Formulation of Topical Flurbiprofen Solid Lipid Nanoparticle Gel Formulation Using Hot Melt Extrusion Technique

Abstract

Hot melt extrusion (HME) has been used for the formulation of topical solid lipid nanoparticle (SLN) gel without using any other size reduction technique including high pressure homogenization or sonication. SLN formulation solely using HME has not been applied to other drugs except IBU. Therefore, the purpose of the present study was to formulate FLB SLN solely using HME technique and evaluate the SLN formulation in inflammation animal model. Stable 0.5% w/v FLB SLN gel with particle size < 250 nm, PI < 0.3 and EE of > 98% was prepared. Differential scanning calorimetry (DSC) thermogram showed that the drug was converted to amorphous form in the HME process. Additionally, rheological studies demonstrated that FLB SLN gel and marketed FLB gel showed shear thinning property. FLB SLN formulation showed significantly (p < 0.05) higher peak force required to spread the formulation as compared to marketed FLB formulation. Stability studies showed that FLB SLN gel was stable for a month at room temperature and 2–4°C. Moreover, in vitro permeation test (IVPT) and ex vivo skin deposition study results revealed that FLB SLN gel showed significant (p < 0.05) increase in drug deposition in dermal layer and drug permeation as compared to control marketed formulation. Further, in vivo anti-inflammatory study showed equivalent inhibition of rat paw edema using 0.5% w/v FLB SLN gel which has 10 times less strength compared to control formulation. Overall, FLB SLN formulation was successfully manufactured solely using HME technique which resulted in enhanced the skin permeation of FLB and superior anti-inflammatory activity.

Introduction

Dermal products are classified into topical and transdermal dosage forms based on their application to produce local and systemic effects, respectively [1]. Topical drug delivery systems are localized drug delivery system for local delivery of therapeutic agents via skin to treat the cutaneous disorder [1]. Topical delivery of lipid nanocarriers has vast pharmaceutical application in delivering diversity of therapeutic agents including biotechnological products and various small active pharmaceutical ingredients (API). These nanoparticles possess numerous merits including enhanced skin hydration and elasticity, skin occlusion, controlled release of API, and improved stability of chemically labile drugs. Moreover, they are alternative colloidal carriers to conventional nanoparticles including polymeric nanoparticles, liposomes, and nanoemulsion [2–10]. Lipid nanoparticles are classified into two types: solid lipid nanoparticles consisting of solid lipid and surfactant and nanostructured lipid carrier consisting of solid lipid, liquid lipid, and surfactant [11, 12]. Solid lipid nanoparticles can be spherical, disk-like in shape or can have flat ellipsoidal geometry. Additionally, loaded API can be present into the solid core or adsorbed on a carrier matrix surface. Manufacturing of these lipid nanoparticles requires high energy to generate nanosized particles because of their hydrophobicity [13–16].

These lipidic nanoparticles are manufactured by exposing them to high energy step including ultrasonic waves (probe or bath sonication) or high pressure homogenization or micro emulsification or solvent displacement [14]. Until today, there are numerous reports published on delivering many APIs using SLNs as carriers for the topical drug delivery system. Pham et al. studied the preparation of curcuminloaded solid dispersion-SLNs system (SD-SLNs) using sonication method in which they showed enhanced curcumin delivery using SD-SLNs topical delivery system compared to SLN [17]. Passos et al. demonstrated development of itraconazole-loaded NLC for the treatment of fungal diseases using sonication method in which they showed enhanced drug deposition in skin because of skin occlusion characteristic of lipid nanoparticles [18]. Geetha et al. reported the formulation of SLNs containing sesamol as an API for skin cancer treatment, in which they showed enhanced skin retention of sesamol and significant reduction in the inflammation in in vivo anticancer study post application of SLN formulation containing sesamol [19]. Additionally, numerous APIs including doxorubicin, benzocaine, human recombinant epidermal growth factor, adapalene, amphotericin B, peptide LL37 and serpin A1, paclitaxel, flutamide, triamcinolone acetonide, aceclofenac, tretinoin, vitamin C, antimicrobial peptide nisin Z, lidocaine and prilocaine, resveratrol, piperine, silybin, fluconazole, and many more have been reported to be delivered using lipid nanoparticulate carrier [14].

