

10 24

52.94 69.47

and increases the thermodynamic activity of the drug as well as skin vesicular partitioning. Thus, it has been concluded that 60 mg of cholesterol is the optimum quantity for proniosomal gel and F5 batch is the optimized batch [16–18]. Characterization of lornoxicam proniosomal gel (LPG) Entrapment efficiency The percentage entrapment efficiency of different proniosomal batches was found to be between ranges of 48.25 and 66.98 %. Percentage drug diffusion For further comparison, in vitro drug diffusion study of lornoxicam proniosomal gel (F5) and pure drug loaded gel (PG) were carried out using dialysis membrane by Franz diffusion cell, results are shown in Table 2. The ideal topical formulation should show a release for sufficiently longer period of time so as to avoid frequent application and better patient compliance. It is clearly evident from the results that plain drug loaded gel diffuses drug in lesser time as compared to proniosomal gel. The lornoxicam proniosomal gel was much slower in diffusing the drug through dialysis membrane. Thus, lornoxicam proniosomal gel (F5) shows a sustained release profile. Characterization of optimized lornoxicam proniosomal gel Particle size and polydispersity index Table 3 and Fig. 1 show the vesicle size and polydispersity index of proniosomal gel batch F5 after hydration with PBS.

Appearance

Size (nm)

Zeta potential (mV)

Polydispersity index

(%) entrapment efficiency

Clear gel

170.98 ± 0.006

−17.95 ± 0.003

0.702 ± 0.02

66.98 ± 0.34

Drug Deliv. and Transl. Res. 100

9

90

8

80

7

70

6

60 5 50 4

40

3

30 20

2

10

1

0 0.5

1.0

5

Scanning electron microscopy The scanning electron micrograph (SEM) of proniosomal dispersion of F5 batch (Fig. 2) shows spherical morphology and size in the nano dimensions. Zeta potential analysis The value of zeta potential was found to be −17.95 mV for optimized batch (F5) as shown in Fig. 3. It indicates prepared proniosomes have sufficient surface charge to prevent aggregation of the vesicles. Differential scanning calorimetry Differential scanning calorimetry (DSC) studies of the lornoxicam proniosomal dispersion was carried out. Lornoxicam shows a sharp melting point at 222.63 °C, depicting a typical crystalline nature of the drug. When incorporated into proniosomal gel, the characteristic endotherm of lornoxicam disappeared. This suggests complete amorphization of lornoxicam when incorporated into the proniosomal gel (Fig. 4a, b). The peak at 56.39 °C is of Lutrol F68.

10

50 100 particle size / nm

500

1000

5000 10000

density distribution q*(x)

cumulative distribution Q(x) / %

Fig. 1 Vesicle size distribution curve of optimized batch (F5)

0

X-ray diffraction X-ray diffraction (XRD) study was in agreement with the results of DSC study. Lornoxicam is a crystalline substance with characteristic peaks at 25.5°, 26.8°, 27.2°, 30.2°, 32.5°, 34.2°, and 38.2° (Fig. 5a, b). The characteristic peaks of lornoxicam were absent in proniosomal gel. Ex vivo skin permeation studies The formulations under study viz. Proniosomal gel (batch F5) and pure drug loaded gel were applied to the dorsal part of the skin which was exposed to donor compartment. The permeation study was carried out up to 24 h. The results of ex vivo skin permeation study shows that pure drug loaded gel gives 67.97 % permeation of drug through rat skin in 6 h as it is has higher permeation and maximum cumulative percent drug release (%CDR) from gel in to receptor medium. Whereas the proniosomal gel formulation (optimized Lornoxicamproniosomal gel (F5)) shows up to 59.73 % of drug permeation through skin at the end of 24 h. Graphical representation of comparative drug permeation of two formulations is shown in Fig. 6. Discussion

Fig. 2 SEM image of optimized formulation batch (F5)

Excipients suitable for topical delivery were used to prepare loaded proniosomal gel. Lutrol F68 and lecithin with excellent skin compatibility were chosen as the surfactants [19]. Cholesterol was used which is an important cell membrane component and its role in the vesicle is to improve the permeability and stability [18, 19]. Incorporation of cholesterol in the niosomes bilayer increases the entrapment efficiency of the drug in the vesicles. Cholesterol increases viscosity of niosomal dispersion and imparts rigidity to the flexible bilayer, which results in the formation of highly ordered structures of surfactants with cholesterol embedded in the bilayer. This further facilitates partitioning of the drug in the bilayer and increases the entrapment of the drug in niosomal vesicles.

