Preparation of 5 aminosalicylic acid –Loaded GG-MMT Nanocomposites5ASA–GG-MMT nanocomposites hadbeen synthesized in two steps. Firstly, Guar gum was to dissolve in water and then to emulsify inthe solution of acetone/ethanol containing 5-ASA with magnetic stirringfollowed by sonication. In the second step, the primary w/o emulsion had beenemulsified in the external aqueous phase of MMT and Pluronic F68 (1%, w/v) toform a w/o/w-emulsion. The intermediate organic phase was separated theinternal water droplets from each other as well as from the external aqueouscontinuous phase. After solvent evaporation the 5ASA–GG-MMT nanocomposites werecollected by centrifugation and washed with double distilled water beforefreeze-drying. 5ASA-GG-MMT NC’s formulations were labeled s code (NC1, NC2, NC3& NC4) (Table 2) 28.
Morphological StudiesTheshape and surface morphology of 5ASS-GG NP’s (NP4) and MMT-5ASA-GG NC’s (NC4) wereobserved by Scanning electron microscopy (SEM) (JELO 5400, USA) ParticleSize, Polydispersity Index (PDI) and Zeta PotentialEvenly dilutedsuspension of 5ASA-GG nanoparticles (NP4) and MMT-5ASA-GG (NC4)nanocomposites had been crammed inthe chamber of a laser diffraction particle size analyzer (DTS Ver. 4.10,Malvern Instruments, Malvern, UK) for examination of PDI and average particlesize. Zeta potential of 5ASA-GG NP’s and MMT-5ASA-GG NC’s were also obtained by using Malvern Zetasizer (DTS Ver. 4.10, MalvernInstruments, Malvern, UK) 29.
DifferentialScanning Calorimetry (DSC)Differential scanning calorimetery (DSC) of pure 5ASA, MMT, GG, 5ASA-GG NP’s (NP4)and MMT-5ASA-GG NC’s (NC4) were estimated by heating thesample from 30 to 400ºC at theheating rate of 10ºC/min, in anitrogen environment (nitrogen gas flow rate of 60.0ml/min). DSC studies hadbeen conducted on NETZSCH DSC 200F3 240-20-427-L Proteus Software 30. PowderX-ray Diffractometry (PXRD)Powder X-ray diffractometry (PXRD)diffraction of formulations (NP4) & (NC4) analyses had been carried out inorder to find out crystalline nature of polymer and drug. Powder X-raydiffraction patterns of samples were completed using power X-ray diffractometer(Bruker, Munich, Germany). X-ray diffraction analysis was observed using anickel-filtered Cu-Ka radiation (a voltage of 40 kV and a current of 20 mA).The scanning rate 2/min over a 2y rangeof 0–40° and with an interval of 0.02? was set 31.
Determinationof Drug Entrapment Efficiency 50mg of 5ASA-GG NP’s and MMT-5ASA-GG NC’s weredissolved in acetone and ethanol (10 ml). Further suspension was centrifuged,and supernatant was decanted than GG was precipitated by adding 10 ml ofethanol. Percentage entrapment of 5ASA was determined using HPLC system(Agilant Technoloogies, 1220 infinity LS, UK) the mobile phase was made of 2 parts i.e. part A phosphate buffer (pH 6.6)and part B (acetonitrile) (77:23 v/v) delivered at a flow rate of 1 ml/min at25ºc, wavelength detector, and a zorbax 5? C18 column (250 × 4.60 mm) at 329nm. The concentration of free drug in the supernatant was taken by comparingthe absorption of the supernatant with standard curve 32.
The amount of drugentrapped into nanocomposites had been calculated as the difference between theamount of drug used for the formulation and the amount of drug in thesupernatant and present as encapsulation efficiency. Encapsulation efficiencieshad been calculated by using formula :- Encapsulationefficiency (%) = totaldrug?freedrug/ total drug ×100 DrugRelease Studies In-vitro drug release of both the formulations(NP4 & NC4) were observed in simulated gastric fluid (SGF) with pH 1.2 andsimulated intestinal fluid (SIF) with pH 7.
