Evaluation of microbeads:

Surface morphology: The surface morphology of coated LR microbeads was examined by method described by Roane, Pepper.¹⁶

Percent yield: Percent yield of this ionotropic method was determined as described by Chavarri.¹⁷ The percent yield was calculated by using following formula:

[Percent yield = (Weight of microbeads formed/ Weight of probiotics + Weight of polymers) × 100]

Entrapment efficiency: The entrapment efficiency was evaluated as per the method described by Chavarri et al. 2010.¹⁷The conical flask containing simulated colonic fluid (SCF) and dried coated microbeads (100 mg) were shaking for 2 hours to disintegrate the microbeads. An aliquot (1 ml) was removed from the mixture and after proper dilutions, they were studied via viable colony count (expressed in a number of spore colony forming units (CFU)) by pour plate method. The entrapment efficiency value was calculated using the following formula and was reported in percent:

[Percent entrapment efficiency = (Practical viable spore count value/ Theoretical viable spore count value) × 100]

Bead size: The dried coated microbeads (20 in number) were evaluated for their size at ×100 magnifications using a calibrated optical microscope having a micrometre scale (Microtech, India). The microbeads were mounted in light liquid paraffin and the diameter of beads was measured and the mean diameter was calculated as per method described by Chavarri et al.¹⁷

Development of new method: In our study, a new method for assessment of in vitro swelling, mucoadhesion, release and viability of probiotics was developed. The hypothesis of new method was based upon our physiological functions that is food and drugs after oral administration first exposed to gastric enviorment (average 2 hours) followed by small intestine (average 3 hours) and colon (average 4 hours) as described by Read et al.¹⁸In previous studies parameters named as in vitro swelling, mucoadhesion, release and viability of probiotics from prepared formulations were assessed after exposure to simulated gastric fluid (SGF) and simulated small intestinal fluid (SIF) separately. Nevertheless, assessment of above stated parameters by separately exposure of formulations to SGF and SIF may not reflect the true simulation of gastrointestinal physiological system and exposure. The upper GI environment exposure prior to colonic environment exposure may affect the encapsulating coat and thereby may affect the properties like mucoadhesion, swelling and release of drug from test formulation. Thus, the reliability on the outcomes of these parameters may remain under questions. Therefore, to overcome these limitations we have developed a new method of assessment of mucoadhesion, in vitro swelling and release of drug from test formulation after sequential exposure to SGF for 2 hours followed by SIF for 3 hours and lastly to SCF for 4 hours. Therefore, as per our new developed method in vitro swelling, release and viability of probiotics, mucoadhesion of prepared microbeads of all formulation batches was assessed by incubating microbeads in SGF for 2 hours followed by incubation in SIF for 3 hours and incubation in SCF for 4 hours. The all stated parameters were also assessed by conventional methods as reported by many authors and results were compared with our new developed method.

In vitro swelling study: The percent swelling of LR microbeads was determined in simulated colonic fluid. Microbeads were incubated in SCF for 4 hours. Hundred mg of microbeads were taken from each formulation batch and were separately incubated in petri dish containing SCF for 4 hours. The percent swelling at the end of 4 hours in SCF was calculated using the following equation.

[Percent swelling = {(Wt of dried microbeads after incubation – wt of microbeads before incubation)/ Wt of microbeads before incubation} × 100]

Ex vivo mucoadhesion test: The mucoadhesive strength of coated LR microbeads was determined by ex vivo mucoadhesion method with slight modification.^19,20 Fresh pieces of goat gastrointestinal tissues such as stomach, small intestine and large intestine were collected from nearest slaughter house and used for assessment of ex vivo mucoadhesion. The mucosal tissues (size approximately 10 cm²) of stomach, small intestine and large intestine were mounted on to glass slide 7.5×2.5 cm size with the help of cynoacrylate glue. Fifty microbeads from each formulation batch were placed over each tissue and immediately mounted on the slide which was hung onto the arm of tablet disintegrating test apparatus. The glass slide with tissue specimen was given a slow, regular up and down movement in the beaker (500 ml) containing SGF for 2 hours, SIF for 3 hours and SCF for 4 hours. The glass slide with LR microbeads on stomach tissue was exposed to SCF, similarly microbeads on small intestinal and colonic tissues were exposed to SIF and SCF respectively. The number of microbeads fell down from the tissue were noted at various time interval. The percent mucoadhesive strength of prepared formulations of microbeads was calculated by using following formula:

