Novel catalase loaded nanocores for the treatment of inflammatory bowel diseases

Abstract

Inflammatory bowel disease (IBD) is an inflammatory disorder of the digestive tract reported to be primarily caused by oxidative stress. In this study, alginate encapsulated nanoceramic carriers were designed to deliver acid labile antioxidant enzyme catalase orally. Complete system was characterized for size, loading efficiency, in vitro antioxidant assay and in vitro release. The prepared nanoceramic system was found to be spherical with diameter of 925 ± 6.81 nm. The in vitro release data followed the Higuchi model in acidic buffer whereas in alkaline pH sustained and almost first order release of enzyme was observed up to 6 h.

Keywords:

• Antioxidant enzymes
• catalase
• hydroxyapatite
• IBD
• nanoceramic core

Introduction

Inflammatory Bowel Disease (IBD) is an autoimmune inflammatory disorder characterized by chronic worsening of gastrointestinal tract. It mainly includes Crohn’s disease (CD) and ulcerative colitis (UC). IBD affected persons are much higher in North America, Northern Europe, and the UK as compared with Africa, Asia, and Latin America. UC is more prevalent in males whereas Crohn’s diseases is more prevalent in women. One of the major causes of IBD is an oxidative stress (Rezaie et al. 2007). Gastrointestinal tract appears as more susceptible region for production of oxidants due to presence of food particles, activated microbes, and immune reactions. Inflammatory situation leads to assimilation of leucocytes and macrophage cells generating tissue damaging reactive oxygen and nitrogen species (Jin et al. 2016, Moura et al. 2015). This chain of reaction further promotes release of T cells and inflammatory cytokines causing inflammatory disorder. T-effector cells produces uncontrolled T-helper 1 and T-helper 2 cells, which results in the production of interleukins and cytokines-IFN-gamma, TNF-alpha, IL-12, and IL-13, IL-5, IL-4, IL-3, which causes crohn’s and colitis inflammation (Singh et al. 2015, Zhang and Yong 2014).

The prime cause of reactive oxygen accumulation is the imbalance in the antioxidant enzymes like catalase, glutathione peroxide, and superoxide dismutase (SOD) (Rana et al. 2014). The imbalance of antioxidant enzymes accelerates the production of reactive oxygen species and reactive nitrogen species aggravating IBD more (Balmus et al. 2016, Kruidenier et al. 2000). The superoxide anion generated is changed into hydrogen peroxide in the presence of antioxidant enzyme SOD, which further breaks down into non-toxic molecules of water and oxygen in the presence of catalase enzyme (Kumagi and Jikimoto 2003). The lack of activities of these enzymes leads to accumulation of these toxic radicals resulting into various diseases such as Alzheimer’s, diabetes, cardiac disorder, multiple sclerosis, arthritis, and IBD (Mc cole and Barrett 2008).

Researchers on the platform of biotechnology manage to advance the treatment by combining biotechnological products like protein and peptides with a novel technology of pharmaceuticals. The major hurdles in use of these peptides as a therapeutic agent is, stability issues in the biological milieu. Thus, to overcome this limitation a nanoceramic system with enteric coating has been designed for delivery of this antioxidant enzyme while retaining its structural conformation (Rawat et al. 2008, Singh et al. 2013).

Catalase (CAT) an antioxidant enzyme has been established to be a potential treatment for oxidative stress and inflammation in IBD. It scavenges free radicals and converts them into hydrogen peroxide molecule, which is further hydrolyzed into a non-toxic molecule of water and oxygen in the presence of antioxidant enzyme SOD (Carmen et al. 2014). Thus, antioxidant enzymes could be variedly used in fibrocystic breast disease, arthritis, chronic bronchitis, carpal tunnel syndrome, and IBD (Greenwald 1990, Ibrahim et al. 2015).

