Formulation, Evaluation and Quality Assessment of Antifungal Cubosomes Drug Delivery System

Fungal infections continue to pose a therapeutic challenge, particularly in patients with compromised immune systems. Although several antifungal agents are available, their clinical performance is often limited by poor aqueous solubility, erratic absorption, and systemic adverse effects. Posaconazole, a second-generation triazole antifungal agent, is widely used for the treatment of invasive fungal infections; however, its low solubility restricts its bioavailability and therapeutic efficiency⁽¹⁾.

In recent years, nanostructured lipid systems have gained considerable attention as carriers for poorly soluble drugs. Cubosomes are discrete nanovesicles that are bi-continuous cubic phase liquid crystals made of water and biodegradable lipid. They have exceptional biocompatibility and bio-adhesive qualities, as well as the capacity to solubilize and encapsulate hydrophilic, hydrophobic, and amphiphilic molecules^(2,3).

In addition to their skin penetration-enhancing qualities, cubosomes are thought to be an excellent option for skin transport due to their resemblance to human skin layers, which has led to an increase in interest in topical therapies. Along with their high interior surface, they also have small pores, which makes them perfect for controlled release, medication payloads, thermal stability, and skin-moisturizing capacity^(4,5).

Cubosomes generally has been incorporated for topical administration i.e. penetration through skin via stratum corneum for antifungal activity. Because it avoids the medication's first pass metabolism and minimizes toxicity and dose-related side effects, the topical route of drug delivery is more compliant than other routes. The process of administration is reversible. The majority of vesicular drugs are ethosomes rather than transferosomes. Due to their weak skin, delivery devices are less appropriate for percutaneous distribution,  permeability, vesicle disintegration, drug leakage, aggregation, and vesicle fusion. A novel kind of carrier system known as "cubosomes" can be used to solve issues. Based on these consideration the present study aims to formulate and evaluate a cubosomes based drug delivery system for antifungal therapy⁽⁶⁾.

MATERIALS:

Posaconazole was selected as model antifungal agent. Glyceryl Monooleate (GMO) was gifted by Mohini Organics Pvt. Ltd. Mumbai which was used by emulsifier. Poloxamer 407 or Pluronics F 127 was purchased from Vishal Chem Pvt. Ltd. which was used as polymeric stabilizer. Polyvinyl alcohol was used as emulsifier stabilizer and distilled water.

PREPARATION OF CUBOSOMES DISPERSION:

Cubosomes were prepared using the top-down method (Emulsification method). A precisely measured amount of glyceryl monooleate (GMO) and different ratios of pluronic F127 were combined and melted at 60˚C in a water bath until Pluronic F127 dissolves entirely in GMO. To the solution mentioned above  adding drug Posaconazole, thoroughly mix it in. Distilled water was gradually heated to 60˚C and added drop by drop in the proper amount to the resulting clear lipid solution by continuous stirring. Following the full addition of lipid phase, it was set aside for a day in order to equilibrium. A two-phase formation occurred system, and stirring upset it. The whole The system was homogenized at 1200 rpm. for one and half hours. The ready Dispersions were kept at room temperature in sealed glass vials. That is protected from the sun and later a evaluation was done. The outcome was a white opaque dispersion in the absence of any aggregates.

Table 1 Composition of  Cubosomes formulations

Note: Posaconazole was used in concentration of 0.35%w/w

EVALUATION OF CUBOSOMES:

Scanning electron microscopy (SEM)

The morphology of cubosomes was examined using scanning electron microscopy (SEM). The optimized posaconazole  cubosome dispersion's shape and surface morphology were examined by SEM (FEI, Tecnai 12, Eindhoven, Netherlands). Using tungsten filament as a source and the program Tecnai Imaging and Analysis (Hillsboro, OR), 50 mg posaconazole  cubosome of lyophilized was utilized at a magnification range of 20x to 3.5 lakhs x, resolution: 0.5nm, and high voltage − 120Kv⁽⁷⁾.

Partical size and  polydispersity index and  zeta potential

Using SZ-100 equipment from HORIBA Ltd. (Tokyo, Japan), dynamic light scattering was used to measure the average particle size and PDI of the cubosomes at a temperature of 25°C and a scattering angle of 90°. To prevent multi-scattering occurrences, batches were diluted with filtered double-distilled water at a concentration of 10% prior to measurement until the proper particle concentration was reached. To evaluate the homogeneity of the particle size distribution, the PDI was examined. Additionally, a zeta potential analyzer more precisely, the SZ-100 from HORIBA Ltd. (Tokyo, Japan) was used to detect the surface charge⁽⁸⁾.

Fourier Transform Infrared Spectroscopy (FT-IR)

FTIR spectra of the optimized posaconazole cubosomes, pure medication, and excipients are obtained and examined for potential incompatibilities⁽⁹⁾.

