Development and evaluation of colon targeted delivery of budesonide polymeric nanoparticles for colitis therapy

Targeted delivery of the drug at site of action in case of In lammatory Bowel Disease like colitis is the big challenge for formulators. The case where conventional drug delivery fails in severe stages of In lammatory Bowel Disease, Nanoparticles is a good dosage form to targeted in lammatory site. The idea of the this researchwas to prepare Nanoparticles with polymer Eudragit S100 and Surfactant Poloxamer containing Budesonide which inally illed in capsules providing immediate release at the ileocecal site, the most affected area in IBD i.e. pH dependent release. Nanoparticles are prepared by nano precipatation technique. Budesonide was used as a drug because of its therapeutic potential for in IBD. This study compares the different ratios of drug to olymer and drug to surfactant with optimized solvent and anti-solvent concentration in preparation of Nanoparticles. Optimized formulation ratio of drug to polymer was 1:2 and drug to surfactant was 1:1. The optimized batch of nanoparticles was illed in capsule and was evaluated for in-vitro studies.


INTRODUCTION
Major population worldwide is getting affected by In lammatory Bowel Disease (IBD) and the prevalence of the disease is increasing day by day (Xavier and Podolsky, 2007). This colitis (in lammation of colon) etiology and pathogenesis are not completely understood. Symptoms of IBD like ulceration, bloody diarrhoea, weight loss and which further lead to complete obstruction of the gas-trointestinal (GI) tract, affect very badly on day today life of people (Frank et al., 2007). Most accepted move towards treating IBD and to reduce the need for surgeries and hospitalizations is by facilitating mucosal healing; induce remission of outbreaks (Xiao et al., 2014). Systemic corticosteroids and mesalazine are used to treat Symptoms of mild-to-moderate IBD (Colombel et al., 2010). These drugs mentioned above are preferred in many cases of pharmacotherapy for colon in lammation consisting of permanent administration of either of drug. It is necessary to take in consideration the quality and severity of adverse effects of these therapeutic regimens. For successful IBD treatment, it is necessary to involve innovative strategies for exact and adequate drug delivery to the in lammed colon for a long-lasting period in a sustained manner to minimize the risk of systemic adverse effects (Lautenschläger et al., 2014). For ef icient treatment of colonic disorder, colon precise drug delivery is necessary. Now a days, it is more complicated to target drug delivery to colon, so new techniques like nanotechnology are emerging out. Nanotechnology is used to aim description, manufacture and application of structure, having nanometer size scale. According to the direction of International System of Units (SI) nanotechnology is typically measured in nanometers scale of 1 billionth of a meter referred as the "tiny science". Minute size molecules and particle act in a different way, as an intact unit in requisites to provide a variety of advantages. Nanoparticles (NPs) are distinct particles of size 1 and 100 nanometres drug transporter .
The drug is attached to a Nanoparticles matrix. Nanospheres and nanocapsules collectively called as nanoparticles. In nanocapsules drug is restricted to a cavity which is enclosed by a distinct polymer membrane, and in nanospheres consist of matrix systems in which the drug is physically and uniformly dispersed. Where conventional techniques reach their limits, nanotechnology provides opportunities for the medical applications (Hua et al., 2015). For colon speci ic drug delivery, now a days we can make use of Eudragit polymers (RS100, L100 and S100) or cellulose acetate phthalate, which are soluble at or above pH 7, or dissolving at pH 6 respectively. In the current study, we used Eudragit S100 to manufacture micro carriers for a de inite colon release of Budesonide. Eudragit, RS100 and RL100 are copolymers based on acrylic and methacrylic acid esters, which has a low level of quaternary ammonium group. As compared to Eudragit -RL (8.8-12% Ammonium groups) Eudragit -RS has a lesser content of charged groups (4.5-6.8%), due to which it is less permeable to water. Due to insolubility at physiological pH the co-polymers will swell form permeable ilm which will facilitate to manufacture sustained release formulation $.E (Kadian and Harikumar, 2009;Paharia et al., 2007)dragit is used in various studies for colon targeting sin c e low content in ammonium group permitting a low solubility in gastric luid.

