Design And Development of Sublingual Films Incorporating R-HCL And Vitamin D-Loaded NLCS

Osteoporosis is a chronic bone condition marked by a progressive reduction in bone mineral density (BMD) and the deterioration of bone microarchitecture, which significantly raises the risk of fractures. It poses a serious public health concern, especially among older adults. In the United States, about 10.3% of individuals over the age of 50 are diagnosed with osteoporosis. The condition becomes increasingly prevalent with age and shows a notable gender disparity approximately 77.1% of women over 80 years old are affected, compared to 46.3% of men. This difference is largely attributed to hormonal changes, particularly the decline in estrogen levels following menopause, which accelerates the process of bone breakdown. Bone remodeling is a continuous and dynamic process involving the coordinated actions of two types of cells: osteoclasts, which break down bone, and osteoblasts, which build new bone. When this equilibrium is disturbed—due to factors such as aging, hormonal imbalances, or long-term use of medications like glucocorticoids—bone mass decreases, making bones more brittle and susceptible to fractures. Estrogen plays a critical role in bone metabolism by modulating osteoclast activity through the RANK/RANKL/OPG signaling pathway. A deficiency in estrogen, commonly seen in postmenopausal women, leads to heightened osteoclast activity and subsequent bone loss. Osteoporosis is typically diagnosed using a bone mineral density scan, most often via dual-energy X-ray absorptiometry (DXA). A T-score of -2.5 or lower is indicative of osteoporosis. Management of the condition involves a combination of drug therapies and lifestyle modifications aimed at reducing the likelihood of fractures. Although estrogen replacement therapy (ERT) has traditionally been a central treatment option, its potential health risks have prompted the exploration of safer alternatives, such as selective estrogen receptor modulators (SERMs), with Raloxifene hydrochloride (R-HCl) being one of the most widely studied agents. Recently, advancements in nanotechnology have led to the development of novel drug delivery systems, including Nanostructured Lipid Carriers (NLCs), which have shown promise in improving the bioavailability and therapeutic potential of R-HCl. These innovative approaches may offer more efficient and safer treatment options for managing postmenopausal osteoporosis, minimizing systemic side effects while enhancing clinical outcomes. ^[1-3]

Materials and Equipment’s

Table 2: List of various equipment items employed during the research work

R-HCl analysis via UV spectrophotometric method

Standard plot of R-HCl in various solvents: UV spectroscopy using methanol, chloroform:methanol (1:1), and PBS pH 7.4 quantified drug concentration and determined extinction coefficients during microemulsion development and NLC characterization through standard plots.

Standard plot in methanol: R-HCl was dissolved in methanol to prepare a 100 µg/ml stock, then diluted (1–10 µg/ml). Absorbance at 284 nm was measured, and specific absorbance calculated using the trendline equation.

Standard plot in chloroform: methanol (1:1):   R-HCl was dissolved in chloroform:methanol (1:1) to prepare a 100 µg/ml stock, diluted (1–10 µg/ml), analyzed at 297.5 nm, and specific absorbance calculated using the trendline equation.

Standard plot in PBS pH 7.4: R-HCl was dissolved in PBS (pH 7.4) to prepare a 100 µg/ml stock, diluted (1–10 µg/ml), analyzed at 289 nm, and specific absorbance calculated using the trendline equation. ^[4-7]

Validation of the analytical method

Linearity: Linearity was assessed for R-HCl (1–10 µg/ml) in methanol, chloroform:methanol (1:1), and PBS using six repeated spectrophotometric analyses.

Accuracy: Accuracy of R-HCl in methanol was evaluated by comparing experimental and nominal concentrations, following US-FDA (2018) ±15% acceptance criteria.

Precision: Precision was evaluated by analyzing 10 µg/ml R-HCl in different solvents (n=6), ensuring results met US-FDA (2018) ±15% criteria.

Inter and intra-day variability: Inter- and intra-day variability of R-HCl in methanol was analyzed at set intervals, assessing accuracy and precision per US-FDA (2018) guidelines (n=3).

Vit D Analysis via UV Spectrophotometric Method 

Standard plot of Vit D in various solvents: A UV spectroscopy method using various solvents was developed to quantify Vitamin D during microemulsion and NLC characterization, determining extinction coefficients from standard plots prepared in triplicate.

