3.1 Preparation of RT-NLC
Different ratios of solid and liquid lipids were used in the formulation of rutin-loaded NLCs. The ratios were chosen based on entrapment efficiency, with a solid lipid-to-liquid lipid ratio of 70:30 being used. RT-NLCs labelled F1-F5 were produced using various concentrations. The resulting RT-NLCs were analyzed for their physicochemical characteristics and stored in appropriate containers.
3.2 Physicochemical properties of RT-NLC
Particle size, size distribution and entrapment efficiency of RT-NLC
The mean particle size of nanoparticles (NLC) ranges from 1-1000 nm. However, for site-specific drug delivery, the particle size range of 50-300 nm is considered good [12, 28]. In the case of RT-NLCs, formulations with codes F1, F2 and F3 have mean particle sizes less than 58 nm - 229.50 nm, which is desirable. However, RT-NLCs with a higher concentration of rutin i.e. 0.25% w/v (F4) have particle sizes on the higher side (> 400 nm). Rutin, with a concentration of 0.5% w/v (F5), showed a mean particle size of 2051.33 ± 158.69 nm. Mean particle sizes greater than 1000 nm indicate that prepared RT-NLC formulations (F5) are not within the permissible range of nonorange. The relationship between particle size and rutin concentration was established, showing an inverse proportionality.
The prepared RT-NLC formulations were further evaluated for polydispersity index (PDI). The RT-NLC formulations with codes F1-F3 had mean PDI values ranging from 0.313 to 0.415. Mean PDI values less than 0.5 indicate a narrow particle size distribution, which is good for the stability of dispersion [28].
The average percentage of Encapsulation Efficiency (% EE) of RT-NLC ranges from 69-72%. To accommodate the lipid into the lipidic matrices, varying concentrations of lipids (soya oils and GMS) were used. The lipid proportion was used in increasing order of rutin dose, i.e., higher lipid proportion was used for RT-NLCs with higher rutin concentrations, and vice versa. The results of physicochemical properties are summarized in Table 5.
Table 5 Characterization of RT-NLC for particle size, PDI, and EE (%)
RT-NLCs Batch No.
|
Conc. (%W/V)
|
Mean Particle Size (nm) ± SD
|
Mean PDI ± SD
|
Mean EE (%) ± SD
|
F1
|
0.05
|
58.00 ± 1.61
|
0.313 ± 0.149
|
69.5862 ± 0.8924
|
F2
|
0.08
|
103.41 ± 4.88
|
0.474 ± 0.032
|
68.8017 ± 1.1155
|
F3
|
0.1
|
229.50 ± 50.58
|
0.454 ± 0.083
|
69.3676 ± 0.5416
|
F4
|
0.25
|
424.46 ± 100.84
|
0.790 ± 0.0613
|
65.0945 ± 7.5251
|
F5
|
0.5
|
2051.33 ± 158.69
|
1.000 ± 0,000
|
72.7777 ± 3.6394
|
3.3 Ex vivo permeation study through the goat cornea
The permeability of the RT-NLC formulations through goat cornea was tested. The results showed that the permeability coefficient values decreased gradually with increasing concentration (Table 6). The particle size of the RT-NLC was found to be correlated with the concentration of rutin used in the preparation process. As the concentration of rutin increased, the particle size also increased. Particle size plays a vital role in determining the permeability of the formulation. Formulation F1 had the smallest particle size and the highest permeability coefficient, while formulation F5 had larger particle sizes in the micron range, resulting in the lowest permeability coefficient [29].
Table 6 Transcorneal permeability of RT-NLC (n=3, mean ± SD)
RT-NLC Formulation code
|
Jss (µg/cm2 h-1)
|
kp × 104
(cm/s)
|
RT permeation
(µg/cm2)
|
F1
|
7.56
|
171.44 ± 13.32
|
159.05 ± 14.02
|
F2
|
10.03
|
163.99 ± 24.92
|
157.38 ± 5.10
|
F3
|
10.45
|
134.99 ± 36.21
|
161.28 ±25.93
|
F4
|
3.43
|
68.99 ± 12.83
|
246.09 ± 43.30
|
F5
|
5.65
|
22.61 ± 2.99
|
259.37 ± 26.08
|
3.4 Optimization of RT-NLCs
RU-NLC formulation F5 has shown a higher entrapment efficiency compared to other formulations. However, its larger particle size and broad particle size distribution, indicating polydispersity, have prevented further study. Similarly, rutin formulation F4 with a particle size greater than 300 nm has also been neglected due to its polydispersity and possible physical stability problems.