Recently, hot melt extrusion technique has been used for production of lipid nanoparticles because of its various advantages including processing the material at controlled temperature and energy required to produce nanoparticles. Shadambikar et al. investigated production of lipidic nanocarriers using HME-HPH method in which they claimed that HME-HPH method can be used as a continuous single-step process over conventional multi-step process [20]. Also, the use of HME along with HPH technique for fenofibrate and HME ultrasonication for lidocaine has been reported in the literature [21, 22]. However, all these studies reporting the use of HME equipment along with HPH technique used the extruder consisting of three mixing zones for the formulation of lipid nanoparticles. Recently, we have developed another method for the preparation of SLN by solely using HME equipment which has only one mixing zone [23]. SLN formulation solely using HME has not been applied to other drugs except IBU. Therefore, the purpose of the present study was to formulate FLB SLN solely using HME technique and evaluate the SLN formulation in inflammation animal model. FLB is a well-known non-steroidal anti-inflammatory drug (NSAID) used in the treatment of severe or chronic arthritis. Oral administration of FLB can cause gastrointestinal irritation and toxicity in liver and kidney. Topical route bypasses the side effects caused by oral route of administration [24, 25]. Additionally, FLB is highly lipophilic API with a log p of 3.82 and with low melting point of 117°C which makes it ideal candidate for lipidic nanoparticulate delivery system [26]. The objective of the present study is to manufacture SLNs containing FLB using a continuous novel HME technique to increase the drug permeation into skin layers with enhanced anti-inflammatory activity in the in vivo carrageen-induced rat paw edema study.

Material and Methods

Materials

Chemicals

FLB was gifted by VWR international (Radnor, PA). Compritol^® 888 ATO (glyceryl dibehenate EP) was donated by Gattefosse (Paramus, NJ). Kolliphor EL and Tween 80 were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile and water were also purchased from Sigma-Aldrich (St. Louis, MO, USA). Distilled water was used throughout the study.

Animals

Sprague–Dawley rats (SD) (Envigo, IN, USA) were used for developing the rat paw edema model. The animals were housed in cages and maintained under controlled conditions of 12:12 h (light:dark) cycle, 22 ± 2°C, and 50 ± 15% RH. The rats were provided with feeding (Harlan Teklad) and water ad libitum. They were housed at Florida A&M University animal labs in accordance with the Guide for the Care and Use of Laboratory Animals and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and were acclimatized to laboratory conditions for a week before the onset of all experiments. The Institutional Animal Care and Use Committee (IACUC) at Florida A& M University, FL, approved all animal protocols that were observed in this study on 10th March (2016) (Protocol number: 016–03. Project number: 861412–3).

Methods

Equipment Configuration

HME equipment (Omicron 10P, Steer America, OH, USA) is configurated into five zones: feeder zone, conveying zone, melting zone, mixing zone, and discharge zone. Three screw designs of HME are available with different diameter (Do:Di) ratios of 1.21, 1.55, and 1.71. The Do:Di 1.21 gives a finer particle size. Solid lipid and API mixture were introduced manually in the feeder zone from the hopper. Surfactant solution was added in the process through mixing zone (Fig. 1).

Fig. 1.

Schematic representation of formulation of FLB SLN using HME technique

Method of Preparation of SLN

Hot‑Melt‑Extrusion Method

Briefly, solid lipid (Compritol 888 ATO) and API (FLB) was mixed and fed through the hopper located in the feeding zone. Once the molten mixture reaches to the mixing the zone, surfactant solution was introduced slowly to form pre-emulsion. This pre-emulsion was then reprocessed through the mixing zone to get the final FLB solid lipid nanoparticle formulation. Further, the SLN formulation was gelled using 1% carbopol. After addition of carbopol, about 0.5 ml of 5% triethanolamine was added for crosslinking carbopol gel. Triethanolamine neutralizes carboxy groups such as carbomer to form a stable polymer structure, so as to achieve the application effect of thickening and moisturizing [27]. Final pH of the SLN formulation was about 4.5.