{Percent mucoadhesive strength = (The initial numbers of beads adhere over the tissue – The number of microbeads fell down from the tissue)/ The initial numbers of beads adhere over the tissue) × 100}

In another set of experiment, mucoadhesive strength of LR microbeads of all formulation batches was assessed by our new developed method. Microbeads (approximate 50) were incubating in SGF for 2 hours followed by incubation in SIF for 3 hours. The microbeads after incubation of SIF were adhered to colonic tissue (large intestine) of goat and were incubated in SCF and thereafter mucoadhesive strength was assessed as per above mentioned formula.

In vitro release/viability study:

The in vitro release study of LR from their microbeads was performed by our new developed method. Microbeads were incubating in SGF for 2 hours followed by SIF for 3 hour and SCF for 4 hours and fluids were stirred at 100 rpm/min at the temperature of 37 ± 2 ºC. At predetermined time intervals (0 minute, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 240 minutes and up to 540 minutes) aliquots of medium were taken out and viability assessment was performed as above mentioned. The viability of released LR at predetermined time intervals was also plotted against the time.¹⁷ One gm of test formulations (LR microbeads) or free LR were added to SGF and it was stirred at a speed of 100 rpm/min at the temperature of 37 ± 2 ºC for 2 hours, SIF for 3 hours and SCF for 4 hours and fluids were stirred at 100 rpm/min at the temperature of 37 ± 2 ºC. The same volume of fresh and sterile simulated gastrointestinal fluids was added to replace the withdrawn samples. The withdrawn samples were subjected to assessment of viable cell count. The viability of released LR at predetermined time intervals was plotted against the time.

RESULTS:

Fabrication of coated LR microbeads: The LR encapsulated microbeads were prepared via ionotropic gelation techniques using variable concentration of pectin and NaCMC as described by many authors.¹⁸ The prepared LR microbeads were coated with CAP and Eudragit S 100 to protect the encapsulated LR from the upper GI harsh environment and release of viable cells at their target site of action that is colon.

Entrapment Efficiency:

The percent entrapment efficiency of LR microbeads with LMP as encapsulating polymer was found in the range 60.33±2.80 to 85.81±1.83 % and with NaCMC as encapsulating polymer was found in the range between 6 2.86±2.09 to 85.06±1.69 %. Our study results indicate that entrapment efficiency of LR microbeads with probiotics: polymer ratio (1:3) was found to be significantly greater than probiotics: polymer ratio (1:1) and (1:2) (Table 2).

Percent yield:

The percent yield value of all the formulations of LR microbeads (LR1 to LR6) which were prepared by LMP as encapsulating polymer was found in the range of 61.46±2.60 % to 83.20±2.88 %. The percent yield of all formulation LR microbeads (LR7 to LR12) of which were prepared by NaCMC as encapsulating polymer was found in the range of 60.41±4.88 % to 82.83±2.84 %. The percent yield value of LR microbeads formulations named as LR3, LR6, LR9 and LR12 were found to be significantly greater than other formulations i.e. LR1, LR2, LR4, LR5, LR7, LR8, LR10, and LR11. Therefore, it was observed that the (percent yield) yield value of LR microbeads was increased with an increase in polymer concentration (Table 2).

Bead Size:

The average particle size distribution of the LR microbeads has been evaluated using optical microscope with calibrated scale. Average size of LR microbeads with LMP as encapsulating polymer was found in the range 843.67±3.21 to 947.33±3.05 µm. Average size of LR microbeads with NaCMC as encapsulating polymer was found in the range 980.67±4.04 to 1100.3±2.92 µm. The results showed the size of the LMP and NaCMC based LR microbeads was increasing with an increase in polymer concentration (Table 2).

