Introduction

The colon targeted drug delivery system (CDDS) is designed to deliver drugs to the large intestine in the lower GI tract. Irritable bowel syndrome (IBS), colorectal cancer, and inflammatory bowel disorders (IBD), which include ulcerative colitis and Chron’s disease, have gained a lot of attention for use as prospective routes of treatment for the local treatment of a number of colonic diseases^{1, 2}. The principal site of organ for the digestion of food eaten orally is the stomach. The biological control of acid secretion by the parietal cell is significant for the use of various medicines to lower gastric acidity ¹.

Colon targeted drug delivery system(CDDS) targets administration of medications into the lower GI tract, particularly in the large intestine. Colon specific targeted drug delivery have attracted a great deal of attention for application as potential route of treatment for the local treatment of a variety of colonic diseases such as irritable bowel syndrome (IBS), colorectal cancer and inflammatory bowel diseases (IBD) which includes ulcerative colitis and Chron’s disease^{1, 2}. Stomach is the primary site of organ for the digestion of orally consumed food. The parietal cell secrets acid and biological regulation of acid secretion has significance for the use of different agents to reduce gastric acidity ¹. Massive amounts of excess gastric acid and pepsinogen are secreted, which damages the gastroduodenal mucosa and causes fatal ulcerations ^2-4. As a result, antacids, proton pump inhibitors, and other types of stomach acid secretion inhibitors can be used as a form of treatment ^5. To ensure a controlled and precise release of the medication, microencapsulation can offer protection to the gastric mucosa ^{6, 30.}

Inflammatory bowel disorders are one of the numerous diseases that can be treated using the GIT microflora of the colon. The prime reason behind the development of colon-targeted medication delivery systems is the modification of pH at various locations throughout the GIT tract ^7-9. The effective development of various CDDS is greatly aided by the promising modulation of pH difference in GIT. Utilizing a pH-sensitive polymer, enteric coating can be created for solid dosage forms such capsules, tablets, and pellets to protect cells from acidic pH ¹⁰. pH-sensitive polymers for colon can be applied for the formulation development which can be specific to gastric pH of the digestive & proximal part of the small bowel whereas disintegrate selectively at intestinal pH¹¹. Eudragit L & S are copolymers of methacrylic acid & methyl methacrylate. Eudragit L is water-soluble at pH 6 & can be used to deliver medications to the small and large intestine ^12-14. The current study focussed on development and evaluation of colon-specific Esomeprazole microcapsules. 

Materials and Methods

Esomeprazole was obtained from Cadila Healthcare Ltd, Ahmedabad, India. Eudragit L100 was from Evonik Industries, India. All chemicals and reagents used were of analytical grades.

Formulations Development using 3² Factorial Design

Specified quantity of Eudragit L100 and Esomeprazole was accurately weighed and dissolved in methanol. As per Table 1, polymer and drug solution was added drop wise to liquid paraffin containing tween 80 as an emulsifying agent. The resultant o/o emulsion was stirred for 3 hours. For three hours, the resulting o/o emulsion was swirled. Vacuum filters were used to filter the developed formulation, which was then washed in petroleum ether and dried overnight.^[15-17] Developed microspheres were evaluated for various characteristics such as particle size distribution, entrapment efficiency, surface morphology, and percent yield. In vitro release studies were performed to evaluate release kinetics and further investigated for the stability of Esomeprazole in UV and solar light. 

Determination of Mean Particle Size

A glycerine based suspension of developed microsphere was subjected for particle size determination using an optical microscope. The size of developed microspheres was detected at 25 °C^{18, 19}. 

Entrapment Efficiency (EE)

Drug entrapment efficiency was investigated by carefully solubilising 100 mg of microspheres in 100 mL of methanol, followed by filteration of the solution. The amount of Esomeprazole in the filtrate was determined spectrophotometrically at 305 nm (Shimadzu 1201). The following equation was used to calculate % entrapment efficiency ²⁰.

Production Yield

The weight ratio of microspheres to loading quantity of the polymer and drug was used to calculate the production yield of microspheres containing a medicament. The production yield was calculated using following equation.

Micromeritic Characteristics of Microspheres

The developed microspheres were evaluated for Micromeritical properties such as particle size, tapped density, true density, compressibility index etc. 

Scanning Electron Microscopy (SEM)

Morphological characteristics of the samples were tested using a scanning electron microscope (Model JSM -5600, JEOL), Japan. Air dried microspheres were covered with platinum for 5-10 minutes using an auto fine covered ion sputter (JEOL-JFC-1600, JEOL, and Japan) and analysed. SEM was operated at a low frequency voltage of approximately 15 KV and load power of approximately 80 MA^{21, 22}.