The objective of this study is to design a nanoceramic system having oligohydroxy coating improve the bioavailability, conformational, and acid stability of enzymes. The nanoceramic sugar coated core gives conformation stability and loading stability to enzymes. This system further encapsulated within alginate coating gives a system longevity and gastrointestinal compatibility (Rawat et al. 2006).

Natural polymers such as cellobiose, trehalose, and alginate were used in the formulation as they are bio-safe; highly inert towards protein drugs; do not need organic solvents; and possess properties of mucoadhesives, biodegradability, low toxicity, low immunogenicity, ready availability, and inexpensiveness (Lee and Mooney 2012).

Materials and methods

Materials

Catalase was purchased from Sigma-Aldrich, japan. Trehalose was purchased from High media Pvt. Ltd. Cellobiose, Sucrose, and Concanavalin-A were purchased from Loba Chemie Pvt. ltd, Mumbai. Sodium alginate, and Calcium chloride dihydrate were purchased from S. D. Fine Chemicals Ltd, Mumbai, India. Calcium phosphate was synthesized in the laboratory. All chemicals used were of analytical grade.

Preparation of nanoceramic core (NC)

Hydroxyapatite (HAP) cores were formulated in a laboratory by wet precipitation method with slight modification. 2.5 ml of ammonium solution was dispersed into 40 ml of 0.32 M calcium nitrate solution under ultrasound irradiation (Ambati et al. 2012, Poinern et al. 2009). 60 ml of 0.19 M KH₂PO₄ solution was added slowly with stirring while, maintaining pH 9 throughout the experiment lead to form white precipitate. The mixture was continuously ultrasonicated ranging from 0 to 500 W (25 kHz) of maximum amplitude for 20 min. The calcium to phosphate ratio was maintained at 1.67 (Ishikawa et al. 1993). The solution was filtered through 0.2-micrometer millipore filter and particles were collected. The temperature was varied as 100 °C, 200 °C, 300 °C, 400 °C for 2 h. Two samples were significant at 100 °C, 200 °C, but 300 °C, 400 °C sample presented insignificant peak of impurities (Poinern et al. 2009).

Adsorption of sugar (cellobiose/trehalose) on nanoceramic core (CNC/TNC)

The ceramic cores of calcium hydroxyapatite (∼500 mg) placed into 10 well-cleaned dried iodine flasks in series. One to ten ml of sugar solution (5 mg/ml of trehalose/cellobiose) was transferred into stoppered iodine flasks and made volume up to 50 ml with distilled water. The well-closed flasks were vigorously shaken for 20 min and poised in water containing trough at room temperature for 1 h with intermittent shaking (Rawat et al. 2008). The sugar-coated ceramics were dried by lyophilization and excess deposition of loosely bound sugar on ceramic removed by washing with fresh distilled water using dialysis method for 48 h with the replacement of water every 6 h (Patil et al. 2004). Then, it was used for structure analysis of sugar coated nanoceramic core. The particle size and shape of prepared nanosystem was determined by TEM (Philips EM268D, The Netherlands).

Characterization of ceramic core

FT-IR spectroscopy

The prepared nanoceramic core (NC) and sugar adsorbed core (CNC/TNC) were analyzed by FTIR spectrophotometer (Shimadzu 8400S). KBr and 1% (w/w) HAP powder compressed for sample disk and infra-red spectra recorded in wave range 4000–400 cm⁻¹ (resolution 4.0 cm⁻¹) (Schimadzu FTIR-8400S, Tokyo, Japan).

X-ray diffraction

Phase and crystalline nature was analyzed by using X-ray diffractometerXRD (XRD-6000, Shimadzu, Japan). XRD was performed in increment of 0.03° 2θ, at angular range 10–50° 2θ.

Particle morphology

The nano size of prepared system was analyzed by TEM (Philips EM268D, The Netherlands). Aqueous dispersion of formulation was negatively stained with phosphotungstic acid (3% w/v, adjusted to pH 4.7 with KOH) and placed the accelerating voltage of 80 kV. The size, polydispersity and zeta potential of the core was measured by zetasizer (Malvern Instruments Inc., UK) after appropriate dilution (1:200) with PBS pH 7.4prior to analysis [20].

