Development And Evaluation of Meloxicam Nanostructured Lipid Carriers for Transdermal Delivery

Osteoporosis is a disorder when the bones become fragile and feeble. Low consumption of calcium raises the risk of fractures, diminished bone density and also cause early bone loss. ⁽¹⁾ Postmenopausal osteoporosis (PMO) is a widespread skeletal condition that increases the risk of fracture due to decreasing bone density or declining bone quality. The female hormone, estrogen plays an important role in maintaining strength of bone. Around menopause, which occurs on average at the age of 50 years, there will be drop in estrogen level in body, leading to increased chances of bone loss. ⁽²⁾ Currently bisphosphonates like Alendronate, Risendronate, Ibandronate are used. However, recent researches showed that upon treatment discontinuation there is a major risk of spinal fractures. ⁽¹⁾ As an alternative treatment for osteoporosis, non-steroidal anti-inflammatory drugs (NSAIDs) are used, along with analgesic and anti-inflammatory effects it has a small protective effect on bones especially in enhancing bone mineral density (BMD). ⁽³⁾ Meloxicam is an oxicam derivative and belongs to enolic acid group of drugs (NSAIDs). It is classified under biopharmaceutics classification system (BCS) II. The partition coefficient (logP) of drug is app = 0.1 in n-octanol/buffer Ph 7.4 with pKa values of 1.1 and 4.2. For oral administration meloxicam tablets are available with a dose of 7.5 mg or 15 mg. However, oral capsules with a single dose of 30 mg shows absolute bioavailability of 89%. ⁽⁴⁾ Administering meloxicam orally can lead to adverse effects like abdominal discomfort, gastric irritations, and indigestion. Prolonged usage of such medication can even result in bleeding and formation of ulcers. In order to overcome these problems transdermal drug administration is a viable solution.  The transdermal approach to drug administration offers several advantages, including its non- invasive nature, which circumvents the need for invasive procedures. This method also side steps the gastrointestinal side effects, making it a preferred choice for addressing both pain and inflammatory conditions. Furthermore, it contributes to a lower occurrence of systemic adverse effects compared to other routes of administration. Various carrier approaches utilized in transdermal drug delivery include solid lipid nanoparticles, polymeric nanoparticles, microemulsions, liposomes, niosomes, nanostructured lipid carriers (NLCs) etc. These approaches offer advantages such as improved targeting of drugs to the skin, better tolerance at the application site, effective incorporation of lipophilic drugs, increased control over drug release rates, and efficient integration of lipophilic medications. ⁽⁵⁾ Biocompatible lipids (both solid and lipid) and emulsifiers constitute nanostructured lipid carriers, which are second generation lipid nanoparticles having sizes that vary from 1-1000 nm. Since nanosized NLC adheres to skin surface and produce an occlusive effect NLC is thought to be an effective tactic for increasing the permeation of drug through the skin. They offer lower toxicity, better bioavailability, biocompatibility, controlled-release, high drug loading capacity and high drug stability. ⁽⁵⁾ By considering all these points, we aimed to develop transdermal delivery system containing NLC loaded meloxicam, this can improve drug’s therapeutic potential.

MATERIALS AND METHODS

MATERIALS

Meloxicam was obtained as a gift sample from Strides Arcolab Bangalore and other chemicals like beeswax, carnauba wax, glyceryl monostearate, oleic acid, poloxamer 188 were purchased from sigma Aldrich Mumbai.

Methods of preparation of nanostructured lipid carriers (NLC)

NLC was prepared by using hot homogenization technique by varying the concentration of lipids and surfactant. Homogenization is carried out by using polytron homogenizer. ⁽⁶⁾

Utilization of experimental design

To optimize both the formulation and processing parameters on the quality criteria via Box Behnken design using Design expert trial version ^{(7, 8)} the factors such as concentration of bee wax, carnauba wax and surfactant was used for the response’s percentage practical yield and drug content. (Table 1)

Evaluation of optimum formulation

Particle size analysis:

The maximum size of nanoparticles should have been of 1000nm. The dynamic light scattering principle was utilized for the particle size analysis from <1nm to >µm. The mean particle size, distribution, and zeta potential were determined by utilizing Horiba SZ-100 nanoparticle dynamic light scattering system. ⁽⁹⁾

Zeta potential:

Nanoparticles require the zeta potential to accurately estimate their surface charge. It is necessary for the physical stability of NLC. HORIBA Scientific measured the zeta potential of the SZ-100. Electrophoretic light scattering at 25°C was utilized to measure particles in triplicate at 90° angle after the samples were carefully diluted with double distilled water. ⁽⁹⁾