Fourier Transform Infrared Spectroscopy (FTIR)

IR spectra of pure drug as well as mixtures of drug and polymer were investigated to determine compatibility of drug with various types of polymers. Spectra of pure drug, polymer mixture, and polymer mixture containing pure drug was compared for compatibility evaluation ²³. 

Differential Scanning Calorimetry (DSC)

API and developed formulation were subjected for analysis to DSC (Shimadzu,DSC 60), which was equipped with a thermal analyzer. Approximately precisely weighed 3 mg of sample was placed in a aluminium pan and hermetically sealed with an aluminium lid. The system was subjected to liquefied nitrogen gas at a flow rate of 50 mL/min, and temperature was raised from 50°C to 400°C at a rate of 10 ° C/min²⁴.

In-vitro Dissolution Studies

USP XXII basket-type dissolution apparatus was used to determine entrapment efficiency of developed microspheres. Phosphate buffer with a pH of 1.2 (900 mL) was maintained at 37ºC with a cycle speed of 100 rpm. A sample volume of 10 mL was taken at one-hour intervals and subjected to spectrophotometric analysis at 290 nm to determine quantity of drug which released from formulation in the dissolution media. The volume of dissolution media was maintained by adding 10 mL of fresh dissolution media after each sampling for a period of two hours to evaluate drug release in an acidic medium. Next to that, phosphate buffer pH 7.4 was applied to study release of drug from formulation in the dissolution media. The results of every test were recorded in triplicate.²⁵.  

Kinetics Modeling of Drug Dissolution Profiles

The zero order, first order, Higuchi and Korsemeyer-Peppas were used to determine the kinetic expression of the drug release with dissolution profile of all batches ²⁶.

Zero Order Treatment

The equation for zero order treatment is represented as,

Where,

Q_t -Quantity of drug released in time (t)

Q_o -Initial amount of drug in solution

K_o -Zero order release constant

First Order Treatment

The equation for the first order treatment is demonstrated as-

Where,

c – Quantity of drug remaining unreleased at time (t)

c₀ – Initial amount of drug in solution

k – First order rate constant 

Higuchi’s Model

The Higuchi equation is denoted as-

Where,

Q_t -Quantity of drug released in time t

k -Higuchi’s constant

A linear association between quantity of drug released (Q) versus square root of time (t^1/2) is witnessed if the drug release from the matrix is diffusion controlled. 

Korsmeyer- Peppas Model

The Korsmeyer-Peppas model communicates drug release exponentially to time. It is expressed by the below equation-

Where,

M_t / M_inf -fractional release of drug

a- Constant dependent on structural and linear characteristics of the drug dosageform

n- Release exponent

The value of n designates the drug release mechanism.

For a slab the value n = 0.5 designated Fickian diffusion and values of n between 0.5 and 1.0 or n = 1.0 indicated non-Fickian mechanism.

Stability Study

The optimized formulations were successfully subjected to stability studies. The stability study was carried out at a temperature of 45 ̊ ± 2 ̊C and a relative humidity of 75 % ± 5 % RH for a period of one month. Evaluations of the formulation’s drug content and in vitro drug release were performed at intervals of 15 days ^{27, 28}. 

Result and Discussion

By using a solvent evaporation approach, Esomeprazole microspheres were developed and subjected to several evaluation criteria. Eudragit L 100 was used to create Esomeprazole microspheres. For maximal drug entrapment and consistent particle size distribution, formulations were prepared using various amounts of polymer in various ratios. With formulation batches containing 1:1 and 1:2 ratios of drug and polymer, uniform microspheres did not developed. Optimum rpm of 800 was found to be suitable with respect to morphology, yield and percent drug entrapment efficiency for formulations developed using drug: polymer ratios of 1:3 and 1:4. 50 mL of liquid paraffin for 1:3 and 1:4 formulations was detected appropriate for desired result. 

Table 1: Formulation Development of Esomeprazole Microspheres.

Statistical Data Analysis

Statistical analysis covers a set of experiment and makes certain accurate and persuasive interpretation of results of a study which determine the response variables, conducting appropriate statistical tests to select best possible model, fitting mathematical models to the data, and determining the values of independent formulation variables to produce optimum response. The present study applied a factorial design which is one of the popular statistical experimental design to optimise the formulation as well as to discover the interactions, if any, between the factors chosen

To study the effects of independent variables on its attributes and performance, a 3² factorial design was applied. The independent variables such as concentration of polymer and rpm at three levels were evaluated for their effect on % drug release, % entrapment efficiency, particle size and % yield. The responses obtained are given in Table 2.