Adsorption efficiency

The adsorption behavior was studied by Freundlich isotherm (log C versus log x/m) and Freundlich-Langmuir isotherm (C versus C/x/m). The Freundlich and Langmuir adsorption parameters were studied for binding constant b, sugar adsorbed/gram of hydroxyapatite Ym, Log K and n (both determined from the graphical data) and correlation coefficient, which signifies adsorption. Sugar adsorption was estimated by concanavalin-A mediated aggregation method. The extent of sugar adsorbed is quantified by recording turbidity at 450 nm of sugar-coated ceramic suspension (100 μg/ml). Similarly, one mL of concanavalin-A solution (10 μg/ml) was added, and absorption was noted at time intervals of 2 mins varied from 2 to 10 min.

Enzyme loading in sugar coated nanocore (ECNC/ETNC)

100 mg of sugar coated NC were dispersed in catalase solution 10 mg/ml in 7.4 pH PBS and kept overnight at 4 °C. Catalase adsorbed ceramic cores (ECNC/ETNC) were washed three times with de-ionized water by centrifugation at 10 000 rpm for 30 min (REMI C-24, Mumbai, India) and stored at 4 °C. The enzyme loaded cores were characterized for morphological characteristics, mean particle size, and size distribution as described before (Loukas et al. 1997, Pandey et al. 2011).

Enteric coating of ceramic cores (AECNC/AETNC)

The enzyme loaded system was encapsulated within alginate polymer by mixing with 2% w/v of alginate solution. CaCl₂ solution (11%) was slowly added for gelation of polymer and stirred for 30 minutes to obtain encapsulated particles. Calcium to alginate ratio was maintained at 0.6% w/w (Rawat et al. 2008). The mean particle sizes, the size distribution of prepared nanoceramic cores were determined by Zeta Seizer (Zeta Nano ZS Malvern, UK).

In vitro catalase assay

The activity of catalase was assessed by using standard H₂O₂ degradation assay. The absorbance of 5 mM hydrogen peroxide PBS buffered solution was read at λmax 242 nm. Catalase loaded nanoparticles were diluted such that concentration of catalase 0.01–0.50 μg/ml correspond to the linear section of the calibration plot where the slope of the decay curve was proportional to the concentration of the catalase added. 2–10 μl of AECNC were diluted to make a total volume of 1.0 ml and similar process for AETNC. The concentration of the H₂O₂ was demonstrated over time and the activity of the catalase was calculated from the slope of the decay curve where (1 activity unit =22 (ΔAbs/t)).

Enzyme loading efficiency (EL)

The formulation (ECNC/ETNC) with known amount of enzyme was kept at constant stirring for 24 h at 4 °C. The stirring solution was centrifuged at 9000 g for 1 h below 4 °C in a cooling centrifuge (IV C1–6363, Remi Instruments, Mumbai, India) (Loukas et al. 1997). The aliquot of the supernatant liquid was estimated by measuring absorbance at 276.8 nm by first derivative method spectrophotometrically (Shimadzu UV-2202).

Equation for calculation: (1)

W1 = The wt. of the enzyme loaded

W2 = The wt. of the enzyme in the supernatant

W3 = The wt. of the coated nanocore of system

In vitro release studies

In vitro release profiles of enzyme loaded nanocores were conducted for 8 h in both acidic HCl buffer (pH 1.2) for 2 h and alkaline phosphate buffer (pH 7.4) for 6 h similar to the GIT passage time. 100 mg of loaded core was suspended in respective buffer (10 ml) on stirring (100 rpm) at a temperature of 37° ± 0.5 °C. At specific intervals, 0.1 ml sample was withdrawn for maintaining sink condition. A sample withdrawn from acidic medium was centrifuged for 10 min at 9000 rpm and protein analyzed in the supernatant. Release models such as first order model, Higuchi model, and Ritger-Peppas model applied to the release data of the delivery system. Results expressed as mean (± SD) of three experiments.

