Table 3 Range of optimized variables

Amount of charge inducer (DCP)

8–16 mg

It was observed that as the concentration of DCP increased, the aggregation reduced. This may be because of the repulsive force developed by negative chargeon niosomes induced by DCP. The results of zeta potential showed that at the concentration of 8 mg and above it attains − 30 mV charge which satisfies the criteria for stable formulation with no agglomeration. Therefore, further formulation optimization was carried out by keep DCP concentration between 8 mg and 16 mg (36.9 mV). Also, the vesicle size decreased comparatively, because of repulsion, which inhibited formation of aggregates

Span 60 to cholesterol ratio

2:2–3:1

It was observed that as the span 60 to cholesterol ratio was increased, the aggregation reduced. This might be due to the increase in the amount of surfactant

Temperature

63 °C ± 3 °C

The phase transition temperature of span 60 is 55 °C, and trials showed lump formation at a temperature below 60 °C. A temperature above 70 °C can cause degradation of the drug. Therefore, the temperature should preferably be maintained in between 60 and 66 °C

Needle size

24 gauge

Initial attempts depicted that there was some effect of higher gauge size needle on vesicle size and polydispersity index. For further optimization, 24-gauge needle was used due to smaller vesicle size and lower PDI when compared with other two sizes (18 and 22 gauge)

Flow rate

2 ml/min

The average vesicle size and PDI showed a non-linear response with flow rate of injection, i.e., change in flow rate does not affect vesicle size and PDI significantly. Hence, for reducing the time for the formulation process, a higher flow rate of 2 ml/min can be used

Sonication

5 min

With increase in the sonication time, decrease in entrapment efficiency and average vesicle size was observed. This was might be due to rupture of niosomes upon sonication for longer duration. Therefore, it was necessary to keep the sonication time constant at 5 min