Entrapment efficiancy (actual factors)

The 3D response surface plots of the factorial model were ploted to demonstrate the effect of the variables on the entrapment efficiency. Figure 1(A) showed the effect of the amount of Eudragit  L100 and rpm on the entrapment efficiency. It was demonstrated that the entrapment efficiency depends upon Eudragit  L100 and rpm. Meanwhile, it was found to be the most important element affecting the effectiveness of entrapment.

Figure 1: (A). Contour plot curve depicting the effect of factorial variables (a)Eudtragit L100 and (b)rpm on the entrapment efficiency. Click here to view figure

Influence of two variables such as polymer concentration and rpm were selected at two levels. According to the surface response graph Figure 1(B), the highest amount of drug entrapment was obtained when both the polymer concentration and the rpm were remained high (280 mg and 800 rpm).

Figure 1: (B). Three-dimensional view of entrapment efficiency with respect to Eudragit  L100 and rpm obtained by D.E.7.0 related to given data. Click here to view figure

On the other hand, very less entrapment was detected when both the variables were applied at low level. Thus from current outcome, it was concluded that both variables had positive effect on percent drug entrapment (Table 2).  

Table 2: Experimental Design of the Optimization of Formulations.

With increased concentration of polymer, viscosity also increases whereas high energy was required for formation of uniform microspheres. Low stirring rate was not sufficient to break emulsion into droplets which was resulted in lump formation. Hence from the above studies it was detected that when polymer concentration increased, stirring rate should be increased for production of uniform and optimum size microspheres.

Drug release (actual factors)

It was concluded that Eudragit  L100 (factor A) and rpm (factor B) had individual effect on drug release.

Conferring to the achieved results, the established models are statistically precise and can be used for next examination.

The obtained graph (Figure 2) showed as the concentration of Eudragit  L100(variable A) increases upto medium level and at that level reduction in drug release was detected. It can be concluded that factor B has significant effect on drug release.

Figure 2: Effect of interaction between Eudragit  L100 and RPM on the drug release. Click here to view figure

Effect of interaction between Eudragit L100 and rpm on the drug release was obtained. Factor A and B have its individual significant effects. Eudragit L100 and rpm at medium level showed significant impact on drug release.

The 3D response surface plots of the factorial model were ploted to demonstrate the effect of the variables on the drug release. Figure 3(A and B) showed the effect of the amount of Eudragit L100 and rpm on the drug release. It was showed that dependency of drug release on Eudragit L100 and rpm. Meanwhile, selected factors showed most significant effect on the drug release. 

Figure 3: (A). Contour plot detected effect of Eudragit  L100 and RPM on the drug release. Click here to view figure

Figure 3: (B). Three-dimensional view of drug release with respect to Eudragit  L100 and RPM obtained by D.E.7.0 related to the given data. Click here to view figure

Approaches For Optimized Solution

Based on acceptance criteria and desirability factor, Design Expert (Version 7.0) software suggested 30 optimum formulations. From the suggested formulations, selected optimized batch (OB1) indicated value of desirability closest to 1, which was considered as most favourable. As per Figure 4, in vitro release of optimized batch was detected with more than 80%. Therefore, from the results obtained for batch 1 were selected from 30 possible solutions as OB1, has been shown in table 3.

Table 3: Suggested Formulations and their Responses by Design Expert7.0.

Figure 4: In-vitro release profile of optimized Esomeprazole microspheres. Click here to view figure

Particle Size

According to Figures 5A and 5B, developed microspheres with a uniform mean particle size distribution were obtained with a particle size distribution up to 210.34 µm. Following Table 4 provided more information on optimised formulation.

Table 4: Evaluation of Optimized formulations.

*OB1= Optimised Batch 1 

Figure 5: (A). Scanning Electron Microscopy of Esomeprazole microspheres prepared with Eudragit  L100 at 1000X. Click here to view figure

Figure 5: (B). Scanning Electron Microscopy of Esomeprazole microsphere prepared with Eudragit  L100 at 2,200 X. Click here to view figure

Percentage Yield

It was found that the batches F3 and F5 simultaneously produced a maximum practical yield of approximately 95.33 and 95.64 percent respectively. ³¹.

Micrometric Properties of Microspheres

The flow characteristics of the developed formulations were determined by angle of repose. All the preparations indicated an angle of repose within the range which is 29.87° to 39.34° which was acceptable for free flowing characteristic ²². 