Scanning electron microscopy (SEM):

To examine optimized formulation’s surface morphology Tescan vega3 scanning electron microscopy was utilized. Gold was utilized for scattering NLC dispersion on a container for samples. The data was collected during purge of argon at a voltage of 10 kV. ⁽¹²⁾

Table No. 01: Experimental Runs

Differential scanning colorimetry (DSC):

DSC investigation confirmed the thermal behavior of pure drugs with lipids. Aluminum pans which are non-hermetically sealed were filled with around 5mg of the sample and drug equivalent formulations were weighed properly. The samples were heated from 0°C - 350°C at a rate of 10°C per minute. During the measurement, nitrogen was continuously purged at a rate of 40ml/min, and thermograms obtained from DSC were measured using the Japanese Shimadzu DSC-60. ⁽¹³⁾

Drug content:

After weighing NLC equal to 20 mg of meloxicam was transferred to 10 ml of the volumetric flask, and dissolved in to 10 ml of DMSO (stock-1), from stock-1 preparation 1ml was diluted to 10 ml DMSO (stock 2) and again 1ml of stock 2 preparation was diluted into 10ml using DMSO (stock3). The UV spectrophotometry was used to detect absorbance at 375 nm. ⁽¹¹⁾

In vitro release studies:

In vitro experiments were performed using the Franz diffusion cell assembly. In a diffusion cell assembly donor and receptor compartments were separated by a cellophane membrane which helps to hold 5mg of drug equivalent weight NLC. Donor compartment contains 1ml of pH 5.5 phosphate. Receptor compartment contains 50 ml of pH7.4 phosphate buffer and stir continuously using a magnetic stirrer at 35°C. To measure drug release rate from NLC, 1 mL of receptor fluid was withdrawn every hour. After appropriate dilution the spectrophotometric approach were used to measure drug concentration at λ max of 375nm. ⁽¹²⁾

Preparation of NLC loaded patch:

The solvent casting procedure was employed for preparing an NLC-loaded patch formulation. In brief, by using magnetic stirrer, HPMC E15(700 mg) was dispersed in 10 ml of water and stirred continuously until a uniform polymeric dispersion was achieved, along with adding 2 ml of polyvinyl alcohol(plasticizer). Finally, MX-NLC were introduced into the polymeric dispersion and mixed well to ensure uniform dispersion. The resulting dispersion was then added to a clean, 57.02 cm2 surface area petri dish. After that, the formulation-containing dish was put into the solvent hood chamber for solvent's rate of evaporation. The dried MX-NLC was obtained once the solvent had completely evaporated. ⁽¹⁴⁾

Evaluation of drug loaded patches

Drug content:

A corresponding amount of 20mg of meloxicam (depending on theoretical drug concentration) was cut out of a 5 cm² NLC-incorporated patch region and it is placed into 10ml volumetric flask and 10ml of DMSO was used to dissolve it (stock-1). For (stock-2) DMSO was used to dilute 1ml of stock-1 preparation to 10ml and again DMSO was used to dilute 1ml of stock-2 preparation to 10ml for preparing (stock-3). UV spectrophotometry was used to detect the absorbance at 375 nm. ⁽¹⁴⁾

Thickness, folding endurance of patches and weight uniformity:

A screw gauge was used to measure thickness at five separate locations. For folding endurance, a patch strip was evenly cut and folded in a repeated manner at the same location until it broke. The average weight was computed by measuring the weight homogeneity from five randomly cut (1 cm by 1 cm) patches.

Moisture content:

Following a 24-hour period at room temperature and individual weight measurements, each created patch was placed in a desiccator with activated silica until a consistent weight was reached.

Moisture uptake:

The weighed patches were kept at room temperature and a relative humidity (RH) of 84% was maintained for 24 hours in a desiccator filled with a solution saturated with potassium chloride. The patches were weighed again after 24 hours, and the moisture uptake percentage below was calculated using formula given below ⁽¹⁴⁾.

Ex vivo permeation studies:

The study was conducted using wister rats that had been approved for use by the institutional animal ethical committee and assigned with ethical approval number (KCP/IAEC/PCOL/PCEU/144/APR2024). To conduct ex-vivo permeation studies modified Franz diffusion cell was used. Donor compartment contains 1ml of phosphate buffer (pH 5.5) and in receptor compartment 50ml of phosphate buffer (pH 7.4) was added. The temperature was maintained at 35°C while the receptor compartment was shaken at 100 rpm using a magnetic stirrer. By extracting 1 milliliter of receptor fluid every hour, the rate of drug release from NLC was calculated. The following formula was used to determine the drug flux (µg/h/cm2) at steady state and the permeability coefficient.