Surface Morphology:

Surface morphology of the microbeads was evaluated by scanning electron microscopy. The majority of the microbeads were observed spherical in shape (Figure 3). However, some of the microbeads were found to be irregular or bit elongated in shape. These changes in the shape might happen at the drying step where microbeads were started to shrink due to water loss. Few pores or cracks were observed over the surface of microbeads which might occur during the drying process (Figure 3).

Table 2: Entrapment efficiency, percent yield and size of LR microbeads Entrapment efficiency:

Formulation code	Entrapment efficiency	Percent yield	Size(µm)
LR1	64.1±3.30	74.37±3.67	843.67±3.21
LR2	70.33±5.5	76.04±2.88	871.67±4.04
LR3	85.63±4.25^a,b	79.11±2.67^a,b	943.33±5.77
LR4	61.00±3.86	77.47±3.88	872.33±2.51
LR5	68.76±3.45	78.15±3.22	882.33±3.78
LR6	84.28±4.43^a,b	80.08±2.87^a,b	947.33±3.05
LR7	64.23±3.85	72.22±3.86	980.67±4.04
LR8	70.87±2.80	74.31±3.12	995.67±5.85
LR9	84.42±5.38^a,b	77.02±2.44^a,b	1100.3±2.92
LR10	63.87±3.51	75.38±3.73	982.33±2.51
LR11	71.34±3.76	76.15±3.22	997.67±4.50
LR12	83.73±4.52^a,b	79.52±2.65^a,b	1050.25±4.5
Note: All data are presented as mean value ±SD & n=3

Each group (n=3) represents mean ± standard deviation. One-way ANOVA followed by Tukey's multiple comparisons test; F (11, 24) = 49.15, p< 0.0001 for evaluating the effect of encapsulating and coating polymers on entrapment efficiency of viable cells. ^ap < 0.05 entrapment efficiency of LR: polymer (1:3) Vs entrapment efficiency of LR: polymer (1:1),^bp < 0.05 entrapment efficiency of LR: polymer (1:3) Vs entrapment efficiency of LR: polymer (1:2)

Percent yield:

Each group (n=3) represents mean ± standard deviation. One-way ANOVA followed by Tukey's multiple comparisons test; F (11, 24) = 17.99, p< 0.0001 for evaluating the effect of encapsulating and coating polymers on percent yield of formulation batches of LR. ^ap < 0.05 Percent yield of LR: polymer (1:3) Vs percent yield of LR:polymer (1:1),^bp < 0.05 Percent yield of LR: polymer (1:3) Vs percent yield of LR: polymer (1:2)

Figure 1: Scanning electron micrographs of LR encapsulated microbeads

A = CAP coated LMP microbeads of LR, B = Eudragit S 100 coated LMP microbeads of LR, C = CAP coated NaCMC microbeads of LR, D = Eudragit S 100 coated NaCMC microbeads of LR.

Swelling index: Swelling studies of LR microbeads was performed to assess the influence of gastrointestinal fluids pH or precisely gastrointestinal environments on swelling of microbeads by incubating in SGF for 2 hours followed by SIF for 3 hours and in SCF for 4 hours. Percent swelling of LR microbeads was very low or negligible in SGF and SIF whereas formulations (LR3, LR6, LR9 and LR12) of LR microbeads have shown good swelling in SCF as compared to formulations (LR1, LR2, LR4, LR5, LR7, LR8, LR10, LR11). Therefore, swelling index of all the formulations has shown increment with the increase of polymer concentration and at colonic pH or environment. Percent swelling of LR microbeads in SCF was given in the (Figure 2).