Preliminary Screening of Lipid, Surfactant, Screw Design, Screw Speed, and Barrel Temperature

Preliminary screening of lipid, surfactant, screw design, screws peed, and barrel temperature was carried in order to obtain SLN of smaller size, narrow PDI, and higher %EE.

Optimization of Barrel Temperature

In order to optimize barrel temperature, the formulation composed of 0.5% w/v FLB, 2% w/v Compritol 888 ATO, 2.5% w/v Kolliphor EL, 2.5% w/v Tween 80, and water (to make up to 100 w/v%) was processed at 90°C, 100°C, 121°C, and 126°C at all zones. Further, particle size and polydispersity index of all the formulations were measured using Nicomp particle size analyzer.

Optimization of Screw Speed

The speed of screws was optimized by processing the SLN formulation at different screw speed including at 50 rpm (revolution per minute), 100 rpm, 125 rpm, and 150 rpm. The pre-emulsion was then reprocessed at 800 rpm. Further, all the formulations were analyzed for their particle size and polydispersity index.

Optimization of Lipid and Surfactant

Compritol 888 ATO and Precirol ATO 5 was used to formulate the FLB SLN. Formulation composed of 0.5% w/v FLB, 2% w/v Compritol 888 ATO/Precirol ATO 5, 2.5% w/v Kolliphor EL, 2.5% w/v Tween 80, and water (to make up to 100 w/v%) was processed at 100°C through the extruder. Further, all the formulations were analyzed for their particle size and polydispersity index.

Optimization of Lipid, Surfactant, and Drug Concentration

Compritol 888 ATO was used in different concentrations of 2%, 2.5%, 3%, and 4% w/v in the formulation to assess the effect of lipid concentration on the particle and polydispersity index of the SLN formulation. Surfactants including Kolliphor RH 40, Kolliphor EL, Tween 20, and Tween 80 were used alone or in different combinations in formulating FLB SLN formulation. Further, all the formulations were analyzed for their particle size and polydispersity index. SLN formulation was also formulated with FLB concentration ranging from 0.125 to 0.5%. Further, all the formulations were analyzed for their particle size and polydispersity index to evaluate the effect of drug concentration on quality of the final formulation.

Physicochemical Characterization of SLN

In order to determine particle size and zeta potential, Nicomp 380 ZLS (PSS.NICOMP, particle sizing systems, Santa Barbara, CA, USA) was used. The total drug content was determined by dissolving 100 μl of the SLN in 900 μl of acetonitrile. From this, 100 μl was removed and diluted with mobile phase. The resulting sample was centrifuged at 12,000 rpm for 15 min, and the supernatant was collected and analyzed by HPLC. The entrapment efficiency was determined as reported [8]. Briefly, SLN was put in the donor compartment of the viva spin centrifuge filter membrane (molecular weight = 14 kD) and centrifuged at 4500 rpm for 15 min. The amount of drug present in the receiver compartment (RC) was determined using HPLC analysis [28–32]. The entrapment efficiency was calculated using this formula:

Entrapment efficiency (%) = (the total drug content – amount of drug in RC)*100/the total drug content.

HPLC Analysis of FLB

Waters HPLC alliance e2695 system with PDA detector with a reverse phase C₁₈ column (Symmetry^®, 5 μm, 4.6 × 250 mm; Waters Technology Corporation, USA) was used for analysis of FLB at wavelength of 247 nm. The mobile phase was acetonitrile and water (pH adjusted to 6 using acetic acid), at a ratio of 70:30 (v/v), flow rate of 0.5 ml/min, retention time of 2.5 min, and injection volume of 50 μl. Data acquisition and analysis were performed using Empower software (Waters Corporation, MA). The calibration curve (peak area vs. concentration) was generated over a range of 5–80 μg/ml and was found to be linear with a correlation coefficient R² = 0.9974. LOD and LOQ was found to be 1.31 μg/ml and 3.97 μg/ml, respectively [33, 34].