Fourier Transform Infrared Spectroscopy (FTIR)

The spectra of pure drug and polymer combination were matched with IR spectra of pure drug. The IR spectra of the drug, polymer and drug with polymer combination were shown in figure 6.

Figure 6: FTIR spectra of Esomeprazole, Eudragit  L 100 and developed formulation. Click here to view figure

In the FTIR spectra of esomeprazole, the CH aromatic stretching was observed in the range of 3100 cm-1to 3000 cm^-1, the CH aliphatic stretching was observed in the range of 2944 cm^-1 to 2844 cm^-1, and the NH stretching was observed at 3414 cm^-1. CH aromatic stretching was observed at 3198 cm^-1, CH aliphatic stretching was observed at 2948 cm^-1, and NH stretching was observed at 3491 cm^-1 when the polymer and medication were combined. The principle peaks that were accomplished for the combinations were almost identical to those that were achieved for the drug. There was no evidence of any variation in the IR spectrum of Drug-Eudragit  L 100. Because there was no significant shift in the absorption bands of the drug and drug polymer combination, it was determined that there was no chance of aninteraction taking place.

Table 5: FTIR spectra interpretation.

Differential Scanning Calorimetry (DSC)

The assessment of the thermograms evidentlyexposed no physical interface between the polymer and the drug in the established formulation (Figure 7). 

Figure 7:  DSC of Esomeprazole and developed formulation. Click here to view figure

In-vitro Release

Theoretical Drug Release Profile of Esomeprazole

Theoretical release rate of Esomeprazole microspheres was considered by using pharmacokinetic parameters.From observation (Figure 8) it was observed that drug release rate of the formulation F5 was found greater in comparison to other formulation. In vitro release data showed that increase in polymer concentration decreases drug release. As the polymer concentration increases, the diffusion path for drug to get release from the matrix increases; that might be the reason for decrease in drug release with respect to enhanced in polymer concentration. Within first hour, 16–25% of drug was released followed by a plateau pattern for 8 hours. The burst release was detecded due to the unentrapped drug presence on the surface of microspheres. Thes burst release was then followed by drug release from microspheres i.e. loaded or entrapped drug for a desired prolonged period of time. 

Figure 8: Comparative dissolution profile of various batches of Esomeprazole loaded microsphere, mean ± SD, n=3. Click here to view figure

Kinetics Models of Drug Dissolution

In-vitro drug release data was subjected to different kinetic equations to witness the mechanism or kinetics of drug release from microspheres. Association measurements of separate batch with functional equation were given in Table 5. All batches displayed higher relationship with Higuchi plot and zero order as compared to first order and Hixon-Crowel whereas predominant drug release was detecded by diffusion type of sustained release mechanism.

Table 6: In-vitro Release Data fitted in to Various Models

 Stability Study

The short term accelerated stability study was accomplished at 40⁰C and 75% RH for 1 month. The optimized batch was verified for its physical presence, drug content and drug release. The optimized batch was found to be stable as per the outcome of data for the stability study after 1 month. Table 6 showed the results obtained in the stability study of the prepared formulation.

Table 7: Stability Study of Optimized Formulation.

*OB1= Optimised Batch 1

Conclusion

The various active pharmaceutical ingredients were compromised by the enormous excess stomach acid and pepsinogen secretion. Therefore, it is crucial to maintain safeguard such pH and acid sensitive candidate in order to facilitate the medication administration. There are so many approaches to protect acid sensitive drug from the gastric envoirnment, but microencapsulation approach provides the controlled drug delivery as well. Microencapsulation is utilised to achieve regulated release of the different drugs, to achieve targeted drug delivery, and to protect sensitive components from the stomach environment. In the recently published research, the solvent evaporation process was used to create Eudragit L100 microspheres that were loaded with esomeprazole. It was determined from the current investigation that the manufactured encapsulated esomeprazole-loaded microspheres proved to be a significant method for improving esomeprazole’s stability in acid. In addition, it was claimed that the created microspheres were particularly effective at protecting the medicine in an acidic environment and made it acid resistant with sustained release activity for duration of 10 hours, increasing patient compliance.

Acknowledgment

Author would like to acknowledge to Trustees, Bhujbal Knowledge City, MET’s institute of pharmacy, Nashik, Maharashtra, India for providing the necessary facilities to carry out current work.

Conflict of Interest

The authors declare no conflicts of interest

Funding support

The authors received no external funding for this research

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