5. These pH values were identifiedbased upon the normal variation of gastrointestinal tract (GIT) in the stomach(pH 1.5), to the colon (pH 7 to 7.8) 33.
10 mg of 5ASA-GG NP’s andMMT-5ASA-GG NC’s were dispersed into 2ml of PBS (pH 1.2) for 2 hrs and it was putinto a dialysis tube (MWCO 2000 Da). Then pH was managed to 7.
5 and the releaseprofile studies were continued for 24 hrs. The dialysis tube was put into 50 mlof aqueous recipient PBS medium (pH 1.2) and (pH 7.4) with continuous stirringat 100 rpm and 37±2 ºC, for the total separation of drug in PBS. At a propertime intervals, the whole medium (50 ml) were replaced with the same volume offresh PBS (pH 1.2 and 7.4) (50 ml) and samples were processed for analysis byHPLC.
In vivo Study These studies were planned according to theguidelines of the CPCSEA, Ministry of Social Justice and Empowerment,Government of India and prior approval from the Institutional Animal EthicalCommittee (Reg. No. 147/PO/a/11/CPCSEA) of Guru Ramdas Khalsa Institute of Science and Technology, Pharmacy,Jabalpur, MP, India. OrganDistribution Study Male albino rats (150-200 g) were selected throughoutthe experiment. Animals were categorized into 4 groups of 6 rats in each. Thegroup I served as control, group II received 5ASA suspension (30 mg/kg), groupIII were introduced 5ASA-GG NP’s (0.5 mg/kg body weight related to 5ASA content)and group IV was given with 5ASA-GG-MMT NC’s (0.
5 mg/kg body weight related to5ASA content) . The formulations were orally administered in suspension formfollowed by enough volume of intake water. Three animals from each group were madeunconscious till their death by deep chloroform anesthesia at 1, 2, 3, 4, 6, 8,10, 12, 14, 16, 20 and 24 hrs after drug administration. The GI tract waseleminated; Stomach, small intestine, and colon were obtained. The luminalcontents were eleminated by applying mild pressure with wet scissors to thetissues. Luminal and organs contents were processed to weigh.
The organs wereincised open longitudinally and washed with saline solution (0.9% NaCl) todetach any remaining luminal contents. The organs (small pieces) were taken forhomogenization by Micro Tissue Homogenizer (Mac, Mumbai, India) at 4 ºC alongwith a small quantity of HPLC grade water. These organs were selected forhomogenization along with a small amount of PBS (pH 7.4); Acetonitrile (1 ml)was cautiously used and added to homogenate and it was kept for 30 minutes.Then it was centrifuged at 10,000 rpm for 5min and supernatant was filtered.This filtrate was used for assay for the drug content by measuring theabsorbance HPLC method. The drug content at different part of GI tract at differenttime period was determined.
TherapeuticActivityMale albino rats (avarageweight 150 g) were utilized for the inflammation model.Animals were treated with customaryexisting conditions such as maintenance of 12h light and 12h dark timings,controlled temperature and humidity (19 ± 29 °C; 35–60% humidity), standardpellet diet, water and beddings in polypropylene caging. Study protocol consisted of division of 24 animalsin four groups each group having 6 animals. details of which are as follows:-Group 1: Animals were keptuntreated and given salines only. Group 2, 3 & 4: Animals weretreated with trinitrobenzenesulfonicacid (TNBS) for induction of inflammation after the following process: firstlycatheterization of animals was done through 4cm intrarectally followed by lightnarcotizing with ether. TNBS (100 ?l) was then introduced in ethanol andapplied in a dose of 160 mg/kg.