Spreadability Test

Spreadability test of FLB gel formulations was performed using TA-XT plus Texture Analyzer in the compression test mode. The rig (TA-425 TTC) spreadability fixture TM and set of precisely matched male and female acrylic 90° cones were used for spreadability testing. The gel formulation was filled into the lower cone with a spatula. Gel was then pressed down gently to remove the air pockets (which are visible through cones), and the surface was leveled using flat knife. Texture analyzer was calibrated before beginning the test. Height was calibrated at 25 mm distance. The probe was then set to travel 24 mm down from a fixed position of 25 mm over the bottom of the lower cone. The final gap of 1.0 mm between the two cones was precisely set with the test speed of 2.0 mm/s for testing the spreadability [35].

Rheological Studies

Viscosity of the gel formulation was measured using AR 1500 Rheometer (New Castle, DE). Approximately 0.4 ml of gel formulation was loaded onto the lower plate of the rheometer and flow sweep measurements with 1000-μm gap, 0.1 to 100 S⁻¹ shear rate, and parallel plate geometry (25 mm diameter) were employed in obtaining the viscosity of each formulation. Rheological study was conducted at standard room temperature (25°C). Results for viscosity were expressed in Pa•s. Both formulations were evaluated using same viscosity parameters [36].

In Vitro Permeation Test (IVPT) and Skin Deposition Studies

Using the dermatomed human skin (0.5 ± 0.1 mm thickness), the deposition study was conducted. The skin was purchased from Platinum Training (Henderson, NV), transported in 10% glycerin solution in saline, and stored at − 80°C. The skin was defrosted and rinsed with distilled water for 15–20 min to remove excess glycerin before all experiments were proceeded. Similarly, to the dialysis membrane released study, the skin was fitted between the donor and the receiver compartment of Franz diffusion cells (Permegear Inc., Riegelsville, PA, USA). The receiver compartment comprised of pH 7.4 buffer and was maintained at 37 ± 0.5°C with continuous stirring at 300 rpm. 0.472 mg of 0.5% w/v SLN gel and 5.005 mg of 5%w/w marketed product (which was sufficient to cover the entire surface of skin in donor compartment) were applied uniformly and the studies were carried out for 24 h. Further, 200 μl of sample was collected at 1, 2, 4, 6, 8, 12, 16, and 24 h and replaced with 200 μl of blank receiving media. Following the IVPT study, the leftover of each formulation was removed from the surface of the skin with a cotton swab, cleaned carefully using 50% v/v ethanol in water, and then the total dosing area (0.636 cm²) was excised using a biopsy punch. Epidermis was carefully separated from the dermis with the help of forceps. Dermis was then minced and boiled with 300 μl PBS (pH 7.4) for 10 min. Similarly, epidermal layer was boiled with 300 μl PBS (pH 7.4) for 10 min. To these samples, 300 μl of acetonitrile was added to solubilize the drug. All the samples were then centrifuged at 15,000 rpm for 15 min. The supernatant was collected and analyzed by HPLC for drug content [37–42].

Anti‑inflammatory Effect of FLB SLN In Vivo

Three groups: no treatment, 0.5% FLB SLN gel, and 5% FLB gel marketed product of SD rats (Envigo, IN, USA) were divide for this experiment. Each group consisted of three animals. Edema was induced by sub-planter injection of 0.05 ml of 1% carrageenan into the right hind paw of each rat and after 30 min, topical treatment of 300 mg approximately of formulation was applied on the inflamed hind paw. The volume of the involved right hind paw of each rat was measured by using a digital plethysmometer (Harvard Apparatus, MA, USA) immediately before and 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after the injection of carrageenan. The edema rate and inhibition rate of each group were calculated using the following equation [36]:

$$\%\text{Edema Rate} (\text{E}\%)=({\text{V}}_{\text{t}}-{\text{V}}_0/{\text{V}}_0)\ast 100$$

$$\%\text{Inhibition Rate} (\text{I}\%)=({\text{E}}_{\text{c}}–{\text{E}}_{\text{t}}/{\text{E}}_{\text{c}})\ast 100$$

where V₀ is the mean paw volume before carrageenan injection (ml), V_t is the mean paw volume after carrageenan injection (ml), E_c is the edema rate of control group, and E_t is the edema rate of the treated group.

