Formulation and Evaluation of Nasal Microspheres

 Mr. Prajwal Gajanan Nakat, Dr. Nitin S. Bhajipale, Prof Dr. Manish R. Bhise

S.G.S.P.S. Institute of Pharmacy Kaulkhed, Akola–444004, (MH) India.

*Correspondence: prajwalnakat7@gmail.com;

DOI: https://doi.org/10.71431/IJRPAS.2025.4802   

INTRODUCTION

Formulation of Rivastigmine Nasal Microsphere

Table: Formulation of Rivastigmine Nasal Microsphere

Characterization of Nasal Microspheres

Percentage Yield (%)

Percentage yield of nasal microspheres was calculated by dividing actual weight of product to total amount of all non-volatile components that are used in the preparation of nasal microspheres and is represented by following formula.⁷³

 Particle Size Analysis

Particle size of the microspheres was determined by optical microscopy. The freshly prepared microspheres were examined on an optical microscope by pre-calibrated ocular micrometre and stage micrometre. The microspheres were suspended in water and a drop of microspheres was taken with a drop of glycerine and covered it with cover slip. Prepared slide of microspheres sample was examined under optical microscope. About 100 particles of each formulation were observed and measured.⁷⁴

Percentage Drug Entrapment Efficiency

To determine the % drug loading efficiency 50 mg of microspheres were taken and crushed using mortar and pestle, and then the crushed powder was transferred into 100 mL volumetric flask. Small quantity of methanol was added to the volumetric flask and the resulting solution was centrifuged for 10 min. Further volume was made up with methanol and filtered. Filtered sample was then further diluted with methanol so that to obtain the solution of desired drug concentration. The absorbance was measured spectrophotometrically at 260 nm. (Shimadzu Model 1601, Japan). The percentage drug loading efficiency of each microspheres formulation batch is shown in Tables.⁷⁵

In Vitro Mucoadhesion Studies

Microsphere mucoadhesion was tested using goat intestinal mucosa. Tissue was cut into 1×1 cm pieces, fixed on glass slides, and 10 mg of microspheres were applied. After 1 hr in a desiccator, slides were placed at a 45° angle and exposed to phosphate buffer (pH 6.8, 37 °C, 1 ml/min). After 1 hr, leached microspheres were collected, dried, and weighed.

% Mucoadhesion = (\frac{\text{Applied wt. – Leached wt.}}{\text{Applied wt.}} \times 100)

Surface Morphology

Surface features of drug-loaded microspheres were analysed using Scanning Electron Microscopy (SEM). SEM provides high-resolution images of particle shape, size, texture, and porosity. Samples were coated with gold/platinum to enhance imaging.

In Vitro Drug Release Studies

Drug release was studied using a Franz diffusion cell with goat nasal mucosa. The receptor compartment contained phosphate buffer (pH 6.8) and a magnetic stirrer. Microspheres (6.25 mg Rivastigmine) were placed in the donor compartment. Samples were withdrawn at intervals, replaced with fresh buffer, and analysed at 260 nm.

Drug Release Kinetics

Drug release data were fitted to various kinetic models:

Zero Order: Release rate is constant and independent of concentration.
Equation: (C = k_0 t)

First Order: Release rate depends on drug concentration.

Equation: (\log C = \log C_0 - \frac{kt}{2.303})

Higuchi Model: Describes diffusion-controlled release from a matrix.

Equation: (Q = k t^{1/2})

Korsmeyer–Peppas Model: Describes polymeric drug release.

Equation: (\frac{M_t}{M_\infty} = k t^n)

• n < 0.45: Fickian diffusion
• 0.45 ≤ n < 0.89: Non-Fickian (anomalous)
• n = 0.89: Case II transport
• n > 0.89: Super Case II transport

 Stability Studies

The accelerated stability studies were carried out according to ICH guidelines on optimized nasal microsphere formulation, which was packed in strip of aluminium foil and this packed formulation was stored in stability chamber maintained at 40^oC and 75% RH (Zone III conditions as per ICH Q1 guidelines) for 3 months. The microsphere was evaluated before and after 1 month for change in appearance, drug content and In vitro release.⁷⁹

RESULTS AND DISCUSSION

1. Identification of Drug

A) Determination of Melting Point

The melting point of Rivastigmine was determined by capillary method, melting point of Rivastigmine was found to be in the range of  123°C to 125°C. Melting point compared with Pharmacopoeial standards that confirmed the purity of drug sample.