In process stability study

Differential scanning calorimetry (DSC)

DSC was performed to study the stability of loaded enzyme over oligohydroxymer coating of the ceramic system. DSC measures glass transition temperature (Tg) i.e temperature at which glassy state melts to a rubbery state. Results were analyzed by studying and interpreting the exothermic graph of plain CAT, ETNC, and ECNC (Shimadzu DSC-60 Systems, Japan).

Gel electrophoresis

SDS-PAGE studies were performed to confirm of presence of enzyme catalase in all the formulation by running plain CAT, ETNC, and ECNC against marker protein. The CAT extracted from 5% (w/v) of sodium dodecyl sulphate in 0.1 N HCl subjected to electrophoresis at 200 V (BioRad), stained by dye Brillant Blue.

Storage stability analysis of formulation

Storage stability studies were conducted as per ICH guidelines at room temperature of (25 ± 2 °C, 60 ± 5%RH) and accelerated condition (40 ± 2 °C, 75 ± 5%RH) for duration of 6 month. The catalytic activity for H₂O₂ observed as mentioned under in vitro catalytic assay section.

Statistical analysis

All values expressed as their mean ± SD ANOVA has been applied and F-value determined. A value of p < 0.05 was considered to be statistically significant.

Result and discussion

Preparation of the nanoceramic core (NC) and their characterization

Various materials such as Tin oxide, diamond, HAP have been utilized as nanocores for implant preparation in bone regeneration, delivery of varied peptides, proteins, and antigen by enhancing its adsorption capacity without altering their structural activity (Goyal et al. 2009, Poinern et al. 2013). HAP was selected for the core preparation due to its biodegradability, stability, safety, and cost (Kossovsky et al. 1994). The hydroxyapatite nanoceramics (NC) were prepared by self-precipitation method. The nanocores (NC) were coated with oligohydroxymer cellobiose and trehalose to form cellobiose coated nanoceramics (CNC) and trehalose coated nanoceramics (TNC), respectively. The oligohydroxy coating gives larger surface area and protective environment for adsorption of protein to form (ECNC and ETNC). Further formed ECNC and ETNC were encapsulated within alginate by gelation technique to protect the enzyme from the acidic environment. The prepared ceramic (HAP) core was analyzed for size by TEM, crystalline nature was assesses and compared at different temperature (200 °C, 300 °C, 400 °C for 2 h) by X-ray diffraction analysis; 2θ angle and phosphate group of calcinied powder was confirm by FTIR. The size of the prepared HAP core (NC) was 198.4 nm as shown in TEM image in Figure 1. XRD analysis of HAP samples prepared at different temperature range showed low intensities of 23.4 θ –23.7 θ. The XRD pattern of the sample at 200 °C reaction temperature was most significant in the 2 h range 26.9 θ –27.4 θ. Among studied range, cores formed at 200 °C has been found to be soft, crystalline and in large quantity. The crystalline nature of HAP (NC) was confirmed by XRD report shown in Figure 2 as it gives intense peak nearby 21–23, 26–31 and 37–39 (2θ) when compared with standard HAP core (Rawat et al. 2008). The FTIR spectroscopy conducted for confirmation of phosphate group of calcined powder showed peak at 507, 604, 945, 964, 1024, and 1184 cm⁻¹, else peak found at 633, 2910, and 3570 cm⁻¹ was due to the OH⁻¹ bending deformation (15–20) as shown in Figure 3 and Table 1. The nanocores coated with oligohydroxymers trehalose (TNC) and cellobiose (CNC) showed increment in size of 340.17 ± 15 nm and 335.17 ± 15 nm, respectively. The increase in size and reduction in zeta potential to slightly negative value was observed after coating of trehalose and cellobiose on nanoceramic core confirming the coating of sugar layer. The size of CAT-adsorbed TNC and CNC was found to be (ETNC) 445.3 ± 20 nm and (ECNC) 450.3 ± 20 nm, respectively. Enzyme loading efficiency of TNC and CNC was found to be 46.64% ± 1.48% and 43.64% ± 1.48%. The alginate encapsulated nanoceramic core showed increment in size. Thus, with each consecutive coating of polymer, enzyme and alginate showed the increase in size of nanosystem, as evident by TEM images from Figure 4 and Table 2. The carbohydrate and catalase adsorption was analyzed and optimized by both Freundlich and Langmuir adsorption isotherm. In case of sugar adsorption, the correlation coefficient of Freundlich isotherm (log C versus log x/m) was found to be in the range of 0.937–0.983, while Langmuir isotherm (C versus C/x/m) was found to be in the range of 0.980–0.999. Thus, adsorption patterns follow both isotherms. The respective values of a constant in the case of both sugars cellobiose and trehalose are mentioned in Table 3. The correlation coefficient of isotherm reveals multilayer adsorption and porous nature of ceramic core. The binding constant (b) of adsorption isotherm of CNC was found to be 6.89 as compared to TNC that is 3.13. Oligomer adsorbed per mg of core (Ym) of TNC was found to be 4.91 compared to CNC having Ym of 3.38. This might be due to strong interaction between sugar molecules or epitaxial attachment of sugar molecule over the core (Goyal et al. 2009). Cellobiose molecules fit well into cavity of ceramic core giving well-packed structure in comparison to trehalose as some aggregation of the polymer was observed in the case of TNC from TEM image. Thus, reduced size of CNC was obtained as compared to TNC. Results obtained from adsorption isotherm revealed more adsorption of enzyme over CNC system as compared to TNC system. The molecular size of ECNC is more than ETNC and confirmed better loading efficiency of CNC as compared to TNC. Glassy matrix of oligomer provides aqueous environment across the enzyme molecule confirming the stability of enzymes. Enzyme molecules adsorbed rather than embedded over glassy matrix retain their activity because of their liberty of mobility. Thus, the system accomplishes both the objective of enhancing stability and activity of the enzyme at target site due to aqueous environment and enteric coating of the system.