Stability Studies

FLB (0.5% w/v) SLN formulation was stored at room temperature and 2–4°C for a month. Further, the formulation was evaluated for particle size, polydispersity index, entrapment efficiency, and drug content. Formulation was also evaluated for sedimentation of particles or phase separation.

Statistical Analyses

Data were presented as the mean ± standard deviation. Statistical differences were evaluated by ANOVA and Student’s t-test. The criteria for statistical significance were set at p < 0.05. Statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA).

Results

Drug‑Lipid Miscibility Study

The miscibility of FLB was assessed by adding 100 mg of the API into 100 mg of Compritol 888 ATO. The mixture was heated for about 10–15 min at 90°C, above the melting point (70°C) of the lipid. The mixture was then visually observed for the transparent molten mixture indicating the API is miscible in the lipid. Data showed that, more than 10% FLB was miscible in the Compritol 888 ATO as the clear transparent mixture was observed in the Eppendorf tube which indicated that drug was completely miscible in the lipid.

Optimization of Barrel Temperature, Screw Speed, and Screw Design

Our results revealed that when the formulation was processed above or below 100°C, sedimentation in the pre-emulsion was observed. Formulation processed below 100°C produced FLB SLNs with particle size of > 500 nm with polydispersity index > 0.4 with sedimentation in the formulation. Additionally, formulation processed above 100°C produced FLB SLNs with particle size of > 400 nm with polydispersity index > 0.4 with sedimentation in the formulation. However, at 100°C, FLB SLNs with particle size of less than 250 nm with polydispersity index < 0.3 were produced. Results illustrated that SLN formulation when extruded at 50 rpm which reprocessed at 800 rpm, the resultant final formulation showed particle size < 400 nm. Further, data showed that SLN formulation when processed at 100 rpm which reprocessed at 800 rpm, the resultant final formulation showed particle size < 250 nm. Additionally, screw speed of 150 rpm for the first run followed by 800 rpm for the reprocessing the formulation did not show significant effect on the particle size and PI.

Optimization of Lipid and Surfactant

Formulation consisting of Precirol ATO 5 as a lipid resulted in a hazy and clumpy suspension. Compritol formulated SLN resulted in the particles with size < 200 nm, using the optimized conditions. The use of Tween 20, Tween 80, Kolliphor EL, and Kolliphor RH40 alone in the SLN formulation resulted in a hazy formulation with sedimented particles. Combination of Kolliphor EL and Tween 80 yielded a stable formulation with particle size < 250 nm.

Concentration of Drug, Lipid, and Surfactant Optimization

Our results showed that, 0.5% FLB, 2% Compritol 888ATO, and 2.5% of each of the surfactants used (Tween 80 and Kolliphor EL) resulted in a formulation with particle size in in the range of < 250 nm. We also observed that, formulations with FLB concentration above 0.5% resulted in sedimentation of particles. Similarly, formulations with lower Tween 80 and Kolliphor EL concentration (below 2.5%) with 0.5% FLB and 2% lipid concentration resulted in cloudy suspension. It was also observed that Compritol concentration above 2% along with 0.5% FLB and 2.5% of each of the surfactants (Tween 80 and Kolliphor EL) resulted in increase in particle size to about 630 nm.

Scanning Electron Microscopy (SEM)

SEM images of FLB-SLN showed that SLN were of particle size below 208.7 nm with spherical shape and smooth surface (Fig. 2A). Our particle size analysis using Nicomp 380 ZLS (PSS.NICOMP, particle sizing systems) also showed FLB SLN with particle size below 250 nm.

Fig. 2.