Drug treatmentGroup 2: Animals were processed for treatmentwith 5ASA solution (at a dose of 30 or100 mg/kg body weight)Group 3: Animals were processed for treatment with5ASA-GG NP suspension (0.5 mg/kg bodyweight related to 5ASA content)Group 4: Animalswere processed for treatment with 5ASA-GG-MMT NC suspension (0.5 mg/kg body weight related to 5ASAcontent)Alladministrations were given once daily for six days.
Further animals were usedto sacrify 24h after the last drug/NP/NC administration and their colons were reselected. Pathophysiological ParametersInflammationintensity was estimated with a medical examination consisting weight loss,stool consistency and rectal bleeding as previously intimated 34, 35.Longitudinal opening of rectal colon tissue samples was performed and luminalcontent was removed after rinsing with iced phosphate buffer.
Further wetweight of tissue and colon was estimated and expressed as colon weight/lengthquotient. Myeloperoxidase activity measurement was done in order to examine theseverity of the colitis since it is a trustworthy index of severity ofinflammation caused by infiltration of activated neutrophils into inflamedtissue. Enzymatic activity was performed to analyse according to a standardmethod 36. StatisticalAnalysis The resultswere outlined as mean values±S.D. For the analysis of statistical significanceANOVA on ranks was used followed by Dunn’s test for all pairwise comparison.
Inall cases, P<0.05 was taken into consideration to be significant. RESULTSAND DISCUSSIONPreparationand Optimization of 5ASA-GG NP's and 5ASA-GG-MMT NC's5ASA-GG NP's were formulated by emulsionwater/oil/water (w/o/w) double emulsion solvent evaporation technique and5ASA-GG-MMT NP's were formulated by emulsion water/oil/water (w/o/w) doubleemulsion solvent evaporation techniques. Formulation NP4 the amount of drug was encapsulatedand increased in polymer content (30 mg) along with encapsulation efficiencywas obtained to be about 62.12% (Table I). NC4 the maximum amount of drug retained was found to be 82.75 % (TableII). Variations in the composition of drug polymer–clay nanocomposites werefurther reviewed in detail.
The drug loading and extent of drug was studied andalso encapsulation in 5ASA–GG-MMT as function of 5ASA content were studied, Theamount of 5ASA that was encapsulated in the 5ASA–GG-MMT nanocomposites enhancesin a linear manner (Table II) with increase in drug to MMT ratio from 1:1 to 2:1,however with further enhancement in drug to MMT ratio up to 3:1, an grow inencapsulation efficiency up to 90.24 % was obtained. This excessive increase ofencapsulation can be attributed to the cationic nature of 5ASA in thenanocomposites, which may enhance the interaction of 5ASA with negativelycharged MMT and polymer resulting in high encapsulation efficiency. Results ofencapsulation efficiency revealed that since formulation NP4 and NC4 furnishedbest performance in encapsulation efficiency, hence they were selected for furthercharacterization studies. MorphologicalStudiesSEM of freshly prepared 5ASA-GGnanoparticles (NP4) and 5ASA-GG-MMT (NC4) presented to be 2µm and 5µm sphericalsmoother surfaces particles. Formulation NP4 had shown smooth surface whereasformulation NC4 shown large irregular traces of particles confirming thepresence of MMT.
(Fig. 1 a, b). PDIand Zeta Potential Determination PDI of 5ASA-GG (NP4) and 5ASA-GG-MMT (NC6)were found to be 0.
064and 0.085, respectively (Fig. 2 a, b). The results werein good achievement with SEM studies, which was also depicted the nanometric size ofnanocomposites. Moreover, the zeta potential of positive values 5ASA-GG (NP4) and5ASA-GG-MMT (NC6) were found 6.48 and 10.07 mV, respectively.
The data clearly wasused to take information that nanocomposites were stable on drug loading andhence could be conveniently accepted as carriers for colon targeting of 5ASA.Particles size distribution pattern has an important role in measuring the drugrelease properties, their feasibility for oral administration and their fateafter in vivo administration. DifferentialScanning Calorimetry In the present study, 5ASA shown sharp endothermicpeak at 280 ºC, which could be corresponded to its melting transitiontemperature Fig. 4 a. In the matter of GG and MMT, endothermic peaks were foundat 81.86ºC and 118 ºC, respectively (Fig.