Entrapment efficiency (EE) and drug loading

The centrifugation method was used to calculate posaconazole cubosomes  percentage entrapment efficiency (%EE). A cooling centrifuge (REMI-C24 BL, Remi Elektrotechnik Ltd., Thane, India) was used to centrifuge a 2 mL sample of the formulation with 5 mL of methanol for an hour at 12,000 rpm. Using a UV-visible spectrophotometer (V-1800, Shimadzu, Japan), the supernatant (1 mL) was diluted to 10 mL with methanol and measured at 259 nm. Conventional equations were used as follow as to compute the %EE and drug loading^10,11:

 % EE = (Amount of drug added – Amount of free drug)/ Amount of drug added x 100 (1)

 % DL = (Amount of drug added – Amount of free drug)/ Amount of lipid added x 100 (2)

In vitro release of posaconazole from cubosomes

Phosphate buffer (pH 7.2) was used to assess the in vitro drug release from posaconazole loaded cubosomes. A dialysis bag (12–14 kDa) containing 100 mg of the freeze-dried formulation was submerged in 900 mL of medium that was kept at 37 ± 0.5 °C while being stirred at 50 rpm. 2 mL samples were taken out at prearranged intervals up to 12 hours, replaced with fresh media, filtered via a 0.22 µm membrane, and spectrophotometrically measured at 259 nm^(8,12).

Release kinetic study

To clarify the release process, the drug release data from posaconazole loaded cubosomes were examined using a variety of kinetic models, such as zero-order, first-order, Higuchi, and Korsmeyer–Peppas equations. The model with the highest correlation coefficient (R²), which indicated the formulation's main release kinetics, was deemed to be the best fit.

MOLECULAR DOCKING

The mechanism of action, binding affinity, binding style, and molecular interactions of the selected drugs were investigated using molecular docking utilizing Schrodinger and Maestro Glide. All compounds were constructed using Maestro build panels, and they were optimized for reduced energy conformers using Ligand preparation (Schrodinger, LLC). With the help of protein preparation experts, the protein X-ray crystal structure (PDB ID:5V5Z) was obtained from the protein data bank and prepared for docking. Grids were then minimized and refined around the structures using the default box size and centered on the ligand. All selected compounds underwent final docking tests utilizing the extra-precision (XP) docking mode on a constructed protein structure grid⁽¹⁸⁾.

Ligand and protein preparation

All target proteins and investigated ligands have to be prepared in order to meet the minimal requirements for further computational computations. The LigPrep tool, which interfaced with the Maestro module of the Schrodinger suite, was used for ligand preparation. 3D structures with all possible tautomers and ionization states at pH 7.0 ± 2.0 of all ligands and reference compounds were constructed and geometrically reduced using the optimal potential liquid simulations (OPLS3e) force field. Schrodinger's multi-step Protein Preparation Wizard was used for protein preparations. First, high-resolution protein crystal structures of the enzyme (PDB ID:5V5Z) complexed with the native ligand fluconazole were looked for in the RCSB Protein Data Bank. Heavy atoms were provided hydrogen after charges and bond ordering were assigned, and all heteroatoms and water molecules were removed while native ligands and metals remained in the active site. The resulting structures were optimized and subsequently reduced using the OPLS3e force field to avoid steric conflicts between atoms.

ANALYTICAL METHOD VALIDATION:

The process of establishing through laboratory investigations that an analytical procedure's performance characteristics satisfy the requirements for its intended usage is known as validation. The planned and methodical gathering of validation data by the applicant to support analytical procedures is the first step in the techniques validation process for analytical processes. Every analytical technique that is meant to be applied to the analysis of clinical samples must be verified. According to ICH requirements, analytical methods are validated⁽¹³⁾.

Selection of wavelength for analysis of Posaconazole

In order to ascertain the wavelength of maximum absorption of posaconazole, the stock solution of 1000 μg/ml was prepared by taking 5mg of the drug in 5ml of methanol. From this stock solution, the concentration of  the drug (10μg/ml) in methanol was prepared and scanned using spectrophotometer within the wavelength range of 200 to 400nm against methanol as blank.

Preparation of standard stock solution

A stock solution was created by dissolving 1 milligram of posaconazole in 10 ml of methanol. Using methanol, appropriate dilutions ranging from 2 to 10μg/ml were used to generate various aliquots of concentration from this standard stock solution (10μg/ml). At 265 nm, the absorbance of the subsequent concentration was measured. The drug's (posaconazole) calibration curve was created using the data.

The UV-VIS Spectrophotometric method was validated  according to the International Conference on Harmonization (ICH) guidelines (2005). The following  characteristics were considered for validation: linearity, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ).

Linearity

The ability of an analytical process to demonstrate the rationale that the observed absorbance is proportionate to the concentration of a sample containing the analyte is known as linearity. Five distinct concentrations (2μg/ml, 4µg/ml, 6µg/ml, 8µg/ml, and 10µg/ml) were tested on three separate days at 265 nm to establish the linearity^(14,15).

Precision

Repeatability (inter-day) and intermediate precision (intra-day) studies were used to assess the method's precision. Three same concentrations (6µg/ml) were taken across two days, and the percent relative standard deviation (%RSD) was computed to determine inter-day precision. The same samples were examined two times during the day for intra-day precision, and the percentage RSD was also computed^(15,16).