Materials
Budesonide was obtained from Wockhardt Ltd. Aurangabad. Eudragit S100 was obtained from Evonik Pvt. Ltd. Poloxamer from Mody Chemi-Pharma. Ethanol and Methanol were obtained from Fisher Scienti ic Pvt. Ltd. Acetone from Fisher Scienti ic Pvt. Ltd. Hydrochloric Acid from Benzer multitech Pvt. Ltd. Tribasic Sodium Phosphate and Sodium Hydroxide from Merck Specialities Pvt. Ltd. All other raw materials used were of pharmaceutical grade.

Pre-formulation
Preformulation parameters like Oraganoleptic properties, Solubility, Melting point for drug and excipients was determined. Compatibility between drug and excipient was determined using FT-IR spectroscopy. DSC was performed to check thermal properties of the drug.

Infra-Red spectra
Infra-Red spectrum of Budesonide IR spectra of Budesonide were taken on IR spectrophotometer by simply placing small amount of drug in powder form on selenium bromide crystal. In a spectrum, peaks were found at 3497.81 for O-H stretching, 2942.03 for C-H stretching.cm −1 . IR spectrum of Budesonide was found as given in Fig

FT-IR spectra of Eudragit S100
FT-IR spectra are used for functional group identiication in compound. A small amount of Eudragit S100 in the form of powder was placed on selenium bromide crystal and spectrum was run. The Infra-Red spectra of Eudragit S100 are as depicted in Fig The IR peaks of Eudragit S100 were compared with standard graph of Eudragit S100 and found to be similar.

FT-IR spectra of Poloxamer
A small amount of Poloxamer in the form of powder was placed on selenium bromide crystal and spectrum was run. The Infra-Red spectra of Poloxamer are depicted in Figure 3.
Infra-Red peaks for Poloxamer were compared with standard graph of Poloxamer and found to be similar.

DSC analysis
The DSC thermogram of Budesonide was obtained to evaluate the thermal behaviour of pure drug in as given in Figure 4. Differential Scanning Colorimetric is a thermo-analytical techniques. A calorimeter gives endothermic and exothermic peaks which indicate heat into or out of test sample of DSC thermograms of Budesonide. Changes in the shape of the peak, shift of peak, absence of endothermic and/or exothermic peaks, generation of new peak shows   Differential Scanning Colorimetric (DSC) was carried out on pure Budesonide. DSC is performed and scan was recorded by keeping parameters like heating rate of 10ºC/min and window kept for temperature was 30 º-300ºC. In this scan reference used was empty standard aluminium pan.
The DSC peak of Budesonide shows sharp endothermic peak onsets from 259.14 and ends at 262.corresponds to standard DSC thermogram of Budesonide.

Compatibility by FT-IR Study
It is necessary to study the compatibility of the excipient with drug. Here drug and excipient is compatible or not was determined with the help of IR spectroscopy. The relative amount of drug and excipient is as given inTable 1.
The compatibility study was performed at the temperature of 55 • C ±2 • C and duration for study was 14 days. Individual drug and Drug: Excipient (as ratio 1:1) was kept in vial which was sealed properly. IR scan were taken for drug, polymer and surfactant prior to initiation of study and these raw materials were kept in vials for the ratio and duration as mentioned above. These glass containers were checked for tests like liquefaction, gas formation, caking and colour change if any. IR scan was taken at last after 14 days of study, spectra given in below Figures 5 and 6.
Both physical and chemical Compatibility study was carried out in the absence of moisture at 40º C in hot air oven for 14 days and found it compatible.

Infra-Red spectrum of mixture
Infra-Red spectrum of Drug+ Eudragit and Drug+ Poloxamer are as given in $ Figures 5 and 6espectively.

Drug+Poloxamer
The drug-excipient mixtures were observed for physical incompatibilities such as colour change, liquefaction, caking, and Gas formation and chemical incompatibilities with the help of FT-IR study. The  Table 2.