Figure 1. TEM image of nanoceramic core.

Figure 2. XRD report of nanoceramic core.

Figure 3. FTIR report of nanoceramic core.

Figure 4. TEM image of trehalose and cellobiose coated nanocores (TNC and CNC) followed by catalase adsorption on both core(ETNC and ECNC) and its nanoencapsulation within alginate polymer (AETNC and AECNC) (A) TNC, (B) CNC, (C) ETNC, (D) ECNC, (E) AETNC, (F) AECNC.

Table 1. Characteristic absorption band of hydroxyapetite core.

Absorption band	HAP (wave no.)
OH^−	3571.9
H2O	3415
CaCOO^-	1384
N-H	3440,3491

Table 2. Morphological characteristics of optimized formulation.

Formulation	Average size (mm)	Zeta potential (mv)	TEM size (nm)	XRD phase analysis
NC	198.4	+ 2.9	208 ± 10	2 ^θ= 23.7 °C, 26.9 °C, 27.4 °C, 31 °C, 45 °C
TNC	498.1	−20	340.17 ± 15	–
CNC	486.1	−12.2	335.17 ± 15	–
ETNC	630.1	−21.5	445.3 ± 20	–
ECNC	560.1	−19.2	450.3 ± 20	–
AETNC	821.1	−23.1	720 ± 20	–
AECNC	830.1	−23.5	740 ± 20	–

Table 3. Reported value of binding constant (b), sugar adsorbed per gram of HAP (Ym), binding efficiency and correlation co-efficient Freundlich (r_F) and Langmuir (r_L) value for adsorption isotherm.