Standard plot in methanol: Vitamin D was dissolved in methanol to create a 100 µg/ml stock, diluted (1–20 µg/ml), analyzed at 265 nm, and specific absorbance calculated using the trendline equation.

Standard plot in chloroform: methanol (1:1): Vitamin D was dissolved in chloroform: methanol (1:1) to prepare a 100 µg/ml stock, diluted (1–20 µg/ml), analyzed at 265 nm, and specific absorbance determined via trendline equation.

Standard plot in PBS pH 7.4: Vitamin D was dissolved in PBS (pH 7.4) to prepare a 100 µg/ml stock, diluted (1–20 µg/ml), analyzed at 265 nm, and specific absorbance calculated using the trendline equation.

Validation of the Analytical Method 

Linearity:  Linearity of Vitamin D (1–20 µg/ml) was evaluated in methanol, chloroform: methanol (1:1), and PBS (pH 7.4) across six replicates.

Accuracy: Accuracy of Vitamin D in methanol was evaluated by comparing experimental and nominal values (1–20 µg/ml) per US-FDA (2018) ±15% criteria.

Precision: Precision of 20 µg/ml Vitamin D in different solvents (n=6) was analyzed, ensuring results met US-FDA (2018) ±15% acceptance criteria.

Inter and intra-day variability: Inter- and intra-day variability of Vitamin D in methanol was assessed at set intervals (n=3) following US-FDA (2018) precision and accuracy guidelines.

Solubility Studies

Solubility in oils and surfactants: This study evaluated R-HCl solubility in various oils and surfactants. After 24-hour mixing and centrifugation, supernatants were diluted and analyzed spectrophotometrically at 284 nm.

Solubility in solid lipids: Solid lipid selection was based on R-HCl solubility observed visually in molten lipids—Compritol 888 ATO®, stearic acid, and GMS—heated in a water bath.

Construction of ternary phase diagram 

NLC formulation began with a pseudo-ternary phase diagram using various surfactant ratios. Lipid phases were heated, mixed, titrated with water, stirred at 80°C, and visually assessed for transparency. The identical methodology was replicated subsequent to the introduction of lipids in summary.

• Pre-weighed test tubes were utilised to prepare mixtures of the lipid phase and surfactant phase at various ratios (w/w) ranging from 10:0 to 0:10.
• The lipid phase was melted by heating above its melting point, then combined with a pre-heated surfactant phase under gentle stirring to form a uniform mixture. This was gradually titrated with distilled water and stirred at 60°C until equilibrium. Transparency was visually inspected, and TriDraw software was used to construct a pseudo-ternary diagram to identify optimal microemulsion regions.

Characterization of microemulsion:  Freeze-thaw cycling involves repeatedly freezing and thawing water. The selected microemulsion series underwent freeze-thaw cycles between 4°C and 40°C for 24 hours each, over a one-week period. This process aimed to observe any physical instabilities, such as phase separation or precipitation, to assess the stability of the microemulsion formulations under varying temperature conditions. _[8-10]

Formulation Studies

Preparation of microemulsion: An o/w microemulsion containing R-HCl and Vit D was prepared through a process involving the melting of solid lipid, the addition of liquid lipid, and the subsequent solubilization of the drug in the resulting mixture to form the lipid phase. The aqueous phase consisted of a combination of surfactant, co-surfactant, and water. The two phases were subjected to a temperature higher than the melting point of the solid lipid and subsequently blended to yield a heated microemulsion.

Preparation of NLCS: NLCs were prepared using the micro-emulsification technique with Compritol® 888 ATO as the solid lipid and oleic acid as the liquid lipid. Drug (30 mg R-HCl and 200 µL Vit D) was added to the melted lipid phase. The aqueous phase, containing Tween 80 and Capryol 90, was heated and mixed with the lipid phase. The microemulsion was stirred at 90°C, then cooled in chilled water and homogenized. Blank NLCs were prepared similarly, without drug addiction. Formulations were stored below 25°C.

Note* Turbid microemulsion was obtained and so NLCs were not prepared.