Formulation
To prepare polymeric Nanoparticles with a required particle size and to attain a constant formulation, the effect of various process variables was investigated. Variety of organic solvents which could dissolve Eudragit S100 and Drug, i.e. ethanol, methanol, acetone and dimethyl sulfoxide (DMSO) were screened for preparation of Nanoparticles (Radhika et al., 2011;Yoo et al., 2015). Ethanol was inalized as organic phase based on initial screening and was used to dissolve Eudragit S 100 and Drug (Sanjay et al., 2016;Leonard, 2012).
Nanoparticles were prepared by using modi ied Nano precipitation method using probe sonication. Required amount of Budesonide and Eudragit S100 were weighed properly and dissolved in Ethanol as organic phase. Then this solution was added drop wise into the water containing Poloxamer as aqueous phase under probe sonication for 10 -15 min. The nano suspension was then subjected to rotavapour for removal of free ethanol. The above mixture placed in to oven for drying the Nanoparticles.
On the basis of literature survey and laboratory work the Nano precipitation method was selected for the preparation of Nanoparticles. Preliminary batches were prepared to whether Nanoparticles formed for this combination or not and if formed at what will be concentration and ration of drug, polymer and surfactant.
With the preliminary knowledge we understand that for preparation of nanoparticles it is necessary to select the proper ratio of the Budesonide and Eudragit S100, it was determined by taking ratio such as 1:1 and 1:2 and 1:3 likewise respectively by taking further given in as Table 3 and which selected on the basis of extensive literature survey for preparation of colon speci ic nanoparticles having high entrapment ef iciency (EE) and optimized particle size. This method is based on trial and error with the base of preliminary batches results.
The amount of Budesonide was kept constant and the ratio of drug to polymer and drug to surfactant was varied as mentioned in above table. All the batches were manufactured according to experimental design described previously. In the batch F1, F4 and F7 the particles were formed so the batch F1, F4 and F7 was selected for entrapment ef iciency and drug release study. Encapsulation ef iciency of Nanoparticle was determined by separating free budesonide from budesonide suspension by centrifuging at 5000 rpm for 15 min (Nikam et al., 2014;Tiruwa, 2016). The clear liquid (supernatant) was taken and diluted with ethanol to calculate un-entrapped drug absorbance measured at 254 nm to calculate encapsulation ef iciency. The percentage of drug entrapment ef iciency was calculated using subsequent Equation $ (Yoo et al., 2015;Lathaeswari et al., 2013) Encapsulation ef iciency % = T otal drug − f ree drug × 100 T otal drug Drug release study

Evaluation of Nanoparticles
The drug release study of budesonide nanoparticles was carried out in dissolution test apparatus II (TDT 08 L Electrolab) at a speed of 50 rpm which contains 900 ml medium at 37 0 C. The capsules are transferred in the medium and samples were taken at certain time interval and analysed by UV at 254nm. The constant dissolution was used by simulating gastric conditions of the gastrointestinal area. For dissolution capsule placed in 700 ml of 0.1 N HCL (pH1.2)

Figure 17: Scanning Electron Microscopy of Batch F1
for 2 hours. At the end of 2 hours 200 ml of tribasic sodium phosphate was added to dissolution vessel pH adjusted to 6.8 for 3 hrs and inally 7.4 by using 2M NAOH (Sanjay et al., 2016).

Particle size determination
Nanoparticle formulation (1ml) was diluted with 10 ml deionized water in a beaker with constant stirring using a glass rod. The resultant solution was then subjected to particle size analysis. With the help of Dynamic light scattering (DLS) technique, using a zetasizer (Nano ZS, Malvern Instruments, UK) the droplet size was calculated (Nikam et al., 2014;Tiruwa, 2016).

Zeta potential
Zeta potential of the formulation was determined with laser diffraction analysis.

Scanning Electron Microscopy
Scanning electron microscopy analysis of the nanoparticle formulation was performed to understand surface morphology of Budesonide loaded nanoparticles.