Formulation	b	Ym	rF	rL
CNC	6.89	3.38	0.966	0.990
TNC	3.13	4.91	0.983	0.980
ECNC	2.69	1.18	0.989	0.991
ETNC	2.14	0.964	0.993	0.996

Analysis of zeta potential (ZP)

Electrostatic charges on the respective surfaces of nanosystem are expressed in the form of ZP, which envisage long-term stability of the nanosystem. Generally, the ZP of a suspension should be either greater than −30 mV or less than +30 mV for the nanoparticles to be stable (Farihurst 2013). Zeta potential obtained for the optimized system at various stages of nanoceramics formulation was determined. ZP of NC found to be +2.9 which were reduced to the negative value, confirming the coating and stability of nanocores with carbohydrate followed by the enzyme. The results of examined size and zeta potential of the successive systems of nanoceramics are illustrated in Table 2 and all data relies upon the standard requirement of zeta potential.

In vitro catalytic assay

The catalase activity of alginate encapsulated nanoceramic systems were evaluated before and after treating them with acidic and basic buffer. The alginate encapsulated system of ECNC and ETNC exhibited reduction in proteolytic activity to be 1.27 ± 0.04%, 1.49 ± 0.05% in acidic buffer and 0.97 ± 0.02%, 0.19 ± 0.04% in alkaline buffer, respectively. The negligible loss of activity of enzyme observed might be due to various processing steps implicated in the formulation development. Free catalase showed complete loss of activity in acidic medium, as it is acid labile molecule whereas in alkaline medium 81.0 ± 3.3% of catalase activity was retained.

In vitro release study

The release mechanism of alginate encapsulated drug delivery system (AETNC and AECNC) in acidic (pH 1.2) and alkaline (pH 7.4) buffer was resoluted and fitted to characteristic drug release kinetics models (Rawat et al. 2008). The cumulative percentage drug release for 6 h of AETNC and AECNC are shown in Figure 5a and b. Release data were fitted to various release models such as first order, Higuchi, and Ritger-Peppas model (Farihurst 2013, Peppas 1985).The graph plotted for first order release kinetics of catalase from AECNC and AETNC in alkaline media was observed to have coefficient (R²) value 0.9664 and 0.9757, and in the acidic buffer, it was found to be 0.6502 and 0.6402, respectively, predicting first order release in the alkaline medium. Initial burst release was observed in the first hour in both acidic and alkaline buffer, as shown in Figure 5a and b. The release data after 1 h were fitted to Higuchi and Ritger-Peppas equation. The (R²) value in Higuchi equation for AECNC was found to be 0.9429 and 0.8609 and for AETNC 0.967 and 0.8763 in acidic and alkaline buffer medium, respectively, which examines diffusion-controlled release between 2 and 6 h in the acidic buffer, whereas in the alkaline buffer, R² value doesn’t favor diffusion pattern. Diffusion control release was obtained due to integrated alginate form. pH sensitivity of alginate gel is responsible for the difference in release patter. In the acidic media (pH 1.2), calcium alginate gel is transformed to alginic acid (unionized form). Integrity of alginate system was maintained all over the passage. Diffusive release of drug may be obtained due to disturbance in ionic linkages causing reduction in gel strength. All this is responsible for decreased release of protein in acidic pH after 2 h. Substituting the release values determined between 2 and 6 h in the Ritger-Peppas equation, the value of a coefficient of determination was about 0.9 in each case. The value of n = 0.43 indicates Fickian (case I) release; > 0.43 but <0.85 for non-Fickian (anomalous) release; and >0.89 indicates super case II type of release. Case II generally refers to the erosion of the polymeric chain and atypical transport (non-Fickian) refers to a combination of both diffusion and erosion controlled– drug release (Peppas 1985, Siepmann and Peppas 2001). The results showed that the Ritger-Peppas model was not suitable for estimating the release kinetics of AECNC and AETNC. It was believed to be due to the immediate release of CAT caused by rapid dissolution of the alginate in the alkaline buffer. At higher pH, alginic acid is converted to the sodium salt of alginate and the matrix gets disintegrated completely, releasing the entrapped core. But the whole protein is not released as a burst due to protein-adsorbed core structure maintaining the sustained effect. This result was attributable to the slightly sustained release of enzyme signifying the mixed type of release pattern. The result obtained has been found to be statistically significant. Recently, Cui et al. and his coworkers demonstrated in mice model that more than 5% of H₂O₂ induces cell death and less than 5% of H₂O₂ inhibits expression of antioxidants enzymes like superoxide dismutase, gluatathione peroxidase and accelerates production of inflammatory cytokines as IL-1β, IFN-γ, TNF-α, IL-6, IL-8 thus contributing to cause of IBD (Cui et al. 2016). In such case, the system encapsulating an antioxidant enzyme catalase could be scale up for safe and sustained delivery at inflamed site to treat bowel disease.