On the other hand, the RT-NLC formulations F1-F3, which have particle sizes in the nano range and narrow size distributions, show good physicochemical properties. Although all three formulations have differences in entrapment efficiency, RT-NLC formulation F3 with a rutin concentration of 0.1% w/v (10 mg/mL) has shown a reasonably good permeability rate (161.28 ±25.93 µg/cm2), which is greater than compared to F1 and F2. Therefore, formulation code F3 with a dose of 10 mg/mL has been selected for further study [30].
3.5 Evaluation of the optimized RT-NLC
RT-NLC formulation (F3) has been studied for particle size analysis, surface morphology study, in vitro cumulative RT release study, and ocular tolerance study.
3.5.1 Mean particle size and PDI of optimized RT-NLC
The optimized RT-NLC's particle size was determined using the DLS method using Malvern’s Zetasizer (Fig. 1). The optimized RT-NLC showed a particle size of 204.5 nm. In contrast, the bell-shaped particle size curve indicated a normal distribution of the particle size. The mean PDI value of 0.458 indicated the particle's monodispersed nature. Overall, the particle size curve of RT-NLC indicated prepared RT-NLC of reasonable physical stability [28].
3.5.2 Morphological evaluation of optimized RT-NLC
The surface morphology of optimized RT-NLC formulations was evaluated using TEM, with particle observation performed on a 500 nm scale. Further, TEM analysis confirmed that the particle sizes of RT-NLCs fall within the nano range and are similar in size to those observed by DLS. In Fig. 2, RT-NLCs exhibited spherical shapes with smooth surfaces [31].
3.6 In vitro evaluation of optimized RT-NLC
Release studies were conducted on optimized RT-NLCs for 24 hours, represented in Fig. 3. The formulation exhibited controlled release due to the presence of a lipidic matrix, which continuously releases the RT from the carrier RT-NLC. Additionally, in vitro release studies were performed on pure rutin drugs using Franz diffusion cells. The results showed that pure rutin remained undissolved for 24 hours and thus failed to release any of its contents.
Several kinetic models were applied to adjust the results of RT cumulative release from RT-NLC to determine the pharmacokinetics and release behaviour. The Higuchi kinetic model exhibited the best correlation data, as shown in Table 7. Fig. 4 represents the Higuchi release order rate for RT-NLC.
Table 7 Values of R2 for various order kinetic models.
Pharmacokinetic model
|
Zero-order kinetics
|
Higuchi
|
Korsmeyer-Peppas model
|
First order kinetics
|
Slope value (R2)
|
0.9273
|
0.9762
|
0.7032
|
0.9457
|
3.7 HET-CAM ocular tolerance of RT-NLC (F3)
The HET-CAM test was used to evaluate the irritancy of RT-NLC formulations for ocular tolerance. The optimized formulations showed no signs of lysis, bleeding, or coagulation, making them non-irritants. The positive control treated eggs had a severe irritant score of 15, indicating that all RT-NLCs were non-irritant for ocular use. The study provides a quick and sensitive method for evaluating irritancy levels of formulations [32, 33]. The results of the HET-CAM test are shown in Fig. 5, whereas the ocular irritancy score is shown in Table 8.
Table 8 HET-CAM test for optimized RT-NLC, positive control and negative control.
Formulation
|
Egg
|
Effect
|
Scoring time (Sec)
|
Overall Score
|
Initial
|
30
|
120
|
300
|
Positive control
(0.1 M NaOH)
|
Egg I
|
Lysis
|
0
|
5
|
5
|
5
|
15
|
Egg II
|
Haemorrhage
|
0
|
5
|
5
|
5
|
Egg III
|
Coagulation
|
0
|
5
|
5
|
5
|
Negative control
(Normal Saline)
|
Egg I
|
Lysis
|
0
|
0
|
0
|
0
|
0
|
Egg II
|
haemorrhage
|
0
|
0
|
0
|
0
|
Egg III
|
Coagulation
|
0
|
0
|
0
|
0
|
Q-NLC (F3)
|
Egg I
|
Lysis
|
0
|
0
|
0
|
0
|
0
|
Egg II
|
haemorrhage
|
0
|
0
|
0
|
0
|
Egg III
|
Coagulation
|
0
|
0
|
0
|
0
|