A SEM image of FLB SLN showing spherical SLN particles with particle size below 250 nm. B DSC thermogram showing sharp endotherm of FLB at its melting point which was disappeared in FLB SLN formulation

Differential Scanning Calorimetry (DSC)

DSC thermogram results revealed that API flurbiprofen showed prominent melting point endotherm peak at 119°C. DSC thermogram of FLB SLN did not show the melting point endotherm peak of drug at 119°C, instead it was slightly increased after processing through the HME extruder (Fig. 2B).

Spreadability Test

Peak force, work area, and stiffness were recorded for both HME-based FLB and marketed FLB gel formulation. Data showed significantly (p < 0.001) higher peak force (668.500 ± 6.082 g), work area (1619.958 ± 106.520 g*mm), and stiffness (286.977 ± 6.246 g/mm) with marketed FLB (5%w/w) gel formulation as compared to FLB (0.5% w/v) gel formulation with peak force of 298.107 ± 27.803 g, work area of 524.455 ± 37.198 g*mm, and peak force of 668.500 ± 6.082 g. High peak force in marketed FLB (5% w/w) gel formulation indicated that the formulation is thicker than the HME-based FLB (0.5% w/v) gel (Fig. 3 and Table I).

Fig. 3.

A Spreadability testing setup using texture analyzer equipment. B Spreadability curve showing significant decrease in peak adhesive force in case of 0.5% w/v FLB SLN formulation as compared to 5% w/w FLB marketed gel formulation

Table I.

Table Showing Peak Adhesive Force, Work Area, and Stiffness of 0.5% FLB SLN Gel and 5% Marketed FLB Gel

Formulation	Work area (g*mm)	Peak force (g)	Stiffness (g/mm)
HME-based FLB gel	524.455 ± 37.198	298.107 ± 27.803	160.708 ± 21.975
Marketed FLB gel	1619.958 ± 106.520	668.500 ± 6.082	286.977 ± 6.246

FLB, flurbiprofen; SLN, solid lipid nanoparticle; HME, hot melt extrusion

Rheological Studies

FLB SLN gel and FLB marketed gel formulation followed a non-Newtonian flow and showed shear thinning property. As shown in Fig. 4, the viscosity was decreased for both the gel formulations as the shear rate was increased. The viscosity of FLB SLN get was measured to be 265 ± 27.3 Pa·s whereas the marketed product was determined to be 2483 ± 155.9 Pa·s at room temperature (25°C).

Fig. 4.

Rheological studies of 0.5% w/v FLB SLN gel and 5% w/w FLB marketed gel formulation showing decrease in viscosity upon increase in shear rate

In Vitro Permeation Test and Skin Deposition Study

Data showed that about 12.10 ± 2.65% drug was permeated from Flub-SLN-G formulation and 7.25 ± 1.57% from the marketed FLB gel at the end of 24 h (Fig. 4). As shown in the figure, Flub-SLN-G showed two fold increase in the drug permeation in the receiver compartment through the skin layers as compared to marketed FLB gel. Results also revealed that FLB SLN gel showed initial 3.12 ± 0.65% drug permeation in the first hour followed by drug permeation in a sustained manner up to 12.10 ± 2.65% at the end of 24 h. FLB marketed gel showed > 3.00% drug permeation in the receiver compartment after 16 h of the skin permeation study (Fig. 5A). Our results revealed that the FLB SLN gel showed about 0.4 ± 0.001% drug deposition in the dermal layer of the skin which was significantly (p < 0.05) higher (about fourfold increase) compared to marketed FLB gel which showed less than 0.1% drug deposition. Data also showed that significantly (p < 0.05) higher amount of drug (1.1 ± 0.02%) was deposited in the epidermis from marketed FLB gel compared to FLB SLN gel which showed about 0.4 ± 0.001% drug deposition (Fig. 5B).

Fig. 5.