Figure 2: Effect of various encapsulating and coating polymers on in vitro swelling of LR microbeads in SCF

Each group (n=3) represents mean ± standard deviation. One-way ANOVA followed by Tukey's multiple comparisons test; F (2, 22) = 1.344, p< 0.2815 for evaluating the swelling index of probiotic:polymer ratio of LR microbeads.^ap < 0.05 percent swelling of LR: polymer (1:3) Vs mucoadhesive strength of LR: Polymer (1:1), ^bp < 0.05 percent swelling of LR: polymer (1:3) Vs percent swelling of LR: Polymer (1:2)

Ex vivo mucoadhesion: Result of mucoadhesion study revealed formulations LR3, LR6, LR9 and LR12 have shown significantly high mucoadhesive strength as compared to other prepared formulations (LR1, LR2, LR4, LR5, L7, LR8, LR10, and LR11) at colonic environment (Figure 3). All the formulations showed very less or negligibl mucoadhesion in SGF and in SIF. Therefore, this experiment also confirmed that mucoadhesive strength of our prepared microbeads formulations has significantly increased with increase in polymer concentration at colonic environment. The results of our new method of sequential exposure also indicate that LR microbeads remain stable in upper gastrointestinal harsh environment and adhere at colonic region a desired site of probiotics action. The values of percent mucoadhesive of all the formulations in SCF after incubation in 2 hr SGF and 3 hours in SIF are presented in (Figure 4)

Figure 3: Effect of various encapsulating and coating polymers on ex vivo mucoadhesive strength of LR microbeads in SCF

Each group (n=3) represents mean ± standard deviation. Two-way ANOVA followed by Tukey's multiple comparisons test; F (11, 144) = 71.60, p< 0.0001 for evaluating the mucoadhesive strength at different intervals and F (5, 144) = 2325, p< 0.0001 for evaluating mucoadhesive strength of various probiotic: polymer ratio of LR formulations. ^ap < 0.05 mucoadhesive strength of LR: polymer (1:3) Vs mucoadhesive strength of LR:Polymer (1:1), ^bp < 0.05 mucoadhesive strength of LR: polymer (1:3) Vs mucoadhesive strength of LR:Polymer (1:2)

Figure 4: Effect of various encapsulating and coating polymers ex vivo mucoadhesive strength of LR microbeads in SCF after incubating 2 hours in SGF followed by incubating in 3 hours SIF

Each group (n=3) represents mean ± standard deviation. Two-way ANOVA followed by Tukey's multiple comparisons test; F (11, 144) = 72.93, p< 0.0001 for evaluating the mucoadhesive strength at different time intervals and F (5, 144) = 993.6, p< 0.0001 for evaluating mucoadhesive strength of various probiotic polymer ratio. ^ap < 0.05 mucoadhesive strength of LR: polymer (1:3) Vs mucoadhesive strength of LR: Polymer (1:1),^bp < 0.05 mucoadhesive strength of LR: polymer (1:3) Vs mucoadhesive strength of LR: Polymer (1:2)

In vitro release and viability study: As release of probiotics assessed in the form of viable cells count therefore, aliquots of each medium were collected during in vitro release study and were also used for viability assessment of LR. The finding of experiments has shown that release and viability of probiotics is significantly higher in SCF compared to SGF and SIF (Figure 5). The results of our new method of sequential exposure also indicate that LR microbeads remain stable in upper gastrointestinal harsh environment and significantly higher number of viable probiotics releases at colonic environment which is a desired site of probiotics action.

Figure 5: Effect of various encapsulating and coating polymers on in vitro release of LR viable cells from LR microbeads in SGF for 2 hours followed by SIF for 3 hours and SCF for 4 hours

Each group (n=3) represents mean ± standard deviation. Two-way ANOVA followed by Tukey's multiple comparisons test; F (3, 88) = 0.06778, P = 0.9769 for evaluating the effect of encapsulating and coating polymers on release of viable cells and F (10, 88) = 1042, P < 0.0001 for evaluating the number of viable cells released at different time intervals. ^ap < 0.05 for evaluating the number of viable cells released at 0 hour Vs and 4 hours onwards

DISCUSSION AND CONCLUSION:

Probiotics are live microorganisms are primarily used for prevention and treatment of antibiotic associated diarrhea (AAD), Clostridium difficile infections (CDI) and chemotherapy induced diarrhea etc.^5,21

In present study, colon targeted microbeads of LR were prepared and assessed for their prevention from upper GI harsh conditions. In this study, ion gelation microencapsulation method was used to prepare colon targeted microbeads of probiotics. This method offers advantages over other methods of microenapsulations such as simple technique and requires no use of organic solvent. The technique involves interaction of a cation (or an anion) with an ionic polymer to generate a highly cross linked structure. However, microbeads of LR with LMP and NaCMC were not previously prepared by ion gelation method. Therefore, in this study, LMP and NaCMC were used as microencapsulating polymers for preparing microbeads of LR with ion gelation method.

LMP an anionic polymer consist of linear chains of (1-4) linked α-d-galacturonic acid units which forms a rigid gel/hydrogel/microbeads (calcium pectinate) by crosslinking with calcium or the multivalent cation. Microbeads of calcium pectinate are stable in low pH solutions but swell in weakly base solutions.²² Similarly, the polyanionic NaCMC crosslinked with trivalent cation i.e. aluminum chloride to form rigid gel/hydrogel/microbeadsand remain stable at low pH whereas, swells at colonic pH.¹⁵ Therefore, in our study, we have successfully prepared microbeads of LR with the use of LMP and NaCMC encapsulating polymers.

In previous studies it is also reported that to an extent, the LMP microbeads encapsulation properties in aqueous media may not be efficiently able to prevent them from avoiding drug release during transit through the upper gastrointestinal tract. Thus making the combined use of other strategies, such as coating with pH-sensitive polymers become an unmet need. Therefore, in our study, we have used Eudragit S 100 and CAP as coating material for colon targeted LMP and NaCMC microbeads. These coating polymers are reported to dissolve at colonic pH and allow releasing drug at their target site i.e. colon.¹³It would be logical to expect a size increase with Eudragit S 100 and CAP coating. However, the increase in microbeads size with coating will not be considered significant as the coating will not be thick enough.

All the formulation of LR microbeads was evaluated for their entrapment efficiency and percent yield. Like previously reported studies, the results of our study have also shown that the entrapment of probiotics depends upon the probiotics polymer ratio and found to be highest at 1:3 probiotics: polymer ratio.²³ The increase in entrapment efficiency with an increase in polymer concentration was attributed due to the availability of excess of polymer to form viscous polymer solution and thereby formed bigger size of microbeads to encapsulate more probiotics.²⁴

The swelling index is defined as the fractional increase in the weight of the microbeads due to water absorption.The swelling of all the prepared microbeads was observed very low or negligible in SGF and SIF however it was observed appropriate in SCF. The swelling behavior of NaCMC and LMP at alkaline pH might be due to the fact that in the alkaline environment, the electrostatic repulsion between carboxylate anions (COO⁻) and the osmotic swelling force within polymeric network controls expansion of the microbeads. However, at low pH (pH 1.2) the swelling index of NaCMC and LMP microbeads was found to be negligible due to less electrostatic repulsion force which might be attributed due to the presence of protonated carboxyl group in polymeric network.²⁵

The observations of our study support the fact of increase of swelling index with the increase of polymer concentration might be due to increase in ionic contribution to the osmotic pressure.²⁶

Mucoadhesion is commonly defined as the adhesion between formulation and the mucosal surface of tissue.²⁷ Mucoadhesive systems are formulated to extend the GI residence time of dosage form and thereby enhance the bioavailability of drugs and provide precise control over drug release rate.In our study, it has been observed that mucoadhesive strength of prepared microbeads formulations was significantly greater at colonic pH as compared to gastric and small intestinal pH. At colonic pH, the mucoadhesion of LR microbeads with LMP polymer was primarily due to the carboxylic group of LMP which can form hydrogen bonds with mucin layer of tissue whereas due to hydrogen bonding between hydroxyl group of NaCMC and mucin layer in case of LR microbeads with NaCMC polymer. The low mucoadhesion of microbeads at gastric and small intestinal pH could be a result of weaker hydrogen bonding formed with mucin layer.