Preparation of Sublingual Film

Sublingual films improve bioavailability by bypassing first-pass metabolism, utilizing water-soluble polymers like HPMC, which act as emulsifiers, stabilizers, and film formers. HPMC films are transparent, odorless, stable, biodegradable, and non-toxic. To prepare fast-dissolving films, varying amounts of HPMC K4M were immersed in phosphate-buffered saline, glycerol added, and agitated at 60°C. The mixture was dissolved in methanol and water, followed by stirring and the addition of hot water to adjust viscosity. The process was optimized by varying film compositions and preparation parameters._[11-12]

Figure 1: Solvent casting in the petri-plate

Formulations F6 and F7 were selected for their solution uniformity. F6 was prepared using the solvent evaporation technique with HPMC and glycerol, stirred for 2 hours, and cast into a 10?cm petri dish. F7 was developed using 2% w/v HPMC K4M and 1% w/v glycerol, stirred for 2 hours with gradual hot water addition. The solution was spread using a Wasag Model 288 Applicator (gap: 900?µm) and dried at room temperature for 24 hours using a TDP/ODF film forming system.

Figure 2: Solvent Casting on Film Former Machine

The film was appropriately trimmed to obtain films with an area of 1 square centimeter each.  Subsequently, these films were employed to ascertain additional characteristics.

Figure 3: Sublingual film of area 1cm²

Characterization of Selected NLC Formulation 

Particle size, Polydispersity index (PDI) and Zeta potential: One-milliliter aliquots were extracted from the non-ionic liquid crystal dispersion and subsequently diluted with 20 millilitres of distilled water. The mixture was then stirred for three minutes. Subsequently, a diluted dispersion of 1 mL was utilised for particle size analysis through dynamic light scattering with a Malvern Zetasizer.

Total drug content (TDC): To determine the TDC of NLC dispersion containing R-HCl and Vit D, 1?ml was disrupted with chloroform:methanol (1:1), diluted to 10?ml, sonicated for 1?hour, and filtered.

Entrapment efficiency (EE): The efficiency of the R-HCl and Vit D dispersion that was prepared was assessed through the use of a dialysis membrane with a pore size of 2.4 nm and a molecular weight cutoff range of 12–14 Kda. The spectrophotometric analysis of drug release into the receptor or release medium was conducted at 284 nm and 265 nm for R-HCl and Vit D respectively using suitable dilutions.

The calculation of entrapment efficiency was performed using equation (1).

Differential scanning calorimetry (DSC): Differential Scanning Calorimetry (DSC) was performed using DSC 60, analyzing samples from 25–300?°C at 10?°C/min to record thermograms via specialized software after proper sealing.

Fourier transform infrared spectroscopy (FTIR): FTIR analysis using Shimadzu FTIR 8400S identified drug-lipid interactions. Samples were pelletized with KBr, and spectra were recorded from 4000–400?cm?¹ to study individual and combined formulation components.

Characterization of Film

pH: Surface pH was measured after swelling the film in phosphate buffer (pH 6.8) for 2 hours using a pH meter.

Total drug content: Drug content uniformity was evaluated by dissolving the film in buffer, filtering, and analyzing R-HCl and Vit D spectrophotometrically.

Thickness: The measurement of film thickness was conducted at three distinct locations using a digital vernier calliper. Subsequently, the arithmetic average was documented.

Weight variation: Weight variation was assessed by randomly selecting and individually weighing three films from each batch using a digital balance.

Tensile strength: Tensile strength was measured using an Instron tester by stretching films between clamps 2 cm apart until breakage, and calculating the maximum force endured using the standard tensile strength formula.

Disintegration time: As per CDER, orally disintegrating tablets must disintegrate within 30 seconds, a standard also applied to fast-dissolving films, typically disintegrating within 5–30 seconds using pharmacopoeial apparatus.

RESULTS AND DISCUSSION

UV-Vis Analysis and Preformulation of R-HCl: Standard plots for R-HCl were generated in three different solvents: methanol, chloroform: methanol (1:1), and phosphate-buffered saline (PBS): methanol with a pH of 7.4. The resulting standard plots were then analysed. Table 5 provides information on the linearity range, and R2 for the standard plots of R-HCl.