A In vitro permeation test (IVPT) showing significant increase in percent drug permeation in case of 0.5% w/v FLB SLN formulation as compared to 5% w/w FLB marketed gel formulation at the end of 24 h. B Skin deposition study showing significant increase in drug deposition in dermal layer in case of 0.5% w/v FLB SLN formulation as compared to 5% w/w FLB marketed gel formulation at the end of 24 h

Anti‑inflammatory Effect of FLB SLN In Vivo

The results of anti-inflammatory study depicted that 0.5% w/v FLB SLN gel demonstrated equal percent edema inhibition as 5% w/w marketed FLB gel. Data showed that FLB SLN gel inhibited about 37.67 ± 6.77% paw edema in the first hour, whereas marketed FLB gel showed 41.55 ± 5.63% edema inhibition which was almost equivalent to FLB SLN gel. Results also revealed that at the end of 24 h, paw edema was reduced to about 62% in both FLB SLN gel and marketed FLB gel formulations (Fig. 6).

Fig. 6.

In vivo anti-inflammatory study showing equivalent edema inhibition effect after application of 0.5% w/v FLB SLN gel and 5% w/w marketed FLB gel

Stability Studies

Results from stability study revealed that FLB SLN gel did not show phase separation or sedimentation behavior at room temperature and 2–4°C after 1 month. There was no significant difference found in particle size, polydispersity index, and entrapment efficiency after 1-month storage at room temperature and 2–4°C. Moreover, the drug content in the formulation was found to be > 98% after a month at room temperature and 2–4°C.

Discussion

There are various studies reporting the use of HME equipment having three mixing zones for the formulation of lipid nanoparticles. Also, the use of HME along with HPH technique for fenofibrate and HME ultrasonication for lidocaine has been mentioned [21, 22]. Recently, we developed another method for the preparation of IBU SLN by solely using HME equipment which has only one mixing zone [23]. SLN formulation solely using HME has not been applied to other drugs except IBU. Therefore, the purpose of the present study was to formulate FLB SLN solely using HME technique and evaluate the SLN formulation in inflammation animal model. It is our hypothesis that a SLN formulation of FLB by HME technology will improve its permeation into skin layers and will have superior anti-inflammatory effect. Results of the current study demonstrated that FLB SLN can be formulated in a single-step HME process with significantly high permeation in dermal layer and superior anti-inflammatory activity compared to marketed gel formulation.

