INTRODUCTION:

Cubosomes are discrete, sub-micron, nanostructured particles of the bi continuous cubic liquid crystalline phase. The term Cubosomes was coined by Larsson, which reflects the cubic molecular crystallography and similarity to liposomes¹. These are nanoparticles which are self-assembled liquid crystalline particles of certain surfactants with proper ratio of water with microstructure.²

Cubosomes are nanoparticles but instead of the solid particles usually encountered, these are self-assembled liquid crystalline particles with a solid like rheology that provides unique properties of practical interest³. Cubosomes are composed of polymers, lipids and surfactants with polar and non-polar components hence said as amphiphilic. The amphiphilic molecules are driven by the hydrophobic effect into polar solvent to impulsively identify and assemble into a liquid crystal of nanometer scale⁴. Cubosomes preparation can be achieved through two approaches top-down (TD) and bottom-up (BU) techniques.⁵ To prepare and characterization of Docetaxel cubosomes. It is used in the treatment of breast cancer. The increasing demand of the efficient delivery of medication with minimum side effects, improved patient compliance has resulted in inventing novel type of dosage form⁶. Docetaxel is a member of a family of drugs called taxanes. This drug works by slowing cell growth. Docetaxel is a taxane derivative similar to paclitaxel. Docetaxel binds to tubulin, the protein component of microtubules, and simultaneously promotes assembly and inhibits them⁷. Stabilization of microtubules leads to inhibition of cell division (mitosis) and tumour proliferation, resulting in cell death. Both docetaxel and paclitaxel bind to the same microtubule site. This medication is used to treat cancer (such as breast, lung, prostate, stomach, and head/neck cancer)⁸.

MATERIALS AND METHODS:

Docetaxel was collected as a gift sample from Aurobindo Laboratories Ltd, Hyderabad, Pluronic F 68, Pluronic F 127, Methanol, and Glycerol monostearate were purchased from AR chemicals.

Methodology:

Determination of λ max:

100mg of Docetaxel was dissolved in 10ml of methanol and further diluted with pH 7.4 buffer, suitable dilutions were made and finally scanned for maximum absorbance using UV spectrophotometer in the range from 200 to 400nm. Average of triplicate readings was taken^9,10.

Docetaxel Calibration Curve:

In present study, the spectrophotometric method was adopted for the estimation of Docetaxel using U.V. spectrophotometer. Preparation of 7.4 pH phosphate buffer: Place 250ml of 0.2M potassium dihydrogen phosphate in a 200ml volumetric flask, add 39.1ml of 0.2M sodium hydroxide and then add water to volume. Preparation of primary stock solution: Take 10mg of drug and add 10ml of 7.4 pH buffer to it. Preparation of secondary stock solution: Take 1ml of primary stock solution and add 9ml of 7.4 pH buffer to it. Standard solutions of different concentration were prepared and their absorbance was measured at 280nm^11,12.

Fourier Transform infrared spectroscopy:¹³

Fourier transform IR spectra were obtained on Shimadzu FT-IR spectrometer. Samples were prepared in KBr disks (2mg sample in 200mg KBr). The scanning range was 450-4000 cm^-1 and the resolution was 4cm^-1.

Method of Preparation:

Bottom-Up Method^:¹⁴

Two solutions were prepared: Solution 1 containing Docetaxel dissolved in ethanol in a glass bottle. Solution 2 consisted of dissolving of varying amounts of GMO, pluronic F127 and pluronic 68 into water. Solutions were initially kept at 40°C under constant stirring for 20min. Afterwards, solution 1 was added drop wise into solution 2. The resulting medium was stirred and heated at 40°C for an additional 10min. Subsequently, the solution was brought to rotary evaporator (IKA works, Guangzhou, China) at 40°C under vaccum where most of the ethanol, and a proportion of the water were removed until less than 10ml solution was left (t=50min). Finally, the medium was stored into 10ml vials and filled with deionized water up to a final volume of 10ml. By this method the formulations of 1:1,1:2,1:3,1:4, ratios were determined. Solution:1 Docetaxel dissolved in ethanol heated at 40^OC. Solution: 2 pluronic F127 and pluronic 68 dissolved in water and Ultra-sonication at 40^OC for 20 min. Later Solution 1 was added into solution 2 then Stirred in rotary evaporator at 40^OC in medium was stored for further studies.