Figure 5. Cumulative % drug release data for 5 (a). Optimized alginate coated nano ceramic formulations of trehalose (AETNC); 5 (b).Optimized alginate coated nano ceramic formulations of cellobiose (AECNC).

In process stability study

In process stability assay was performed by studying Glass transition temperature (Tg) of carbohydrate and protein with the help of DSC in exothermic graph. DSC thermogram of formulation containing enzyme i.e. (TNC, CNC) and free catalase was studied and the difference between Tg of different formulations were found to be insignificant as compared to plain CAT (Figure 6). The results of DSC support the efficiency of a carrier system for providing in-house stability to protein loaded over carbohydrate layer providing an aqueous environment and maintaining its structural confirmation, during and after the processing of formulation.

Figure 6. DSC thermogram of a ceramic system, (a) Plain CAT, (b) CAT adsorbed over TNC, (c) CAT adsorbed over CNC.

SDS-PAGE analysis of catalase observed through electrophoresis in sequential lane of SDS-PAGE of each formulation in range of 43 KD, which corresponds to marker protein in range of 43 KD. Thus confirms stability of catalase in each formulation by observing band of catalase compared with plain catalase in same molecular weight range.

Storage stability analysis

Results of stability studies showed that plain-CAT completely lost its activity. Ceramic system ECNC and ETNC lost around ±1.5% and ±1.9% of protein activity respectively from the first month to 6 months. This loss was also marginal in case of accelerated conditions where ECNC lost 1–8% and ETNC lost 1–12% activity around the 6 months as shown in Figure 7(a) and (b). The difference in activity observed in the system may be due to ease of availability of enzyme molecule from ETNC system for activity as compared to ECNC. Based on results obtained, the prepared systems were stable under both normal and accelerated conditions.

Figure 7. Storage stability data 7 (a): Effect of storage on % residual CAT content of Plain CAT, TNC and CNC stored at 25 ± 2 °C, 60 ± 5% RH; 7 (b): Effect of storage on % residual CAT content of Plain CAT, TNC and CNC stored at 40 ± 2 °C, 75 ± 5% RH.

Conclusion

The ceramic systems were successfully formulated. Cellobiose coated NC were found to be more efficient compared to trehalose in terms of stability and enzyme loading efficiency as compared to TNC. Thus, cellobiose based NC is the complete solution for maintaining the conformational integrity of protein, protecting it from the acidic environment with ease of loading and controlled release of enzyme all over the GIT for management of IBD. Future investigations need to be done to establish the clinical utility of the formulation.

Funding information

The authors thank UGC-MRP41–748-2012, DST-FIST, and UGC-BSR F(0).7–341/2011 for financial assistance relating to this work.

Acknowledgements

Authors are thankful to School of Studies Physics, Pt. Ravishankar Shukla University Raipur for XRD studies; All India Institute of Medical Sciences, New Delhi for Transmission Electron Microscopy; Director, University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur (C.G.), India for providing all necessary facilities for carrying out this work.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.