Figure 4: Calibration curve of R-HCl in methanol with concentration range (1-10 µg/ml)

Figure 5: Calibration curve of R-HCl in chloroform: methanol (1:1) with concentration range (1-10µg/ml)

Figure 6: Calibration curve of R-HCl in buffer (PBS 7.4): methanol (1:1) with concentration range (1-10µg/ml)

Validation of Analytical Method

Linearity: Solutions of varying concentrations (1-10 µg/mL) of R-HCl were prepared in respective solvents and their absorbance was measured (n=6). The results Figure 15, 16 and 17 showed a linear relationship between concentration and corresponding absorbance values upon repeated observations. 

Accuracy: Accuracy of the UV spectrophotometric method of analysis for R-HCl in the methanol was assessed. Accuracy of ±15% is considered to be satisfactory as per the US-FDA guidelines (2018), and the values obtained presently were found to fall within these limits, thus validating the test.

Precision: The UV spectrophotometric method of analysis for R-HCl was sufficiently precise as indicated by a small S.D ≤0.0137 in the repetitive determinations of the same concentration (10 µg/mL) of R-HCl in different solvents. The results were within the limits defined by US-FDA guidelines (2018) (≤15%).

Inter- and intra-day variability: Different concentrations of the drug were prepared in methanol and analysed at intervals of 0, 6, 24, 36,48 hours. The inter- and intra-day variability in precision and accuracy was found to be in accordance with US-FDA guidelines (2018) (≤15%) average inter and intraday variability was reported as shown in Table 6.

Table 7: Maximum wavelength and extinction of absorption coefficient of Vit D in different solvent systems in concentration range (1- 20 g/ml) 

Figure 7: Calibration curve of Vit D in methanol with concentration range (1-20 µg/ml)

Figure 8: Calibration curve of Vit D in chloroform: methanol (1:1) with concentration range (1-20 µg/ml)

Figure 9: Calibration curve of Vit D in buffer (PBS 7.4): Methanol with concentration range (1-20 µg/ml)

Validation of Analytical Method

Linearity: Solutions of varying concentrations (1-20 µg/mL) of Vit D were prepared in respective solvents and their absorbance was measured (n=6). The results Figure 18,19 and 20 showed a linear relationship between concentration and corresponding absorbance values upon repeated observations. 

Accuracy: Accuracy of the UV spectrophotometric method of analysis for Vit D in the methanol was assessed. Accuracy of (±15%) is considered to be satisfactory as per the US-FDA guidelines (2018), and the values obtained presently were found to fall within these limits, thus validating the test.

Precision: The UV spectrophotometric method of analysis for Vit D was sufficiently precise as indicated by a small S.D ≤0.0137 in the repetitive determinations of the same concentration (20 µg/mL) of Vit D in different solvents. The results were within the limits defined by US-FDA guidelines (2018) (≤15%).

Inter- and intra-day variability: Different concentrations of the drug were prepared in methanol and analysed at intervals of 0, 6, 24, 36,48 hours. The inter- and intra-day variability in precision and accuracy was found to be in accordance with US-FDA guidelines (2018) (≤15%) average inter and intraday variability was reported as shown in Table 8.

Solubility in solid lipids:

Figure 10: Solubility studies of R-HCl and Vit. D in solid lipids

Solubility in liquid lipid

Figure 11: Solubility studies of R-HCl and Vit. D in liquid lipids

Figure 12: Solubility of R-HCl and Vit. D in different surfactants

Screening of co- surfactant: surfactant ratio: The solubility of R-HCl and Vit D was determined in various proportions of co- surfactant: surfactant. Results are illustrated in Figure 12. The ratios were taken and given below in Table 12.

Figure 13: Solubility of R-HCl and Vit. D in various proportion of co- surfactant: surfactant

Pseudo-ternary phase diagram was constructed to identify the microemulsion region and to optimize the concentrations of the selected vehicles (Compritol®888 ATO: Oleic acid, Tween 80: Capryol 90, water) for the development of NLCs.

Figure 14: Pseudo Ternary Phase Diagram

The selected composition yielded a significant microemulsion region. Higher concentrations of surfactant were required to produce fine and stable emulsions was due to the fact that smaller the desired globule size, greater the surface area and hence, greater the amount of surfactant required to stabilize the oil globules.

