Mucoadhesive chitosan-dextran sulfate nanoparticles of acetazolamide for ocular hypertension

2.1 Material

AZ, chitosan (CS) high molecular weight (CS-HMW), and CS low molecular weight (CS-LMW) were purchased from Sigma Aldrich (India). Dextran sulfate (DS) was purchased from High Media (India), and mannitol was purchased from SD-Fine (India).

2.2 Methods

2.3 In vivo studies

2.4 Ex vivo mucoadhesion studies

A pig mucin (Himedia, Mumbai, India) suspension was prepared in 0.05 m saline phosphate buffer (pH 7.4). Placebo CS NPs and drug-loaded CS NPs were mixed with 1 ml mucin suspension and incubated at 37°C for 30 min and kept for 24 h at room temperature. The samples were centrifuged in a cooling centrifuge (12,000 rpm, 30 min); the supernatant was collected; and free pig mucin was quantified by using a UV spectrophotometer (Perkin Almer) at 251 nm. The binding efficiency of mucin with CS NPs was calculated by using the following equation [11]:

2.5 Stability studies on optimized formulation

Accelerated stability study conditions, i.e. 40°C and 75% relative humidity (RH), were imposed on the lyophilized nanoformulation after packing in screw-capped glass bottles wrapped with aluminum foil, and samples were withdrawn at different time intervals (0, 1.5, 3, and 6 months) for determining drug content for long term stability studies. The lyophilized nanoformulation was stored at room temperature, and samples were withdrawn at different time intervals (0, 3, 6, and 12 months).

2.6 Statistical analysis

The whole data were statistically analyzed by using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA), and p<0.05 was considered significant. The adopted statistical methods include ANOVA followed by Dunnett’s test or Student’s t-test.

3.1 Percent drug entrapment

In the present study, the percent drug entrapment (±SD) for the AD1, AD2, and AD3 formulations was found to be 56.23±0.090, 53.32±0.030, and 38.53±0.023, respectively, and for AD4, AD5, and AD6, the percent entrapment was 62.45±0.05, 62.33±0.02, and 46.63±0.02, respectively. It was observed in formulations that as we increased the DS/CS ratio, the percent entrapment of acetazolmide decreased, and that is attributed to decreased relative DS concentration [12].

3.2 Particle size and ζ potential

The polyelectrolyte ratio affects the ζ potential of NPs. Measurement of ζ potential is done to determine the surface charge of NPs. The ζ potential of NPs is greatly influenced by the composition of the particle and dispersion medium, and it also gives an idea about the electrical potential of the particles. The individual values for particle size, ζ potential, and PDI of all the formulations are listed in Table 2. The particle size of the formulations is within the range of 149.7–330.73 nm, whereas the ζ potential and PDI were found within the range of 12.43–36.46 and 0.257–0.362, respectively. All of the NPs were found to have a positive charge value, with the highest charge value of 36.46 for the AD2 formulation, whereas PDI is least (0.257), which is the indicator of good stability and dispersion homogeneity [13]. Also, as the corneal mucin layer is negatively charged, the positively charged NPs will be mucoadhesive and will increase the ocular bioavailability of the drug.

3.3 Particle morphology

The shape of the particles was analyzed by using TEM, which revealed a discrete spherical shape (Figure 2). The TEM images revealed the particle size of the optimized formulation (AD2) to be <200 nm. The result of TEM matched with the results of particle size measurement done with Zetasizer.

Figure 2:

TEM image of the optimized nanoformulation.

The prepared particles were spherical or subspherical in shape, having a smooth surface devoid of cracks. Particles with sharp edges and angles cause more irritation as compared to isometric particles with obtuse angles and edges. Therefore, the optimized formulation is not supposed to cause irritation on the ocular surface [14]. Due to the strong surface charges, the particles are stabilized against agglomeration as they were observed separated from each other.

3.4 PXRD studies

The X-ray diffraction patterns of samples are presented in Figure 3. AZ exhibited the characteristic diffraction peaks at 9.08°, 9.9°, 16.38°, 19.84°, 19.94°, 19.96°, 21.66°, 29.96°, and 30.14° 2θ, while the diffractogram of mannitol showed the characteristic peaks at 10.36°, 14.56°, 18.76°, 20.4°, 21.08°, 23.38°, 25.82°, 28.2°, 29.46°, 31.68°, 33.52°, and 38.6° 2θ. Sharp peaks, present in the diffraction pattern of AZ and mannitol, confirmed their crystalline structure, while the diffractograms of CS and DS indicated an amorphous structure. The diffractogram of the nanoformulation exhibited the significant peaks that were corresponding to the crystalline structure of mannitol, and no peaks corresponding to AZ was obtained, which indicated the drug’s presence in the amorphous state inside the polymer [14].

Figure 3:

PXRD of (A) drug, (B) DS, (C) CS, (D) mannitol, and (E) formulation.

3.5 FTIR-ATR studies

The FTIR spectrum (Figure 4) of AZ gave signals at 3296.88 and 3174.70 cm^-1 from the N-H stretching of the secondary amine. The presence of absorption at 1678.21 cm^-1 was due to the C=O stretching of the carboxyl groups. The characteristic peaks at 1173.81 cm^-1 were attributed to the S=O stretching of sulfonyl groups. S-N stretching absorption was observed at 907.12 cm^-1. Characteristic CS peaks were observed at 1645.68 cm^-1 for the amide I band (C=O stretching), 1565.97 cm^-1 for the amide II band (N-H in plane deformation coupled with C-N stretching), and 1075.08 cm^-1 (C-O-C stretching). The FTIR spectra of DS shows S=O vibration at 1233.97 and 991.82 cm^-1 and O-S-O vibration at 815.64 cm^-1. The mannitol spectrum showed peaks at 3394.12 cm^-1 for OH stretching at 1419.19 cm^-1 for OH in plane bending, at 1078.92 cm^-1 for C=O stretching, and at 701 cm^-1 for OH out-of-plane bending for alcohol. However, the spectrum of nanoformulations did not show any characteristic peak of AZ, which may be due to excess dilution of AZ.