Jss = steady state flux (µg/h/cm²⁾

Q = Quantity of drug that passing through the membrane at a given moment (t)

A = Area of exposed membrane (cm²)

C0-Ci = Concentration of the drug on the donor and receptor side of the membrane respectively (cm/h) ^(15,16)

Study of anti-inflammatory effect:

The anti-inflammatory activity was studied via carrageenan induced rat paw edema method. For a week before starting the experiment, the animal was housed in an animal house, providing food and water without any restrictions. The rats were split into two groups, each with six rats. Prior to each experiment at an interval of 1 hour, 0.1ml of 1% carrageenan in saline was injected into the rat’s right plantar side paw to cause edema.  Paw volume will be measured immediately after carrageenan injection and at 1, 2, 3, and 4h interval using plethysmometer against the control group. 0.1ml of 1% carrageenan in saline was given to group 1, which served as a positive control. For group 2 0.1ml of 1% carrageenan in saline was given followed by NLC formulation was administered to rat paws plantar area.

Fourier transforms infrared spectrum (FTIR) studies:

FTIR studies was performed for the meloxicam pure drug, NLC formulation and NLC-meloxicam patch formulation. Analysis of samples was done by the potassium bromide pellet method in the IR region between 4000-400cm^-1 using Thermo- Nicolet 6700 which is an IR spectrophotometer.

Stability studies:

The NLC-incorporated patch formula was tested for three months at 25 ^O± 2^O C/60 % RH ± 5% and 40 ^O± 2^O C/75 % RH ± 5% in stability investigations of NLCs. At the halfway point of the trial period, the formulations, drug content and physical nature was examined.

RESULTS

Drug content and Percentage yield:  

F14 formulation has shown highest drug content and percentage yield of 95.09±0.012% and 92.01±0.017 respectively. This is because of having a high surfactant content in this composition. Hence this formulation is selected for further studies.

Table No. 02: Data of drug loading and percentage yield

3D Surface Plots:

Figure 01: 3D Surface plots

Contour plots:

Figure 02: Contour plot

Particle size and Zeta potential:

Figure 03: Particle size and Zeta potential

Scanning electronic microscopy:

Figure 04: SEM images of optimized formulation

Differential scanning calorimetry:

Figure 05: DSC of (A) Pure meloxicam, (B) Nanostructured lipid carriers of meloxicam

In-vitro drug release:

A comparative in-vitro drug release studies has been conducted between pure meloxicam and meloxicam loaded in NLC. A 24h study was conducted and at the end of 24h it has been found that NLC loaded with meloxicam has showed a drug release of 92.41±0.24, whereas pure meloxicam has showed a drug release of 59.71±0.26.

Figure 06: Graphical representation of comparative invitro drug release

Evaluation of patch

The pharmaceutical composition of the patch formulation drug content was determined to be 94.9±0.06, the thickness 0.110±0.08, the folding endurance 236.2±0.05, the weight variation 192±1.3, and the moisture content 1.5±009.

According to 24h release studies, drug's penetration rate from the NLC patch was 92.14±0.08%, in comparison with commercialized meloxicam patch which demonstrated a permeation rate of 90.15±0.007. NLC formulation demonstrated action for up to 24 hours, confirming the patch's extended activity. The commercial formulation, on the other hand, had higher action during the first eight but at 24 hours it showed less activity.

Table No. 05: Ex-vivo skin permeation tabular column

Figure 07: Ex-vivo skin permeation studies graphical representation

All observed ranges were within the stretching ranges hence there is no incompatibility between drug and excipients. Both NLC-MX formulation and NLC-MX patch shown the characteristic peaks.

Figure 08: FTIR spectrum of (a) meloxicam pure drug, (b) NLC- meloxicam optimized formulation, (c) NLC- meloxicam patch formulation

This test was conducted for 90 days at 25°C ± 2°C/ 60% ± 5% RH and 40°C ± 2°C/75% ± 5% RH on the ideal NLC-MX and NLC-MX loaded patch. The physical appearance result indicates that the attributes remain unchanged at the above-mentioned storage conditions. It demonstrates that more stable conditions are provided by 25°C ± 2°C RH than by 40°C ± 2°C RH.

Table No. 06: Stability studies

Anti-inflammatory studies:

By observing the data obtained, at the end of 24 hours meloxicam transdermal patches has successfully reduced the inflammation in mice.

Figure 09: Analysis of anti-inflammatory effect