Preparation of R-HCl and Vit D loaded NLCs by micro emulsification method:

• Micro-emulsification method followed by high-speed homogenization was used.
• Compritol®888 ATO (8%) was chosen for its stability and ability to form small particles.
• Tween 80 (11%), Capryol 90 (44%), and water were heated together to 82–85°C.
• Compritol®888 ATO was melted separately at the same temperature.
• Oleic acid (2%), R-HCl, and Vitamin D were added to the melted lipid.
• The aqueous mixture was gradually added to the hot lipid mix under magnetic stirring.
• A clear microemulsion was formed.
• This hot emulsion was poured into cold water (2–5°C) and homogenized at 13,000 rpm for 20 minutes.
• The final dispersion was refrigerated before characterization and evaluation.

Figure 15: Steps for preparation of NLCs

Characterization of R-HCl and Vit D Loaded NLCs

Particle size and Polydispersity index (PDI): Particle size analysis was carried out using Malvern's Zetasizer by differential light scattering technique. Figure 16(a) shows the particle size distribution of the prepared formulation. Mean particle size of NLCs was 81.3 nm and PDI was found to be 0.192 which indicated homogeneity of the prepared formulation (less than 0.5).

Zeta potential: Zeta potential is an indicative of stability of colloidal dispersion. Aggregation of the particles is avoided by inducing/acquiring surface charge on the particles. Surfactant and/or coating material adsorption is also responsible for inducing surface charge. Potential of the NLCs was measured using Malvern Zetasizer 90S (Malvern Instruments Ltd.). Measurements were done at 25°C and the electric field strength was 23.2 Mv. The observed zeta potential of the tested formulation exhibited a tendency towards neutrality, with a recorded value of around -0.69 mV as shown in Figure 16(b).

Total drug content (TDC): TDC of R-HCl and Vit D in NLC dispersion was found to be 85.76 ± 0.24 % and 80.24 ± 0.45% (n=6) which confirmed minimum drug loss during formulation. High values of TDC are indicative of the insignificant losses incurred during the processes of preparation of R-HCl and Vit D NLCs by micro-emulsification technique.

Entrapment efficiency (EE): EE of the NLCs formulation was determined by dialysis bag method was found to be 91.41 ± 0.28 % and 79.56 ± 0.42 % respectively for R-HCl and Vit D (n=6) Determining EE is of prime importance in NLCs it influences the release characteristics of drug molecule. High EE values indicate the suitability of the components and their relative proportion in the formulated NLCs which was similar to the study reported by Murthy et al., 2020.

Figure 16: Depicts the characterisation of NLCs of R-HCl and Vit D

(a) Particle size and PDI (b) Zeta potential

DSC thermogram: DSC is a thermoanalytical technique that measures the difference in the amount of heat required to maintain the sample and reference at same temperature as a function of temperature and time. DSC measures the heat flow when the sample is heated or cooled. DSC thermogram of R-HCl, Compritol®888ATO and Vit D showed endothermic peaks at 266°C,72.93°C and 167.99°C corresponding to their melting points as depicted in Figure 17(a), (b) and (c) respectively. DSC thermogram of physical mixture is shown in Figure 17(d) observed with sharp peaks at 266.15° C,72.93°C and 167.99°C representing melting points of R-HCl, Compritol®888 ATO and Vit D respectively. Thermogram of NLCs Figure 17(e) showed an endothermic peak at 72.94°C, representing the melting point of Compritol®888 ATO but the absence of endothermic peak within the melting range of R-HCl and Vit D indicates either solubilization or conversion of R-HCl and Vit D from crystalline to amorphous form in the solid and liquid matrix. Similarly, Murthy et al. prepared R-HCl NLCs and observed peak broadening for glyceryl behenate in the melt dispersion which showed the reduction in crystallinity of glyceryl behenate.

Figure 17: DSC thermogram of (a) R-HCl (b) Compritol®888 ATO (c) Vit D

(d) Physical mixture (e) NLCs loaded with R-HCl and Vit D

FTIR spectra: FTIR spectroscopy is used to investigate the interactions between lipid, drug and other excipients. The FTIR spectrum of R-HCl, Compritol®888 ATO, Vit D, Physical mixture, NLCs, HPMC, Film mixture and sublingual film obtained are given in Figure 18. The major peaks observed in the IR spectra of Compritol®888 ATO, R-HCl, Vit D and HPMC are given in Table (13-16) respectively.