B) Solubility

Rivastigmine was found to be very soluble in water, soluble in ethanol and acetonitrile, slightly soluble in n-octanol, and very slightly soluble in ethyl acetate

C) UV-Spectroscopy (Determination of λ max)

The solution containing 10 ug/ml of Rivastigmine in phosphate buffer pH 6.8. was prepared and scanned over 200-800nm against phosphate buffer pH 6.8. solution as a blank using Shimadzu UV spectrophotometer. The maximum wave length was observed at 260 nm, which match with reported wave length. The λ max of rivastigmine was shown in figure 7.1.

Figure: UV Absorption Maxima of Rivastigmine at 260 nm

2. Standard Calibration Curve of Rivastigmine

The stock solution is used to prepare 2 to 16 μg/ml of Rivastigmine in 6.8 pH phosphate buffer and analysed at 260 nm. The graph v/s concentration was plotted and data was subjected to linear regression analysis. The data of absorbance shown in table 7.1 and figure 7.2. The standard calibration curve of Rivastigmine in the concentration 2 μg/ml to 16 μg/ml was straight line. The absorbance increased with increased in concentration. Thus the standard curve follows the Beer-Lamberts Law.

Table: Standard Calibration Curve of Rivastigmine in Phosphate Buffer pH 6.8

Figure: Standard Calibration Curve of Rivastigmine in Phosphate Buffer pH 6.8

3. Drug Excipients Compatibility Studies (FT-IR)

Both the polymer and pure drug's infrared spectra are examined. It has been found in this investigation that there is no chemical interaction between the polymer and Rivastigmine. The major peak in the drug and polymer mixture's infrared spectra was found to remain unchanged, indicating that there was no physical interaction due to bond formation between the two substances.

Figure: IR Spectra Of Pure Drug Rivastigmine

Figure: IR Spectra of Rivastigmine Carbopol Microsphere

4. Characterization of Rivastigmine Nasal Microspheres

Rivastigmine-loaded nasal microspheres were successfully formulated using varying concentrations of HPMC K4M (R1–R3) and Carbopol (R4–R6), with the drug-to-polymer ratios set at 1:1, 1:1.5, and 1:2 respectively. The batches were evaluated for percentage yield, particle size, drug content, entrapment efficiency, and mucoadhesion strength

Table: Characterization of Nasal Microspheres (F1 to F6)

(Values are average SD±, n=3)

4.A) Percentage yield

The percentage yield of all batches ranged from 62.56% (R1) to 72.18% (R3) for HPMC-based formulations and from 63.67% (R4) to 70.34% (R6) for Carbopol-based formulations. An increasing trend was observed with rising polymer concentration in both groups. The maximum yield was observed in R3, suggesting that higher polymer levels facilitate better encapsulation and recovery. Formulation R3 showed highest yield while formulation R1 showed lowest yield. It was found that average percentage yield was greater than 50 % for all the batches which shows the suitability of this method for preparation of microspheres. The results were showed in table 7.2. and figure 7.5

Figure: Percentage yield of formulations R1 to R6

4.B) Particle Size Analysis

Particle size of the microsphere is the crucial parameter during the formulation.  The particle size ranged from 24.61 ± 3.16 µm (R4) to 31.41 ± 2.46 µm (R3). A clear trend of increasing particle size with increasing polymer concentration was noted. This can be attributed to the increased viscosity of the polymer solutions, which leads to the formation of larger droplets during microsphere formation. Carbopol-based microspheres generally exhibited slightly smaller particle sizes compared to their HPMC counterparts at similar ratios. Formulation batch R4 showed lowest particle size, while batch R3 showed highest particle size of microsphere. All batch formulation showed the size of microsphere in micron range, showing effectiveness of method and technique. The results were showed in table 7.2

Figure: Average Particle Size Analysis of Microsphere Formulation R1 to R6

4. C) Drug Entrapment Efficiency and Drug Content

The DEE showed a steady increase with polymer concentration, ranging from 74.62 ± 1.64% (R4) to 81.30 ± 1.66% (R6) for Carbopol formulations, and from 75.25 ± 1.78% (R1) to 79.10 ± 2.19% (R3) for HPMC-based microspheres. The highest DEE was observed in R6, indicating that Carbopol at higher concentrations enhances entrapment, likely due to its strong gel-forming ability and higher viscosity that minimizes drug loss during preparation. It was also observed from the results that, as the polymer concentration increases, the entrapment efficiency was also increases, this could be because of more encapsulation efficiency with higher polymer weight.  An increase in polymer concentration in the internal phase shows increase in drug loading. This may be due to increase in viscosity of internal phase which reduces the migration of drug in aqueous phase, thus entrapping greater amount of drug.