Table-1: Formulation development of Docetaxel cubosomes

Ingredients (mg)	F1	F2	F3	F4	F5	F6	F7	F8
Docetaxel	20	20	20	20	20	20	20	20
Pluronic F127	20	40	60	80	-	-	-	-
Pluronic F68	-	-	-	-	20	40	60	80
Glycerol Monooleate	10	20	30	40	10	20	30	40
Ethanol	20	20	20	20	20	20	20	20

Evaluation of Docetaxel loaded Cubosomes:

Size and size distribution:¹⁵

Size and size distribution studies were done for cubosomes prepared by Thin Film hydration. The cubosomes suspension (100mg) was hydrated in a small glass test tube using 10ml of pH 7.4 phosphate buffer solution. The dispersion was observed under optical microscope at 40X magnification. Size and size distribution of 200–300 cubosomes were noted using calibrated stage and ocular micrometers (Elico Instruments, Hyderabad).

Entrapment efficiency:^16,17

To 0.2g of Cubosomes, weighed in a glass tube, 10ml phosphate buffer pH 7.4 were added. The aqueous suspension was then sonicated. Cubosomes containing Docetaxel were separated from untrapped drug by centrifugation at 9000rpm for 45 min at 4⁰C. The supernatant was recovered and assayed spectrophotometrically using UV spectrophotometer. The encapsulation percentage of drug (EP) was calculated by the following equation:

EP = [(C_t – C_r)/ C_t] * 100.

Where, Ct is concentration of total Docetaxel and Cr is concentration of free Docetaxel.

In vitro drug Release Study:^13,18

Ex vivo release studies were carried out using unjacketed vertical franz diffusion cells with a diffusion surface area of 6.154 cm² and 20ml of receptor cell volume. Prior to the study, the dialysis membrane was soaked in phosphate buffer pH 7.4 Formulation equivalent to 5mg of Docetaxel was placed in the donor compartment. The receptor compartment consisting of PB pH 7.4 (containing 0.02% w/v of ethanol to retard microbial growth) was maintained at 37±2°C under constant stirring up to 24 hrs. The donor chamber and the sampling port were covered with lid to prevent evaporation during the study. Aliquots of 5ml were withdrawn periodically at different time intervals (0, 2, 4, 6, 8, 10, 12 Hrs) and replaced with equal volume to maintain constant receptor phase volume. At the end of the study, the samples were suitably diluted and the amount of drug was determined spectrophotometrically.

In Vitro Drug Release Kinetics:^19,20

The results of in vitro release profile obtained for optimized formulation was plotted in modes of data treatments as follows: Zero-order kinetic model (cumulative percent drug released versus time), First order kinetic model (log cumulative percent drug remaining versus time), Higuchi’s model (cumulative percent drug released versus square root of time) and Peppa’s model (log cumulative percent drug released versus log time)

Stability Studies:^21,22

The formulations stored in glass vials covered with aluminum foil were kept at room temperature and in refrigerator (4^OC) for a period of 90 days, samples were withdrawn and hydrated with phosphate-buffered saline (pH 7.4) and observed for any sign of drug crystallization under optical microscope. Furthermore, the samples were also evaluated for particle size and percent retention of Docetaxel.

RESULTS AND DISCUSSION:

Docetaxel Calibration Curve studies:

Fig-1: Calibration curve of Docetaxel in pH 7.4 Buffer

FTIR Studies:

FT-IR Spectra of Docetaxel and F4 formulation were recorded. All these peaks have appeared in formulation and physical mixture, indicating no chemical interaction between Docetaxel and polymer. It also confirmed that the stability of drug during process.