It is also reported that increasing the concentration of the NaCMC in the formulation increases the bonds forming groups, thus increasing the mucoadhesive force of the formulations. Similarly in our study the mucoadhesive strength was found to be higher with increased polymer concentration (probiotics polymer ratio 1:3). The potential use for mucoadhesive systems as drug carriers lies in its prolongation of the residence time at the absorption site, allowing intensified contact with the epithelial barrier.²⁸ In AAD, there is an unmet need of colon targeted mucoadhesive viable probiotics delivery systems.

Viability of probiotics from formulated microbeads and free probiotics were assessed in SGF, SIF and SCF in our study. The results have shown less viability of probiotics from formulated microbeads in SGF which was corresponding to less release of probiotics in SGF medium. The viability of free probiotics also dramatically decreased in SGF due to the unfavorable conditions such as low pH and presence of proteolytic enzymes. The reports of previously published studies also support our findings that the viability of free probiotics is less in SGF as compare to SCF.²⁹Contrary to viability in SGF, the viability of free probiotics in SIF was improved and it was suspected due to less degradation of probiotics in favorable pH of SIF. However, the results of test formulations in SIF were found to be similar to viability in SGF and also corresponding to less release of probiotics in SIF. The viability of probiotics from formulated microbeads and free probiotics was observed appropriate in SCF. The appropriate viability of free probiotics and test formulations in SCF was attributed due to least degradation of free probiotics and appropriate release of probiotics from test formulations.²

CONCLUSION:

In present study, microbeads of LR encapsulated LMP and NaCMC microbeads were developed using the ionotropic gelation method. Microencapsulation of probiotics was done for their protection from the harsh gastric environment and release of the entrapped probiotics at the colonic site. The prepared microbeads were coated with Eudragit S 100 and CAP separately to ensure the prevention of probiotics release during transit of upper gastrointestinal region. The study showed that the Eudragit S 100 and CAP coating provides protection to the loaded probiotics from the gastric acidic environment. The viability of free probiotics has observed significantly greater in SCF as compared to viability in SGF and SIF. This lesser viability in SCF and SIF was attributed due to degradation of free probiotics in upper GI harsh condition. The microencapsulated probiotics has shown greater viability in SCF as compared to SGF and SIF. The lesser viability in SGF, SIF and appropriate in SCF might be due to the fact of lesser degradation of probiotics microbeads in upper GI harsh conditions. Also among formulated microbeads LR3, LR6, LR9 and LR12 has shown promising result as compare to other formulated microbeads. Therefore, it was concluded that the encapsulation of probiotics in coated microbeads (LR3, LR6, LR9 and LR12) could be a promising strategy to deliver them safely and effectively into the targeted colonic region.

ACKNOWLEDGEMENT:

Authors are grateful to NDRI Karnal Haryana, Wadia Institute of Geology, Dehradun, Shri Guru Ram Rai Institute of Technology and Sciences, Dehradun, India for providing support to conduct the study.

CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

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Received on 26.05.2019 Modified on 05.07.2019

Research J. Pharm. and Tech. 2019; 12(12): 6049-6056.

DOI: 10.5958/0974-360X.2019.01050.3

Formulation code	Ratio of probiotics and encapsulating Polymers			Coating Polymer
Formulation code	LR	LMP	NaCMC	CAP (%)	Eudragit S 100 (%)
LR1	1	1	-	5	-
LR2	1	2	-	5	-
LR3	1	3	-	5	-
LR4	1	1	-	-	5
LR5	1	2	-	-	5
LR6	1	3	-	-	5
LR7	1	-	1	5	-
LR8	1	-	2	5	-
LR9	1	-	3	5	-
LR10	1	-	1	-	5
LR11	1	-	2	-	5
LR12	1	-	3	-	5