Drug content varied between 72.35 ± 1.23% (R2) and 85.26 ± 1.17% (R6). In both polymer systems, an increase in drug content was observed with increasing polymer concentration. Notably, Carbopol-based formulations (R4–R6) exhibited higher drug content compared to HPMC-based batches, possibly due to stronger drug-polymer interaction and better encapsulation capabilities of Carbopol.

4. D) In Vitro Mucoadhesion Studies

Mucoadhesion is a critical parameter for nasal formulations. All formulations were tested for in vitro mucoadhesion studies and showed excellent muchoadhesive strength. It was noted that with increased in amount of polymer, the mucoadhesive strength of microparticles was increased. In this study, mucoadhesion ranged from 80.34 ± 1.65% (R1) to 89.51 ± 1.67% (R6). Carbopol-based formulations demonstrated superior mucoadhesive properties compared to HPMC-based ones at equivalent concentrations. This is attributed to Carbopol’s high density of carboxylic groups, which form hydrogen bonds with mucin, enhancing adhesion to the nasal mucosa. Among all, R6 exhibited the highest mucoadhesion, making it a promising candidate for nasal delivery of rivastigmine. Among the formulations batch R6 showed showed highest muchoadhesive strength, which cleared that the microparticles remain adhered for a prolonged period

4. E) Surface Morphology Study

The surface morphology of the drug loaded optimized microsphere (R6) was investigated by scanning electron microscopy. The microspheres have seen to be spherical morphology and smooth outer surface. The microsphere was also seen to non-aggregated, which reflects the effective methodology of microspheres formulation. The results of SEM of optimized batch R6 was shown in figure 7.7. 

Figure: SEM Image of Rivastigmine Loaded Optimized Nasal Microsphere (R6)

4. F) In Vitro Drug Release Study

In order to study the effect of different polymer concentration and to compare the drug release pattern of all batches of nasal microsphere prepared using two polymers: HPMC K4M and Carbopol, in different ratios with the drug was subjected to in vitro release study. In-vitro drug release studies were performed using Franz diffusion cell in 6.8 pH phosphate buffer for the period of 12 hr. The results of cumulative drug release were showed in table 7.3. The graph was plotted between percentage drug release and time and it was showed in figure 7.8. The in vitro drug release profiles of Rivastigmine nasal microspheres were studied over a 12-hour period. The formulations were

The drug release from batch R1 (1:1) was the fastest among all formulations, with 96.64% ± 2.18 release within 6 hours. Increasing the concentration of HPMC K4M slowed the release rate slightly. Batch R2 (1:1.5) showed 97.42% ± 1.41 release at 8 hours, while batch R3 (1:2) extended the release to 98.28% ± 1.20 at 10 hours. This delayed release with increasing polymer concentration can be attributed to the higher viscosity and thicker gel barrier formed by HPMC K4M, which slows drug diffusion. HPMC is known for its swelling and controlled release properties, and this is evident in the sustained release profile observed in R3.

Carbopol-based microspheres formulation’s (R4 to R6) showed a more prolonged and sustained release profile compared to HPMC-based formulations. Batch R4 formulated with drug to ppolymer ratio (1:1) released 86.2% ± 2.56 drug at 7 hours, and batch R5 (1:1.5) released 96.45% ± 1.66 at 10 hours. The most sustained release was observed in R6 (1:2), with 99.82% ± 2.89 drug release at the end of 12 hours. The slower release in Carbopol formulations is attributed to its higher viscosity and strong mucoadhesive properties, which help form a tighter gel matrix around the drug particles, thereby reducing the rate of drug diffusion. Additionally, Carbopol swells significantly upon hydration, further impeding drug release. From the study it was observed that HPMC K4M provided a relatively faster and moderate sustained release, making it suitable for shorter-duration nasal drug delivery. While Carbopol, on the other hand, exhibited more extended and controlled release, ideal for formulations targeting prolonged therapeutic effect.

From the dissolution study it was concluded that, polymer concentration and type significantly influence the drug release profile of rivastigmine nasal microspheres. Among all, carbopol base nasal microsphere batch R6 exhibited the most sustained release up to 12 hours, coupled with excellent mucoadhesion and entrapment efficiency. Hence, batch R6 emerges as the most promising formulation for controlled nasal delivery of Rivastigmine.

