Formulation and Evaluation of Nanosponge Hydrogel for Transdermal Release of Curcumin

Regardless of lot of efforts still dangerous human diseases like cardiovascular diseases, cancer, and neurological diseases etc. have not decreased till now. Scientists have discovered numerous smart drugs which target on specific signaling pathways that cause a sudden change in the body and trigger a disease. But considering above points smart drugs are often costly and have numerous side effects. So, there is a need of medications that is of low cost, can target various signaling pathways, should have fewer problematic consequences, should be easily available in the daily life routine for avoiding and treating various human diseases^(1)(2).

Curcumin is a polypotent or multiactioned molecule that can interact at various target site^(3).It is a nutraceutical that is a polyphenol which possess antioxidant, anticancer, anti-inflammatory, anti-microbial, and wound healing properties^(4). It’s a traditional drug that has been used from decades for prevention and treatment of various diseases. Numerous preclinical studies have been conducted on Curcumin for past three decades^(5).Curcumin is derived from a rhizome called Curcuma Longa^(6).Curcumin is a lipophilic molecule and can pass through the cell membrane easily .It is used as spices and as a natural food coluring agent^(7).Important molecules for curcumin binding include transcription factors, inflammatory mediators, and enzymes such as histone acetyltransferase, reductase and protein kinase. Curcumin is a potent epigenetic regulator in various disease like cancer. Additionally, by inhibiting the phosphorylase kinase enzyme it can modify number of proteasomal pathways^(8).

Curcumin in Breast Cancer: In breast cancer there is over activation of nuclear factor kappa B  which is responsible for survival, proliferation and metastasis of cancerous cell. Curcumin inhibits nuclear factor κBp65 factor which is a key factor for NF –Κb pathway. This reduces breast cancer ability to move, migrate, grow and invade to other tissues. Another breast cancer factor is HER2 protein which promote tumour growth. Curcumin reduces HER2 protein levels and reduces activation and phosphorylation of key growth pathways like AKT and MAPK and it also reduces RON mediated invasion.^(9)(10)(11)

Curcumin has poor aqueous solubility and low bioavailability. Its poor bioavailability is due to its fast metabolism, poor absorption and fast elimination. To overcome this problem there are various promising approaches like, nanoparticles, liposomes, micelles, nano sponges, micro sponges and phospholipid complexes .^(12-14)

To overcome this problem Nano sponges, act as best carrier as they can efficiently solubilize poorly water-soluble drugs and can give a prolonged drug release. Nanosponges can be beneficial for both hydrophobic and hydrophilic drugs because of their internal hydrophobic cavities and external hydrophilic nature⁽¹⁵⁾.

Nanosponges are prepared by various methods namely Hypercrosslinked β Cyclodextrin method, Emulsion Solvent Diffusion method, Quasi emulsion method, Ultrasound Assisted synthesis, etc. Nanosponge is a drug carrier that uses polymers to entrap the drug which reduce its degradation and provide a sustain and controlled drug release at the site of action⁽¹⁸⁾

The most widely used technique for creating nanosponges is the quasi-emulsion solvent diffusion method, which allows for dissolving a water- insoluble  polymer in an organic solvent and then making it solubilize in an aqueous phase with a surfactant which is hydrophilic. The organic solvent is then removed by constant stirring and leaving behind spherical particles. The spheres are formed by polymer with internal and external pores⁽¹⁶⁾.There are factors that affect pore formulation polymer ratio, organic solvent, emulsifier concentration, stirring rate.⁽¹⁷⁾

We have developed a formulation of nanosponges by using ethyl cellulose and hydroxypropyl methylcellulose as polymers loaded with the selected drug curcumin. Here we have changed the concentration of ethyl cellulose and observed the drug entrapment efficiency of the nanosponges. Additionally, the influence of polyvinyl alcohol with various concentration which is used as an emulsifier was observed.

MATERIALS AND PROCEDURES         

Materials

Curcumin was purchased from  LOBA chemie  pvt ltd, Mumbai, Hpmc K100 and Dichloromethane was purchased from Solanki Excipients, Pune, Ethyl Cellulose, Polyvinyl alcohol, Carbopol 940, Triethanolamine, Methyl Paraben, Propylene glycol was used from the research lab of the institute.