Accuracy

The degree of agreement between the values that are recognized as a conventional true value and the value discovered is a measure of an analytical technique's accuracy. The accuracy tests were performed in triplicate at three distinct concentrations (4.4µg/ml, 6µg/ml, and 8.4µg/ml) made from the stock solution, and the standard curve was used to assess their strengths^(15,16).

Limit of detection ( LOD)

The detection limit of an analytical method is defined as the lowest amount of analyte present in a sample that can  be detected but not necessarily quantitated as an exact value15. The limit of detection was performed from the standard curve.

LOD = 3.3 S/M

Where, S is the standard deviation of the absorbance of the sample and  M is the Slope of the calibration curve.

Limit of quantification (LOQ)

Limit of Quantification (LOQ) can be defined as the lowest amount of analyte present in a sample that can be quantitatively determined with suitable precision and  accuracy15.  Limit of quantification was based on the standard deviation of the response and the slope of the corresponding curve using the following equation:

 LOD = 10 S/M

Where S is the standard deviation of the absorbance of  the sample and M is the Slope of the calibration curve⁽¹⁷⁾.

Robustness

The robustness of an analytical procedure is the measure of its capacity to remain unaffected by small, but deliberate, variations in method parameters and provides an indication of its reliability during normal usage.

Ruggedness

The (intra-laboratory tested) behavior of an analytical process when small changes in the environmental and/or operating conditions are made (generally used term).

RESULT:

Scanning electron microscopy (SEM)

The morphology of cubosomes was examined using scanning electron microscopy (SEM). The optimized posaconazole  cubosome dispersion's shape and surface morphology were smooth and devoid cracks giving them good appearance. The SEM data obtained on the drug  loaded cubosomes are showed figure1.

Figure 1 SEM image of prepared drug loaded cubosome dispersion

Partical size and  polydispersity index and  zeta potential

The particle size and polydispersity index of drug loaded cubosomes dispersion has examined by the dynamic light scattering the particle size was obtained 126.4 nm and the polydispersity index (PDI) was 0.546 and it was shown in figure 2. Zeta potential was analyzed and obtained a surface charge -37.2Mv and electrophoretic mobility was -0.000288 cm/Vs  as shown in figure 3

 

 

Figure 2 Particle size and polydispersity index                                           

 

Figure 3 Zeta potential

 

 

Figure 4 FTIR spectra of drug

 

 

Figure 5 FTIR spectra of cubosomes

 

 

Figure 6 Overlay of physical mixture of drug and cubosomes

 

 

Figure 7 %Entrapment efficiency of drug loaded cubosomes

Table 3 In vitro release profile of all Posaconazole Cubosomes optimized formulation over 24 hours

Time(hour)	1	2	3	4	5	8	12	24
Plain drug F0	2.56±  0.21	4.82±  0.31	9.26±  0.34	16.44±  0.14	22.18±  0.12	27.46±  0.02	30.21±  0.25	36.32±  0.15
F1	6.8±  0.35	12.56±  0.78	27.58±  0.21	59.12±  0.24	64.45±  0.25	74.92±  0.05	84.2±  0.12	92.36±  0.23
F2	9.22±  0.68	15.53±  0.22	31.6±  0.54	62.14±  0.35	68.2±  0.23	78.4±  0.56	85.36±  0.36	93.21±  0.52
F3	8.44±  0.78	16.24±  0.28	30.46±  0.56	62.18±  0.34	70.1±  0.56	79.55±  0.45	86.37±  0.15	94.56±  0.25
F4	7.42±  0.15	16.23±  0.36	28.5±  0.57	61.18±  0.02	72.47±  0.74	80.56±  0.25	89.42±  0.25	96.36±  0.29
F5	8.57±  0.43	19.48±  0.23	35.88±  0.23	63.62±  0.05	72.5±  0.56	81.54±  0.56	90.23±  0.29	101.36±0.30
F6	8.46±  0.45	18.32±  0.01	38.36±  0.25	64.16±  0.23	74.2±  0.57	83.33±  0.34	91.2±  0.58	102.37±0.51
F7	8.7±  0.98	18.18±  0.56	26.49±  0.15	65.25±  0.25	75.88±  0.54	83.68±  0.25	91.36±  0.25	102.31±0.58
F8	10.6±  1.23	22.24±  0.78	38.54±  0.02	57.3±  0.56	74.6±  0.02	85.7±  0.51	91.68±  0.12	103.36±0.45
F9	11.74±  0.36	26.2±  0.85	38.94±  0.01	63.7±  0.12	80.8±  0.45	90.65±  0.25	100.25±  0.13	106.3±0.36

 

 

 

Figure 8 %Cumulative in vitro drug release study

	(A)
(B)

Figure 9 Docked images of the (A) Posaconazole and standard (B) fluconazole in a 2D and 3D interacting diagram for in protein (PDB ID: 5V5Z).

 

 

Figure 10 Calibration curve of posaconazole

Accuracy:

CONCLUSION

The developed cubosomal formulation showed good compatibility and stability as confirmed by FTIR analysis. Molecular docking supported the drug’s interaction with the target site. The validated UV method met all required criteria for linearity, precision, and sensitivity. Overall, the study confirms the reliability of the formulation and analytical approach for antifungal applications.

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