In vitro drug release diffusion studies:

Table-3: In vitro drug release studies of Docetaxel Cubosome Formulations

Time	F1	F2	F3	F4	F5	F6	F7	F8
0	0	0	0	0	0	0	0	0
1	12.36	10.26	9.63	11.54	10.28	9.15	8.26	9.14
2	17.58	20.21	18.26	21.15	19.86	13.56	11.28	17.1
3	25.86	29.25	22.56	28.49	28.46	25.48	24.15	25.49
4	35.61	32.19	30.15	38.7	35.84	32.56	31.28	32.4
5	40.28	35.68	35.26	50.24	39.48	38.64	39.46	48.24
6	42.23	38.48	40.26	51.75	46.89	45.81	42.18	50.94
7	51.18	45.86	53.26	66.74	50.18	49.38	48.63	63.27
8	64.31	49.68	65.15	82.44	63.15	55.18	52.18	69.32
9	71.25	55.15	70.15	91.44	75.63	59.16	60.15	71.25
10	79.56	57.63	78.56	95.8	80.26	63.25	62.35	78.26
12	85.61	58.91	85.38	99.37	85.46	70.73	63.89	85.73

Fig-8: Zero order kinetics studies of optimized F4 Docetaxel Cubosome Formulation

Fig-9: First order kinetics studies of optimized F4 Docetaxel Cubosome Formulation

Fig-10: Higuchi model kinetics studies of optimized F4 Docetaxel Cubosome Formulation

Fig-11: Korsmeyer peppa’s studies of optimized F4 Docetaxel Cubosomal Formulation

Table-4: Stability studies of optimized formulation F-4 Cubosomal Formulation

F. Code	Parameters	Initial	1^st Month	2^ndMonth	3^rdMonth	Limits as per Specifications
F-4	25⁰C/60% RH % Release	95.40	95.38	95.30	95.21	Not less than 85%
F-4	30⁰C/75% RH % Release	95.40	95.37	95.28	95.19	Not less than 85%
F-4	40⁰C/75% RH % Release	95.40	95.32	95.22	95.15	Not less than 85%

Stability studies:

There was no significant change in physical and chemical properties of the formulation F-4 after 3 months. Parameters quantified at various time intervals were shown. (Table-4).

SUMMARY:

Cubosomes can be formed by simple combination of biologically compatible lipids (GMO) and water and are thus well suited for pharmaceutical and body tissue. The ability to form cubosomes during manufacture offers enhanced flexibility for product development. The above research specifies cubosomal utility as controlled release drug carrier. Our results suggest that cubosomes formulation is an ideal candidate for many cubosomes required in various applications. In vitro study revealed that cubosomes formulations F1(1:1) containing 10mg F 127 shown better releases than other dispersion. Cubosome formulation prepared by GMO, pluronic F-127 shows good cubic structure, satisfactory entrapment efficiency (96%) and drug release (95.40%). As GMO concentration increases entrapment efficiency and drug release are increased but the prepared formulations are stable. In conclusion cubosomes are promising control release vehicle for the effective drug delivery of Docetaxel. Although they possess advantageous characteristics, there is still a long way to go before their clinical application.

CONCLUSION:

From the current work it was evident that Docetaxel cubosomes were formulated and evaluated for their properties. The developed cubosomes exhibited characteristics which are in accordance with the official limits. All the formulations were found to be In accordance with respect to their Particle size, Drug Loading and Entrapment Efficiency. Among all the formulations the F4 Docetaxel cubosomes formulation produced maximum drug release compared to other formulations which was considered as optimized formulation. The optimized formulation drug release data was subjected to release kinetics; from the release kinetics data it was evident that the formulation showed zero order diffusion type of drug release. Hence Docetaxel cubosomes could be formulated for sustained release and targeted drug delivery for better release and improved drug action for gastric cancer therapy.

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Received on 08.04.2021 Modified on 28.04.2021

Res. J. Pharma. Dosage Forms and Tech.2021; 13(3):179-184.

DOI: 10.52711/0975-4377.2021.00032

Formulation	Drug Entrapment Efficiency
F1	76.16±0.15
F2	79.23±0.20
F3	82.78±0.23
F4	90.15±0.27
F5	83.99±0.19
F6	79.74±0.22
F7	78.93±0.25
F8	71.45±0.18