Drug Deliv. and Transl. Res. Fig. 3 Zeta potential graph of optimized formulation batch F5

Fig. 4 a DSC thermogram of pure drug Lornoxicam and b optimized formulation batch F5

Drug Deliv. and Transl. Res.

a 4500

Intensity 2

4000 3500 3000

Intensity

2500 2000 1500 1000 500 0 30

40

50

60

70

80

2 Theta (Degree)

b 1400

Intensity 2

1200

Intensity

1000

800

600

400

200 30

40

50

60

70

80

2 Theta (Degree0

Fig. 5 XRD graph of a pure drug lornoxicam and b in formulation batch F5

Increased hydrophobicity and rigidity of the bilayered vesicles imparts stability and reduces permeability of the niosomes thereby increasing the entrapment efficiency. Increasing cholesterol concentration beyond 80 mg showed decrease in entrapment of the drug. Higher amounts of 80

LOR proniosomal gel Pain drug loaded gel

% CDR

60

cholesterol compete with the drug for packing space within the bilayer and effluxes the drug when the amphiphiles assemble into the vesicles. It also disrupts the regular bilayer structure and causes leakage of the drug [20, 21]. DSC and XRD studies were performed for solid-state characterization of LPG. DSC and XRD studies revealed loss of characteristic peaks of lornoxicam when incorporated into bilayer vesicles. This indicates complete amorphization of the Lornoxicam when incorporated into bilayer vesicles. The amorphous nature of the drug shows high permeation and deposition than its crystalline form, which further aids in efficient delivery of drug on topical application. As the LPG semisolid gel like colloidal network that aligns itself in the direction of applied shear showing pseudoplastic behavior (a shear thinning system showing decrease in viscosity on application of shear stress) which facilitates its topical application [22–25]. LPG showed better spreadability over pure drug loaded carbopol gel. Skin permeation studies were performed on Wistar rat skin. Pure lornoxicam gel is a carbopol-based gel of lornoxicam representing a simple, aqueous system with lornoxicam dispersed in the matrix. Permeation of lornoxicam from pure drug-loaded gel is thus determined by permeation properties of the drug. Improved skin permeation of the drug from LPG can be justified by several mechanisms. Non-ionic surfactants in the formulation act as permeation enhancer. The surfactants in vesicular form decrease the crystallinity of the intracellular lipid bilayers of the skin and thus enhance drug deposition. Increased solubility of the lornoxicam enhances its skin permeation. Vesicular nature of proniosomes after hydration enhances the permeation of the drug through the skin Pure lornoxicam gel is a hydrophilic gel system, without a lipid or oil component, while LPG is comprised of cholesterol and hydrophobic surfactants of vesicular size on hydration in the nanometer size range. This offers extensive surface area which forms a superior occlusive film on the skin surface and thus prevents the transepidermal water loss. It further improves hydration status of the skin and enhances skin permeation of the drug. Decreased particle size, decreased degradation, sustained release, and amorphization of the drug improve hydration status of the skin and its pharmacodynamic activity on topical application.

40

Conclusions

20

From the current study, it can be concluded that formulation of proniosomal gel using coacervation phase separation method proved to be a sound approach to obtain stable proniosomal gel of lornoxicam. F5 batch of lornoxicam-loaded proniosomal gel shows highest entrapment efficiency as compared to other batches and optimum particle size of vesicles.

0 0

10

20

30

Time (Hrs)

Fig. 6 Ex vivo skin permeation study of lornoxicam proniosomal gel (batch F5) and pure drug loaded gel

Drug Deliv. and Transl. Res.

SEM study reveals the formation of vesicles with spherical and smooth nature. Zeta potential analysis result adds to stability of optimized proniosomal gel formulation against aggregation. Optimized proniosomal gel formulation aspect had the desired properties with respect to their clarity, appearance, uniformity, and consistency. In vitro drug diffusion and skin permeation indicated that the effect of the drug was prolonged by prepared optimized proniosomes. Thus, proniosomes proved to have the potential for transdermal drug delivery of lornoxicam drug over conventional gel formulations. Overall, transdermal drug of delivery for lornoxicam has been successfully developed.

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Compliance with ethical standards 14. Conflict of interest disclosure The authors declare that there is no conflict of interest involved with this manuscript. 15. 16.

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