Pre-formulation studies showed that screw speed and temperature in the extruder has significant effect on the quality of the final product. Results also revealed that concentration of drug, lipid, and surfactant significantly affected the particle size of PI and entrapment efficiency of the final formulation. Similar observations were seen in our previous studies on IBU SLN gel developed using HME [23]. Patil et al. and Bhagurkar et al. have also demonstrated in their studies on formulation of lipid nanoparticles, where they showed particle size and entrapment efficiency of SLNs are affected by concentration of drug, lipid, and surfactant [20, 21]. Factors to optimize for stable formulation include the following: first, screw speed (100 rpm for first run followed by 800 rpm for the second) which leads to particles of about 175 ± 20 nm. Second, the screw configuration contributed to the stable formulation and was optimized for Do/Di of 1.21. Third, the temperature was optimized to process the formulation to achieve the lower particle size. Even though temperature is an important component for obtaining desirable particle size, other factors contributed to the melting of the API. Thus, the choice of suitable lipids with solubility enhancing capability is very important. Compritol 888 ATO is known not only to hold the API but also to help in its solubilization along with adequate temperature [43]. Various studies have reported the use of Compritol 888 ATO as their lipid choice including Hassan et al. who developed acyclovir SLNs for enhancing oral bioavailability of acyclovir and Yong-Tai et al. who studied transdermal delivery of aconitine SLNs with enhanced permeability of aconitine [44, 45]. Moreover, the choice of right surfactants, Kolliphor EL (HLB 14) and Tween 80 (HLB 15), helped in enhancing the solubility of FLB and preventing the oil–water phase separation in the SLN formulation [46]. SEM images showed that HME process could produce the FLB SLN spherical particles with particle size < 250 nm which indicates that HME can be applied to deliver variety of thermostable API by manufacturing their SLN formulation. Rheological studies showed that both FLB SLN and marketed SLN gel showed decrease in viscosity on increase in shear rate which indicates that both the formulations showed shear thinning property. Inverse relation between shear rate and viscosity has also been reported by Khan et al. in their study on argon oil and simvastatin combination-loaded SLN hydrogel formulation [47]. Spreadability and rheological properties of a semisolid formulation are inter-dependent. Spreadability is inversely proportional to the material’s yield stress, the minimum shear stress necessary to initiate the flow [48]. Our spreadability results revealed that (0.5% w/v) FLB-SLN-G showed significantly less stiffness and peak force as compared to control marketed FLB (5% w/w) formulation which indicates that our HME-based SLN formulation was easy to spread on the skin with significantly less resistance to apply. This suggested that higher the viscosity, lower the force required to spread the semisolid formulation on the skin [35]. Spreadability also affects the drug transport across the skin as seen in our in vitro permeation testing (IVPT) study which showed significantly higher FLB deposition in dermal layer of skin in case of FLB SLN formulation, whereas the control 5% w/w FLB showed no drug permeation in the dermal layer of the skin. Our stability data demonstrated that FLB SLN gel did not show phase separation and drug degradation at room temperature and 2–4°C after a month which indicates that the formulation is stable. Enhanced solubility of FLB in an ester of mono-di and triglycerides lipid along with right combination of high HLB O/W surfactants and gelling agent might have led to stabilization of the FLB SLN formulation [49]. DSC thermograms demonstrated that FLB SLN gel showed slight increase in the drug meting point endotherm peak which indicates that drug and lipid interaction might have to lead to conversion of crystalline form a drug to the amorphous form. Similar results were obtained by numerous researchers including Mancini et al. who studied etomidate and ibuprofen combination SLN gel thermal properties and found increase in melting point of a drug after incorporation in SLNs [50]. In vitro skin deposition data revealed that FLB SLN gel showed less drug deposition in epidermis with significantly (p < 0.05) enhanced deposition in dermal layer of the skin compared to marketed gel formulation which could be because of small SLN particles and high surface area. A study by Pham et al. who developed IBU-loaded SLN hydrogel showed significantly high flux of IBU from IBU SLN gel as compared to marketed product Nurofen (5% IBU) (as positive control) and IBU suspension gel (as a negative control) [51]. Mancini et al. also studied ibuprofen skin permeation from etofenamate and ibuprofen combination SLN gel in in vitro skin permeation studies. Their results revealed that unlike marketed gel formulations Ozonol (5% IBU) (control), SLN gel formulation showed significantly higher skin permeation in in vitro studies [50]. Anti-inflammatory study demonstrated that both 0.5% w/v FLB SLN gel and 5% w/w marketed SLN gel (control) formulation inhibited the paw edema to 62% which indicates that even though FLB SLN gel strength was 10 times lower than that of marketed gel, it could reduce the carrageenan-induced rat paw edema to 62% like marketed gel formulation. Jain et al. studied formulation of SLN gel for transdermal delivery of FLB and evaluated the formulations in in vivo rat paw edema study. Their results showed that FLB SLN gel exhibited significant reduction in rat paw edema as compared to FLB suspension (control) [52]. Similar observations are reported in another in vivo study by Pham et al., where IBU SLN gel showed higher anti-inflammatory activity on inflamed rat paw as compared to marketed product Nurofen (5% IBU) [51]. Collectively, FLB SLN gel was successfully formulated solely using HME technique and skin deposition and permeation was significantly enhanced with superior anti-inflammatory effect as compared to marketed FLB formulation.

Conclusion

FLB SLN using HME method was successfully formulated with particle sizes below 250 nm. The lipid of choice, Compritol 888 ATO and the surfactants used (Tween 80 and Kolliphor EL) helped in the solubilization of FLB leading to enhanced entrapment of FLB in SLN formulation. Skin deposition studies showed enhanced FLB deposition of 0.5% w/v FLB gel in dermal layer as compared to 5% w/v FLB marketed product. Anti-inflammatory study revealed that 0.5% w/w FLB gel showed similar effect like 5% w/w FLB marketed gel formulation.