Method of Preparation for Curcumin Nanosponges⁽¹⁹⁾

Nanosponges were formulated by quasi emulsion diffusion method

Preparation of Internal phase: Varying ratio of Ethyl cellulose (EC), Hpmc K100 and Curcumin were dissolved in 20 ml of Dichloromethane(DCM).This solution forms the polymeric organic phase(Internal Phase)

Preparation of External Phase: Different concentration of Polyvinyl alcohol (PVA) were dissolved in 100 ml of  Distilled water. This formed the aqueous (external phase),acting as emulsifying medium.

Emulsification: Under continuous stirring the internal phase was gradually introduced to the external phase, stirring was done under high-speed homogenizer at 5000 rpm.

Solvent Diffusion and Evaporation: Stirring was continued for 4 hours at room temperature to allow evaporation of DCM, leading to formation of nanosponges.

Filtration and washing: The formed Nanosponges were filtered using Whatman cellulose filter paper. They were washed with double distilled water to remove excess PVA and other residues.

Drying: The nanosponges were dried at 40 ? C for 24 hours to ensure removal of residual solvents.

Factorial batches with 3²  factorial designs

Table no 2.1:Variables in 3² factorial designs

Response Variables:

Y1=Percentage yield, Y2=Entrapment Efficiency

Characterization of Curcumin loaded Nanosponges (Cur-Ns)

1. Visual Appearance:

The nanosponges were observed for their colour and shape

2. Theoretical Yield:

Theoretical yield =Actual amount of drug added + Actual amount of polymer added

3. Practical Yield:

Determination of dried nano sponges recovered from the batches

4. Percentage Yield^(46)(47)(48):

The weight of dried nanosponges (W1) recovered from batches and the total of the original dry weight  of  beginning material (W2) were used to compute the production yield percentage(wt/wt) using the formula below

% Production yield = W1/W2 × 100

5. Particle size analysis

Determination of the average particle size of nanosponges was carried out by Horiba Scientific SZ-100 For the determination of particle size  the prepared formulations were suitably diluted with 2 ml of ethanol and diluted up to 10 ml with distilled water. The Horiba Scientific SZ100 was used to determine the particle size of the nanosponge after the dilution.

6. Analysis of Zeta Potential⁽²³⁾

The Light scattering method was used to determine the prepared nanosponges Zeta potential. The sample must be completely transparent in order to determine the zeta potential. When the solution is prepared for analysis, it should be carefully into the cuvette to prevent bubbles from forming on the cuvette walls. It might also be possible to get rid of the bubbles by gently titling the cuvette or tapping it on a hard surface. After that the electrode was dipped within the sample solution containing cuvette. Bubbles in between the electrodes should be avoided. It is possible to insert the solution containing cuvette into the device. Zeta potential. analysis was performed using the same tool that was utilized for particle size analysis.

7. Index of Polydispersity

Using the Dynamic light scattering(DLS),the produced nanosponges was calculated for its Polydispersity index(PI) .The sample must be crystal clear to very slightly hazy for the DLS technique to work. If the mixture is excessively white it should be further diluted before performing DLS size assessment because it is cloudy. When the solution is prepared for analysis ,it should be carefully transferred into the cuvette to prevent bubbles from forming on the surface of the cuvette. The cuvette holding the solution can be inserted into the device once it is homogeneous and prepared for DLS measurement.

8. Entrapment Efficiency^(20)(21)(22)

Entrapment efficiency = Actual drug loading / Theoretical drug loading     ×    100

9. In Vitro drug release study⁽²⁴⁾

A six-vessel USP class II dissolution apparatus(Curio 2020+paddle apparatus) was used to assess the invitro drug dissolution from nanosponges. With paddle rotation set at 50rpm,a 10 mg drug equivalent sample of nanosponges  was kept at 37± 0.5 °C in 500 ml of phosphate buffer (pH 7.4).For total of 24 hrs ,a 5 ml of sample was taken at prearranged intervals. After every sampling the dissolving media was  refilled with freshly  prepared phosphate buffer to preserve the sink condition. A UV visible spectrophotometer set to 425 nm was used to analyse the samples in triplicate, and the % drug release was computed using a number of standard solutions

10. Scanning electron Microscopy or surface morphology

Particle size distribution, surface topography, roughness, and the morphology of the sectioned surface  have all been determined using scanning electron microscopy. Due to its ease of use and simplicity in sample preparation, SEM is most likely the most widely used technique for characterizing drug delivery system. SEM experiments were conducted in Infinite Biotech, Sangli.

