Preparation of 5 aminosalicylic acid –
Loaded GG-MMT Nanocomposites
5ASA–GG-MMT nanocomposites had
been synthesized in two steps. Firstly, Guar gum was to dissolve in water and then to emulsify in
the solution of acetone/ethanol containing 5-ASA with magnetic stirring
followed by sonication. In the second step, the primary w/o emulsion had been
emulsified in the external aqueous phase of MMT and Pluronic F68 (1%, w/v) to
form a w/o/w-emulsion. The intermediate organic phase was separated the
internal water droplets from each other as well as from the external aqueous
continuous phase. After solvent evaporation the 5ASA–GG-MMT nanocomposites were
collected by centrifugation and washed with double distilled water before
freeze-drying. 5ASA-GG-MMT NC’s formulations were labeled s code (NC1, NC2, NC3
& NC4) (Table 2) 28.
shape and surface morphology of 5ASS-GG NP’s (NP4) and MMT-5ASA-GG NC’s (NC4) were
observed by Scanning electron microscopy (SEM) (JELO 5400, USA)
Size, Polydispersity Index (PDI) and Zeta Potential
suspension of 5ASA-GG nanoparticles (NP4) and MMT-5ASA-GG (NC4)
nanocomposites had been crammed in
the chamber of a laser diffraction particle size analyzer (DTS Ver. 4.10,
Malvern Instruments, Malvern, UK) for examination of PDI and average particle
size. Zeta potential of 5ASA-GG NP’s and MMT-5ASA-GG NC’s were also obtained by using Malvern Zetasizer (DTS Ver. 4.10, Malvern
Instruments, Malvern, UK) 29.
Scanning 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 the
sample from 30 to 400ºC at the
heating rate of 10ºC/min, in a
nitrogen environment (nitrogen gas flow rate of 60.0ml/min). DSC studies had
been conducted on NETZSCH DSC 200F3 240-20-427-L Proteus Software 30.
X-ray Diffractometry (PXRD)
Powder X-ray diffractometry (PXRD)
diffraction of formulations (NP4) & (NC4) analyses had been carried out in
order to find out crystalline nature of polymer and drug. Powder X-ray
diffraction patterns of samples were completed using power X-ray diffractometer
(Bruker, Munich, Germany). X-ray diffraction analysis was observed using a
nickel-filtered Cu-Ka radiation (a voltage of 40 kV and a current of 20 mA).
The scanning rate 2/min over a 2y range
of 0–40° and with an interval of 0.02? was set 31.
of Drug Entrapment Efficiency
mg of 5ASA-GG NP’s and MMT-5ASA-GG NC’s were
dissolved in acetone and ethanol (10 ml). Further suspension was centrifuged,
and supernatant was decanted than GG was precipitated by adding 10 ml of
ethanol. 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 at
25ºc, wavelength detector, and a zorbax 5? C18 column (250 × 4.60 mm) at 329
nm. The concentration of free drug in the supernatant was taken by comparing
the absorption of the supernatant with standard curve 32. The amount of drug
entrapped into nanocomposites had been calculated as the difference between the
amount of drug used for the formulation and the amount of drug in the
supernatant and present as encapsulation efficiency. Encapsulation efficiencies
had been calculated by using formula :-
efficiency (%) = total
drug/ total drug ×100
In-vitro drug release of both the formulations
(NP4 & NC4) were observed in simulated gastric fluid (SGF) with pH 1.2 and
simulated intestinal fluid (SIF) with pH 7.5. These pH values were identified
based 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 and
MMT-5ASA-GG NC’s were dispersed into 2ml of PBS (pH 1.2) for 2 hrs and it was put
into a dialysis tube (MWCO 2000 Da). Then pH was managed to 7.5 and the release
profile studies were continued for 24 hrs. The dialysis tube was put into 50 ml
of aqueous recipient PBS medium (pH 1.2) and (pH 7.4) with continuous stirring
at 100 rpm and 37±2 ºC, for the total separation of drug in PBS. At a proper
time intervals, the whole medium (50 ml) were replaced with the same volume of
fresh PBS (pH 1.2 and 7.4) (50 ml) and samples were processed for analysis by
In vivo Study
These studies were planned according to the
guidelines of the CPCSEA, Ministry of Social Justice and Empowerment,
Government of India and prior approval from the Institutional Animal Ethical
Committee (Reg. No. 147/PO/a/11/CPCSEA) of Guru Ramdas Khalsa Institute of Science and Technology, Pharmacy,
Jabalpur, MP, India.