4 b, c. In case of 5ASA-GG endothermof guar gum and 5ASA was observed 78 ºC and 275 ºC Fig. 4 d. The DSC thermogramof 5ASA-GG-MMT, endothermic peaks were observed 80 ºC, 116 ºC and 278 ºC whichshowed presence of GG, MMT and 5ASA respectively Fig. 4. e.
Endothermic peak at 280 ºC, indicating that the drug was found tobe present in crystalline form inside the nanoparticles (Fig. 4 d, e. Theresults are in good agreement with previous studies 37.
PowderX-ray DiffractometryThe PXRD diffractograms were shown inFig. 4. 5-ASA that had shown characteristic intense peaks between 2y values of5°,16°, 23°, 31°, 39° and 40°, which had been confirmed its crystalline nature(Fig. 4a). The XRD diffractogram of GG and MMT had no sharp peak and it wasindicated the amorphous nature of GG and MMT (Fig. 4b, c). However, the diffractogramsof 5ASA GG and 5ASA-GG-MMT were shownthe typical crystalline peaks of 5-ASA associated with the crystalline drugmolecule, it was suggested that the formulations were suitable for sustainedand prolonged release (Fig. 4d, e).
The results were in good agreement withpreviously reported studies 38. In-vitroDrug ReleaseFormulations NP4 & NC4 had beenchoosen for in-vitro drug release characterization studies. In vitro drugrelease studies were done in gastrointestinal fluids of different pH at 37±0.5ºC.
Release in simulated gastric (Hcl, PH 1.2), (PBS, PH 6.8) and intestinalfluid (PBS, PH 7.4) were performed. Formulation NP4 showed 12.
01%, 26.32% and82.41% in pH 1.2, 6.
8 and 7.4 upto 24hrs respectively. Similarly formulationNC4 furnished drug release of 20.
12%, 49.05% & 97.32% pH 1.2, 6.8 and 7.
4up to 24hrs respectively. Formulation NC4 shown more than 90% of release incontrast to formulation NP4, the rationale behind this may be attributed to thepresence of MMT which significantlyenhances the level of absorption of the drug along with this presence of MMTalso proffered the controlled behavior of 5ASA release. This may also beconcluded by the barrier properties in the path offered by high amount of MMTlayers to release the drug in both the release media of drug until it entersinto the colon. OrganDistribution StudyOrgan distribution study of optimizedformulations were performed in albino rats for establishing its targetingpotential in colon. 5ASA suspension was comprised in the study for the comparisonof results. The results that was found indicate that maximum (84.
27±2.32%) drugconcentration was seen in stomach 1 hrs after oral administration of 5ASA suspension.Moreover, a minimal drug concentration was seen in the small intestine (9.
21±2.11%)and no drug was present in colon. At the end of 4 hrs, 40.
31±1.02% drugconcentration was seen in small intestine while the colon was found to be stilldevoid of drug. The relatively sharp was found to be decreased in the drugconcentration in stomach at time intervals (2–4 hrs) could be attributed todrug absorption through the stomach, systemic distribution and intestinal drugtransit.
After 6 hrs, maximum percentage of drug concentration was seen in thecolon 9.43±2.10% (Fig. 6). In case of nanocomposites (5ASA-GG-MMT NC’s), wasenterically protected with MMT, no drug concentration was regained from smallintestine even after 4 hrs (Fig. 6). However, the drug concentration wasreobtained only in small intestine after 5 hrs from 5ASA-GG-MMT NC’s.
Theformulation could have entered into ileum after 5 hrs and due to which3.42±0.02%, drug concentration was regained from small intestine after 5 hrs.In colon, abundant microflora and extended transit time was exploited fortargeted drug release and prolonged drug exposure.