Table 13: Characteristic peaks in FTIR spectra of Compritol® 888 ATO

Table 14: Characteristic peaks in FTIR spectra of R-HCl

Table 15: Characteristic peaks in FTIR spectra of Vit D

Table 16: Characteristic peaks in FTIR spectra of HPMC

The characteristic peaks of R-HCl appeared at 3143.17, 1642.13, 1595.97 1465.90 and 950 cm⁻¹ Figure 18 (A) which were associated with – amine stretching, –C=O stretching -C O-C stretching, S-benzothiophene group and benzene ring respectively. Compritol®888 ATO indicated the –OH stretching at 3420.6 cm⁻¹ and aromatic –CH stretching at 2919.00 cm⁻¹ Figure 18(b). Vit D indicated the –CH3 stretching at 2905.01 cm−1, ester stretching at 1895.07 cm⁻¹, aromatic cyclohexene stretch at 1639.30 and carbonyl stretching at 1082.00 Figure 18(C). The peaks of R-HCl, Compritol and Vit D were retained in case of their physical mixture with a slight shift indicating no chemical interaction between them Figure 18(D). On the other hand, the characteristic peak of R-HCl and Vit D was lacking and –OH stretching of Compritol®888 ATO shifted to 3438.49 cm⁻¹ in case of NLCs Figure 18(E). This confirmed the successful incorporation of R-HCl and Vit D into the NLCs. Annu et al. have developed the chitosan nanoparticle. The result reported in the FT IR study stated that the characteristic peaks of the drug in nanoparticles were absent compared to drug alone spectra, and it could be the possible reason for entrapment of the drug within the nano formulation. HPMC indicated –OH stretching at 3432.49 cm⁻¹, -OH bending at 1377.25 cm−1, C-H stretch at 2922.27 cm⁻¹ and C-O stretch at 1064.88 cm⁻¹ Figure 18(F). The peaks of R-HCl, Compritol, Vit D and HPMC were retained in case of the physical mixture of components of film with a slight shift indicating no chemical interaction between them Figure 18(G). On the other hand, the characteristic peak of R-HCl, Vit D and Compritol®888 ATO were lacking and –OH stretching of HPMC shifted to 3434 cm⁻¹ in case of film indicating entrapment of the NLCs within the sublingual film Figure 18(H).

Figure 18: Infrared spectra of (A) R-HCl, (B) Compritol®888 ATO, (C) Vit D, (D) Physical mixture of drug, lipid and Vit D, (E) NLCs, (F) HPMC, (G) Physical mixture of drug, lipid, Vit D and HPMC, (H) Sublingual Film

Characterization of Sublingual Film

pH: The pH value of the film that was prepared was determined to be 6.8 ± 0.2, close to neutral. The findings indicate that the surface pH of the film falls within the typical pH range of healthy human saliva which is between (6.3 - 7.3). At this pH, the films are less likely to irritate the mucosal lining of the oral cavity, and therefore, they should be fairly comfortable.

Total drug content: The total drug content of film was calculated for film of area 1cm2. Total drug content of the film (n=3) was found to be 89.43 ± 0.59 % and 84.67 ± 0.32% for R-HCl and Vit D respectively.

Thickness: The measurement of the film's thickness yielded a value of 100µm. In a study conducted by Najafi et al., a mucoadhesive buccal film of glipalamide was formulated, and the thickness of the film was reported to be 90 ± 1.6 µm.

Weight variation: The weight of the film with a surface area of 1 cm2 was determined to be 52.8 ± 0.55 mg.

Tensile strength: The film's tensile strength was determined to be 5.453 N/mm2 (Figure 19). Salehi and colleagues conducted a study in which they formulated a sublingual film containing rizatriptan benzoate. The researchers reported a tensile strength ranging from 2.33 ± 0.37 to 12.01 ± 0.049 MPa, indicating favourable tensile strength properties of the film. According to Salehi and Boddohi's (2017) findings, an increase in the concentration of HPMC K4M and glycerol resulted in an increase in tensile strength

Figure 19: True Stress Vs True Straing