11. DSC(Differential Scanning Colorimetry)

Mettler Toledo(*SW920) Differential Scanning Colorimeter using aluminium pan equipped with an intercooler and a refrigerated cooling system was used to analyze the thermal behaviour of Curcumin which shows an endothermic peak at 184°C

Formulation of hydrogel of optimized batch of Curcumin nanosponge⁽²⁵⁾

A precisely weighed amount of Carbopol 940 was dispersed in approximately 20 ml of distilled water and allowed to hydrate for 24 hours. After complete soaking, the dispersion was neutralized using triethanolamine (TEA) under continuous stirring to facilitate gel formulation.

In another container, the required quantity of methyl paraben and the drug-loaded nanostructure(NS)-equivalent to the desired topical dose ,were dissolved in propylene glycol with gentle stirring to ensure homogeneity. This solution was then gradually added to the pre-neutralized Carbopol dispersion while stirring continuously.

The combined mixture was subjected to further mixing for 20 minutes to achieve a uniform gel base. The resultant dispersion was then kept undisturbed for  60 minutes at room temperature to allow complete hydration and swelling of the gel components.

All prepared gel formulations were allowed to equilibrate for a minimum of 24 hrs. at room temperature before proceeding with viscosity measurements and other physicochemical evaluations.

Table 2.3.Formulation table for hydrogel

Evaluation of hydrogel .

1] Physical inspection: The colour, homogeneity, consistency and appearance of the produced hydrogel compositions were assessed visually

2] pH: A digital pH meter was used to determine the pH values of 1% aqueous solutions of the produced gel

3] Viscosity: The Brookfield – DV-II + Pro Viscometer was used to measure the viscosity of the hydrogel that were manufactured. Spindle no.7 was rotated at 0.5 rpm to test the apparent viscosity at 25°C

4] Spreadability: It was determined by wooden block and glass slide apparatus. It consists of two parallel-placed platforms to hold the glass slides. It is because of these parallel-placed platforms that the upper slide will be pulled in a straight line when force is applied. A pulley is centrally attached to the upper slide. Scale is fixed on one of the platforms to measure the time taken to move the slide a fixed distance. The pan was filled with weights weighing around 20 g and the amount of time it took for the removal of upper slide to disengage from the fixed slides recorded.

5] Ex-vivo permeation investigation: Franz diffusion apparatus was used to conduct this experiment. The vertical type Franz diffusion cell was designed, fabricated, and validated, before diffusion study, the cello phonic membrane of approximately 2.5cm³ area was taken and these slices were hydrated in phosphate buffer (pH-7.4) overnight before use. After assembling the entire cell,100 mg of the gel sample was put on the membranes surface which is connected to the donor compartment’s bottom. The receptor’s capacity was maintained at 25 ml. The way the cell was put together ,the membrane was drained to the surface of the permeation fluid(phosphate buffer 7.4)was kept at 37±1°C constantly swirled using a magnetic stirrer at 50 rpm.1 ml sample was removed and observed in UV spectroscopy for drug content. Following each sampling, fresh buffer was then was then added back to replenish the fluid . For every gel formulation samples were taken at 0.5,1,2,3,4,5,6,7,8 hours at every sample the total amount of medication that has diffused out through the membrane was determined and noted. (cumulative percentage of drug release)

RESULT AND DISCUSSION

Characterization of Curcumin Nanosponges

Table 3.1 Depict Theoretical yield, Practical yield, Percentage yield, Entrapment Efficiency

Particle Size, Zeta potential, Polydispersity index and DSC and SEM

Fig. 3.1 Particle Size, Zeta Potential, Polydispersity Index

Fig 3.2 DSC of Curcumin

Fig 3.3 SEM of Nanosponge

Fig 3.4 SEM of Nanosponge

In vitro drug drug release

Evaluation of Optimized batch of  Hydrogel

Fig 3.6 Visualisation of Gel

1) Spreadability :

Table 3.3 Spreadibility data

Observation- Higher concentration of Carbopol 940 reduces spreadability

2) pH: (for topical application pH should be 4.5-7)

Table 3.4 pH data

3) Viscosity

Table 3.5 Viscosity data

Observation: G1 has low concentration of Carbopol so its depicting low viscosity,G2 has high concentration of Carbopol depicting high viscosity

4) Ex-vivo permeation study

Table 3.6 Ex-vivo permeation data

Fig 3.7 Ex vivo permeation study