Male albino rats (150-200 g) were selected throughout
the experiment. Animals were categorized into 4 groups of 6 rats in each. The
group I served as control, group II received 5ASA suspension (30 mg/kg), group
III 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 to
5ASA content) . The formulations were orally administered in suspension form
followed by enough volume of intake water. Three animals from each group were made
unconscious 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 was
eleminated; Stomach, small intestine, and colon were obtained. The luminal
contents were eleminated by applying mild pressure with wet scissors to the
tissues. Luminal and organs contents were processed to weigh. The organs were
incised open longitudinally and washed with saline solution (0.9% NaCl) to
detach any remaining luminal contents. The organs (small pieces) were taken for
homogenization by Micro Tissue Homogenizer (Mac, Mumbai, India) at 4 ºC along
with a small quantity of HPLC grade water. These organs were selected for
homogenization 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 the
absorbance HPLC method. The drug content at different part of GI tract at different
time period was determined.
Male albino rats (avarage
weight 150 g) were utilized for the inflammation model.
Animals were treated with customary
existing conditions such as maintenance of 12h light and 12h dark timings,
controlled temperature and humidity (19 ± 29 °C; 35–60% humidity), standard
pellet diet, water and beddings in polypropylene caging.
Study protocol consisted of division of 24 animals
in four groups each group having 6 animals. details of which are as follows:-
Group 1: Animals were kept
untreated and given salines only.
Group 2, 3 & 4: Animals were
treated with trinitrobenzenesulfonic
acid (TNBS) for induction of inflammation after the following process: firstly
catheterization of animals was done through 4cm intrarectally followed by light
narcotizing with ether. TNBS (100 ?l) was then introduced in ethanol and
applied in a dose of 160 mg/kg.
Group 2: Animals were processed for treatment
with 5ASA solution (at a dose of 30 or
100 mg/kg body weight)
Group 3: Animals were processed for treatment with
5ASA-GG NP suspension (0.5 mg/kg body
weight related to 5ASA content)
Group 4: Animals
were processed for treatment with 5ASA-GG-MMT NC suspension (0.5 mg/kg body weight related to 5ASA
administrations were given once daily for six days. Further animals were used
to sacrify 24h after the last drug/NP/NC administration and their colons were reselected.
intensity 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 luminal
content was removed after rinsing with iced phosphate buffer. Further wet
weight of tissue and colon was estimated and expressed as colon weight/length
quotient. Myeloperoxidase activity measurement was done in order to examine the
severity of the colitis since it is a trustworthy index of severity of
inflammation caused by infiltration of activated neutrophils into inflamed
tissue. Enzymatic activity was performed to analyse according to a standard
were outlined as mean values±S.D. For the analysis of statistical significance
ANOVA on ranks was used followed by Dunn’s test for all pairwise comparison. In
all cases, P<0.05 was taken into consideration to be significant. RESULTS AND DISCUSSION Preparation and Optimization of 5ASA-GG NP's and 5ASA-GG-MMT NC's 5ASA-GG NP's were formulated by emulsion water/oil/water (w/o/w) double emulsion solvent evaporation technique and 5ASA-GG-MMT NP's were formulated by emulsion water/oil/water (w/o/w) double emulsion solvent evaporation techniques. Formulation NP4 the amount of drug was encapsulated and increased in polymer content (30 mg) along with encapsulation efficiency was obtained to be about 62.12% (Table I). NC4 the maximum amount of drug retained was found to be 82.75 % (Table II). Variations in the composition of drug polymer–clay nanocomposites were further reviewed in detail. The drug loading and extent of drug was studied and also encapsulation in 