Figure 4:

FTIR images of (A) drug, (B) DS, (C) CS, (D) mannitol, and (E) formulation.

3.6 DSC

Figure 5 represents the DSC thermograms of different samples. AZ exhibited a sharp endotherm at 261°C corresponding to the melting point of AZ. The thermal curve of CS revealed a broad endotherm without any sharp peak, while the thermogram of DS showed an exotherm at 206.78°C. On the other hand, an endothermic peak at 167.04°C was observed for mannitol. A small endotherm at 167°C was displayed in the thermogram of nanoformulations (AD2) corresponding to the endothermic peak of mannitol, while no endotherm of the drug was observed indicating decreased crystallinity in the formulations or because of the very small quantity of drug in the freeze-dried formulation [14].

Figure 5:

DSC of (A) drug, (B) DS, (C) CS, (D) mannitol, and (E) formulation.

3.7 In vitro drug release

Figure 6 represents the comparative in vitro release profile of different CS-based nanosuspensions. The dialysis method was used for studying AZ release in Sorenson’s phosphate buffer (pH 7.4), which served as the release medium. The NP formulations made with CS-LMW (AD1, AD2, and AD3) showed 43.81%, 39.20%, and 38.32% drug release, respectively, in 8 h. On the other hand, nanoformulations made with CS-HMW (AD4, AD5, and AD6) showed 45.83%, 44.45%, and 43.71% drug release, respectively, while from the 0.1% aqueous drug solution (control), 98.72% drug was diffused. The results suggest that entrapment of the drug in the NPs hinders drug release. On increasing the polymer concentration or increasing the drug/polymer ratio, sustained drug release was obtained. The optimized formulation showed initial burst release for the first 2 h of release study (22.52%), followed by sustained release. For determining the release mechanism and regression coefficients (R²), different mathematical kinetic models like Korsmeyer-Peppas, Higuchi, Hixson Crowell, zero order, and first order were applied over the release data of the nanoformulations [15]. The release of AZ from CS NPs linked best to Korsmeyer-Peppas, which can be established by comparing the values for the regression coefficient (R²) of zero order (0.8670), first order (0.8764), Higuchi (0.9480), Korsmeyer-Peppas (0.9674), and Hixson Crowell (0.8900). The diffusion exponent of the Korsmeyer-Peppas equation, i.e. “n” indicated that the release of AZ from CS NPs is by a dual mechanism of dissolution and diffusion, i.e. anomalous, as its value was found within the range of 0.5–1.0. The initial fast release of AZ can be attributed to the rapid hydration of NPs due to the hydrophilic nature of CS and DS, as well as the drug present on the surface of the particles. The sustained effect can be explained by the presence of drug within the core of the particles. The release medium penetrates into the particles and dissolves the entrapped drug, which further diffuses out into the dissolution media.

Figure 6:

In vitro release profile of AZ from CS NP formulation. *Statistically significant (p<0.05) compared with the aqueous solution, as determined by one-way ANOVA followed by Dunnett’s test. Each bar represents the mean±SD (n=3).

3.8 Ex vivo transcorneal permeation; percent corneal hydration

A 2.5 times enhanced transcorneal permeation (statistically significant, p<0.05) of drug was observed with the optimized nanoformulation (AD2), across excised goat cornea, in comparison to the control formulation (0.1% aqueous solution) (Table 3, Figure 7). The possibly positive charge and small size contributed toward the enhanced corneal uptake. The normal range of corneal hydration (75–80%) was observed, which indicates the eye-friendly nature of the optimized formulation [16].

Figure 7:

Ex vivo transcorneal permeation of different formulations.

3.9 In vivo ocular hypotensive efficacy studies

The observation suggested that the hypotensive activity of the drug-loaded NP formulation (AD2) was comparable to that of the plain drug solution (Figure 8). In case of plain AZ solution, the IOP was decreased for a period of 2 h and then started rising, whereas in case of nanoformulations this fluctuation was not observed: the IOP was below the normal level throughout the period of study, i.e. 5 h. The results suggested that the AZ-loaded nanoformulation produced a significant and prolonged reduction in IOP throughout 5 h. This overwhelming superiority over the plain AZ solution was further magnified when single instillation was considered.

Figure 8:

Ocular hypotensive activity of AZ from the aqueous solution and optimized CS nanoformulation.

3.10 Determination of ocular irritation index

The formulation was found non irritant as Iirr was found to be zero.

3.11 In vitro mucoadhesion studies

The mucoadhesive strength or bioadhesive force of CS NPs (AD2) was determined by the adsorption of CS NPs on pig mucin glycoprotein, and found to be excellent, i.e. 91.59±1.48% for AZ-loaded CS NPs. It is due to hydrogen bond formed between the positive-charge amino group of CS and the oligosaccharide chains of mucin [11].

3.12 Stability studies

Under an accelerated condition (i.e. 40°C/75% RH) for 6 months, the nanoformulation (AD2) had 96.08% AZ content; on the other hand, the content was 95.21% after 12 months storage at room temperature.