Determination of absorption maxima (λ max ) and preparation of calibration curves
The calibration curve for Budesonide using double beam UV spectrometer The calibration curve for Budesonide in Ethanol, 0.1 N HCl, pH 6.8 and pH 7.4 buffers was prepared by plotting absorbance versus concentration at practically obtained λ max 254 nm. Calibration curve was plotted in replicate manner. Ranges selected for concentration were of 10, 20, 30, 40, 50 µg/ml. The calibration curve of Budesonide in 0.1 N HCl of pH 1.2 and phosphate buffer of pH 6.8, pH 7.4 were found as shown in Figures 7, 8, 9 and 10 respectively.
From the calibration curve of the Budesonide the regression was found to be 0.947 in 1.2 pH HCl buffer and 0.945 in 6.8 pH phosphate buffer and 0.964 in pH 7.4 buffer. The regression shows that the drug was pure.

Percent Entrapment Ef iciency (EE)
The range of percent EE was established from 80% to 90 %. EE of optimized formulation was found to be 89.52%. Trial batches result prove that, polymer concentration shows positive in luence of the entrapment ef iciency as batch F1, F4, F7 show % EE 89.52%,81.30%,85.50% have more polymer content.

Drug release pro ile (In-vitro) of optimized batches
The drug release pro ile was also performed on optimized batches (Batch F1, F4 and F7). Release pro ile was studied for twelve hours. The release curve is shown in Figure 11.
Above data clearly show that the batch F1 and F7 show good value of In-Vitro Drug Release and entrapment ef iciency so, two batches F1 & F7 were taken for further evaluation for determination of particle size and zeta potential.

Determination of particle size
Particle size analysis of nanoparticles for the optimized batches F1, F7 is 318.8 nm and 523.4 nm shown in Figures 12 and 13 respectively.

Zeta Potential Determination
Zeta potential of Nanoparticles from optimized batches F1 and F7 is shown in Figures 14 and 15 respectively.

Filling of Nanoparticles in capsules
On the basis of experimental design and evaluation of the Nanoparticles optimized batch i.e. F1 batch Nanoparticles were illed in capsule i.e. inal dosage form and these illed capsules drug release pro ile were tested up to 12 hours in triplicate. Drug release data of capsule containing nanoparticles is shown in Figure 16.
From the dissolution study it was clear that batch F1 shows the good drug release in colonic region.
The batch F1 A, F1 B and F1 C contains Eudragit S100 in 100mg. The result was good because it shows less than 11% drug release in stomach i.e. 9.01%, 9.88% and 9.11% respectively which is advantageous and achieves our objective to target colon.

Scanning Electron Microscopy
The Scanning Electron Microscopy analysis of optimized batch (batch F1) demonstrates that nanoparticles are round in shape. The Budesonide entrapped Nanoparticles do not show presence of cavities. The results of Scanning in Electron Microscopy are shown in Figure 17.

Stability study
The capsule containing Nanoparticles were stored in a container at 25ºC ±5ºC and looded solution of sodium chloride to attain relative humidity of 60% ±5%. Study was continued for a duration of 6 month and time points for testing were 1 month, 3 months and 6 months. This optimized formulation was tested to determine whether there is any signi icant change in appearance and other evaluation parameters like drug release.
Appearance and in-vitro drug release shows there is no signi icant change in the results comparable with initial results of optimized batch i.e. Batch F1.

CONCLUSIONS
In the treatment of IBD targeted delivery to site of action is advisable, so in this regard considerable progress has been made and novel technologies like nanoparticles were approached. In current study we developed polymeric nanoparticles of Budesonide to target colon in the treatment of colitis. The optimized nanoparticles formulated to minimize drug release in the stomach and release at site of treatment i.e. in colon in treatment of IBD. In vitro release pro ile demonstrated maximum release of budesonide from the formulated polymeric Nanoparticles. The present study has brought our potential of polymeric Nanoparticles formulation (Nanoparticles in capsule) in ulcerative Colitis. Extensive clinical studies will further substantiate the merit of this novel formulation.