Table: In-vitro dissolution studies of Rivastigmine Nasal microspheres

(Values are average SD±, n=3)

Figure: In Vitro Dissolution Profile of Formulations R1 to R6

5.  Drug Release Kinetics

In order to determine the drug release kinetics of optimised batch formulation and to describe the model which best suited for drug release, the in-vitro release data of formulation F6 was subjected in various models such as zero order, first order, higuchi and korsmeyer peppas kinetics models. Model fitting release profiles of formulation F5 was showed in table 7.4.  The highest correlation coefficient (r² = 0.994) was observed for the Zero Order model, indicating that the drug release from F6 followed zero-order kinetics, where the drug is released at a constant rate independent of concentration. This is considered ideal for controlled release formulations, especially in systems like nasal microspheres, where sustained and predictable release is desirable for prolonged therapeutic effect.

The Higuchi model also showed a good fit (r² = 0.919), suggesting that diffusion plays a significant role in the drug release mechanism. However, since the Zero Order model had a superior correlation, it can be inferred that the release is governed more by the formulation matrix properties than by simple diffusion.

The Korsmeyer-Peppas model exhibited an r² value of 0.983 with an n value of 0.723, which lies between 0.5 and 1.0, indicating an anomalous (non-Fickian) transport mechanism. This implies that the drug release is controlled by a combination of drug diffusion and polymer matrix relaxation/erosion mechanisms. The poor fit of the First Order model (r² = 0.474) suggests that the release is not concentration-dependent, further supporting the Zero Order and anomalous release mechanisms.

From the study it was concluded that, formulation F6 follows Zero Order release kinetics, suggesting a constant and controlled drug release. The Korsmeyer-Peppas model further indicates an anomalous (non-Fickian) mechanism, which is favorable for achieving sustained drug delivery through nasal microspheres. This combination of release patterns makes F6 a promising formulation for effective and prolonged intranasal delivery of rivastigmine.

Table: Model Fitting Release Profile Of Formulations R6

Figure: Cumulative % Drug Release V/S Time (Zero Order Kinetics) Of Batch R6

Figure: Log Cumulative % Drug Retained V/S Time (First Order Kinetics) Of Batch R6

Figure: Cumulative % Drug Release V/S Square Root Of Time (Higuchi Release Mechanism) Of Batch R6

Figure: Log Cumulative % Drug Release V/S Log Time (Korsmeyer-Peppas Release Mechanism) Of Batch R6

6. Stability Studies:

The accelerated stability studies were carried out according to ICH guidelines optimized formulation F6 was packed in strip of aluminum foil and this packed formulation was stored in stability chamber maintained at 40^oC and 75% RH (Zone III conditions as per ICH Q1 guidelines) for 3 months. The microspheres were evaluated before and after specified period of time for change in appearance, drug content, percentage muchoadhesion and In vitro release. After a period of 3 months, the sample were observed for any change on appearance. It was observed that microsphere was devoid of any change in color or appearance of any kind of spot on it. It was also noted that microsphere was free of any kind of microbial or fungal growth

bad odour. The drug content of formulation F6 after 3 months was found to be 83.20 ±1.12% which shows there was small decrease in drug content but difference is insignificant. In vitro drug release of optimized formulation F5 after stability period was found as 97.66±1.80%. The % muchoadhesion of optimized formulation F5 was found to be 87.41±0.83% after stability period. Thus from the stability study data it was confirmed that, the optimized batch F5 showed very negligible changes in results after stability period and hence found to be stable. The data of stability study was given in table 7.5

Table: Stability Studies of Microsphere Formulations R6

CONCLUSION:

From the extensive analysis and results obtained in this study, it can be concluded that Rivastigmine nasal microspheres formulated using Carbopol 934P, especially the F6 formulation (1:2 drug-to-polymer ratio), offers a promising platform for nasal drug delivery. It demonstrated High drug entrapment efficiency and drug content, Excellent mucoadhesive properties, Sustained drug release for up to 12 hours, Favourable zero-order kinetics indicating controlled release and Stability under accelerated conditions

These characteristics suggest that the F6 formulation is an effective carrier for intranasal delivery of Rivastigmine, potentially improving patient compliance and therapeutic outcomes in the management of diseases like Alzheimer's. Future in vivo studies and clinical evaluations are warranted to confirm the efficacy and safety of this delivery system in biological environments.

ACKNOWLEDGMENT: We are thankful to the management and Principal of S.G.S.P.S. Institute of Pharmacy Kaulkhed, Akola for their support. I am grateful to my guide and faculty members for their guidance.

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