After entry of nanocompositesin colon, the release of drug was found to be continuously increased. Theseresults were in well agreement with those we had observed during in vitro drugrelease studies. The peak drug concentration in colon observed with 5ASA-GG-MMTwas 70.09±6.34%.
Therewere significant differences in the drug concentration after 12 hrs from5ASA-GG-MMT NC’s in comparison to 5ASA-GG However, the drug concentration wasfound to be continuously declined and after 20 hrs, 27.23±0.56%, drugconcentration was regained in colon (Fig. 6). Thus, higher drug concentrationwas found with these colon-specific nanocomposites at all time points. The drugconcentration was found to be still detectable in the colon after 24 hrs in thecase of colon-specific nanocomposites. The drug concentration was recovered incolon and it was found 14.46±2.
22% from 5ASA-GG-MMT NC’s after 24 hrs. Thishigh concentration in the colon might be attributed to protection of the core5ASA-GG from the environment of the upper part of GI tract by the MMT, that iswhy preventing drug release in the stomach and small intestine. 5ASA-GGNP & 5ASA-GG-MMT NC Therapeutic ActivityIn response to examine the therapeutic activity,the explanatory result of 5ASA-GG NP’s & 5ASA-GG-MMT NC’s systems were studiedon a pre-existent inflammatory model in rats. After induction of experimentalinflammation, there was a substantial increment in clinical outcome index inresponse to intestinal inflammation within 24hrs. All the 5ASA containing formulations shownreduction in the inflammation, differences became statistically significant onday 6. Similar results were obtained in response to administration of oralsolution of 5ASA at 30 mg/kg. Results of clinical activity index, and colonweight/length ratios were estimated to be lower in contrast to the inflammatorycontrol (Fig.
7). All 5ASA loaded formulations were shown a myeloperoxidaseactivity considerably different from the untreated group which revealed similartherapeutic effects with 5ASA-GG NP’s and 5ASA suspension at 30mg/kg (Fig 7).In comparison to 5ASA-GG NP’s, 5ASA-GG-MMT NC’s had been shown better performance in reducingthe inflammation i.e. (27.2±2.1 U/mg tissue for 5ASA-GG-MMT NC’s &15.
8±21.8 for 5ASA-GG NP’s ), while oral solution at the lower dose presentedthe same level of therapeutic effect compared to 5ASA-GG NP’s (Fig.7 ).
CONCLUTIONSIn this study an oral controlled drugdelivery system for 5ASA loaded GG nanoparticles and 5ASA–GG-MMT nanocompositeswas developed by w/o/w double emulsion solvent evaporation technique. Aboutnanoparticle 62.12% entrapment efficiency and 79.23% release and nanocomposites82.75% entrapment efficiency and 95.
45% release were achieved for the highlyhydrophilic drug, 5ASA. The presence of drug within the 5ASA–GG nanoparticlesand 5ASA–GG-MMT nanocomposites also confirmed by DSC data. The observedmorphology and structure of the 2µm-5µm spherical 5ASA–GG nanoparticles and 5ASA–GG-MMTnanocomposites (confirms by SEM data).The drug release profile of GG was found to be pH dependent, the presence ofMMT platelets within the 5ASA–GG formulations results in controlled and higher% release of drug. Results of therapeutic activity shown that 5ASA–GG-MMTnanocomposites performed better in reducing the inflammation in contrast to5ASA-GG nanoparticles and 5ASA suspension. Therefore, it can be said that thesynthesized formulations have high potential as a controlled drug deliverysystem for 5ASA. Organ distribution studies were found to be revealed that thedrug concentration was observed higher in the colon tissue, with low systemicexposure to the drug.
However, proposed system was found to be reduced the sideeffects of the drug caused by its absorption from the upper part of the GItract. The new colonic delivery system significantly was found to improve theefficacy of 5ASA in the healing of induced colitis in rats. The describedsystem was provided therefore this may bevery useful for clinical treatment of human colonic inflammatory bowel disease.