5ASA–GG-MMT as function of 5ASA content were studied, The amount of 5ASA that was encapsulated in the 5ASA–GG-MMT nanocomposites enhances in 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 in encapsulation efficiency up to 90.24 % was obtained. This excessive increase of encapsulation can be attributed to the cationic nature of 5ASA in the nanocomposites, which may enhance the interaction of 5ASA with negatively charged MMT and polymer resulting in high encapsulation efficiency. Results of encapsulation efficiency revealed that since formulation NP4 and NC4 furnished best performance in encapsulation efficiency, hence they were selected for further characterization studies. Morphological Studies SEM of freshly prepared 5ASA-GG nanoparticles (NP4) and 5ASA-GG-MMT (NC4) presented to be 2µm and 5µm spherical smoother surfaces particles. Formulation NP4 had shown smooth surface whereas formulation NC4 shown large irregular traces of particles confirming the presence of MMT. (Fig. 1 a, b). PDI and 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 were in good achievement with SEM studies, which was also depicted the nanometric size of nanocomposites. Moreover, the zeta potential of positive values 5ASA-GG (NP4) and 5ASA-GG-MMT (NC6) were found 6.48 and 10.07 mV, respectively. The data clearly was used to take information that nanocomposites were stable on drug loading and hence could be conveniently accepted as carriers for colon targeting of 5ASA. Particles size distribution pattern has an important role in measuring the drug release properties, their feasibility for oral administration and their fate after in vivo administration. Differential Scanning Calorimetry In the present study, 5ASA shown sharp endothermic peak at 280 ºC, which could be corresponded to its melting transition temperature Fig. 4 a. In the matter of GG and MMT, endothermic peaks were found at 81.86ºC and 118 ºC, respectively (Fig. 4 b, c. In case of 5ASA-GG endotherm of guar gum and 5ASA was observed 78 ºC and 275 ºC Fig. 4 d. The DSC thermogram of 5ASA-GG-MMT, endothermic peaks were observed 80 ºC, 116 ºC and 278 ºC which showed presence of GG, MMT and 5ASA respectively Fig. 4. e. Endothermic peak at 280 ºC, indicating that the drug was found to be present in crystalline form inside the nanoparticles (Fig. 4 d, e. The results are in good agreement with previous studies 37. Powder X-ray Diffractometry The PXRD diffractograms were shown in Fig. 4. 5-ASA that had shown characteristic intense peaks between 2y values of 5°,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 was indicated the amorphous nature of GG and MMT (Fig. 4b, c). However, the diffractograms of 5ASA GG and 5ASA-GG-MMT were shown the typical crystalline peaks of 5-ASA associated with the crystalline drug molecule, it was suggested that the formulations were suitable for sustained and prolonged release (Fig. 4d, e). The results were in good agreement with previously reported studies 38. In-vitro Drug Release Formulations NP4 & NC4 had been choosen for in-vitro drug release characterization studies. In vitro drug release studies were done in gastrointestinal fluids of different pH at 37±0.5 ºC. Release in simulated gastric (Hcl, PH 1.2), (PBS, PH 6.8) and intestinal fluid (PBS, PH 7.4) were performed. Formulation NP4 showed 12.01%, 26.32% and 82.41% in pH 1.2, 6.8 and 7.4 upto 24hrs respectively. Similarly formulation NC4 furnished drug release of 20.12%, 49.05% & 97.32% pH 1.2, 6.8 and 7.4 up to 24hrs respectively. Formulation NC4 shown more than 90% of release in contrast to formulation NP4, the rationale behind this may be attributed to the presence of MMT which significantly enhances the level of absorption of the drug along with this presence of MMT also proffered the controlled behavior of 5ASA release. This may also be concluded by the barrier properties in the path offered by high amount of MMT layers to release the drug in both the release media of drug until it enters into the colon. Organ Distribution Study Organ distribution study of optimized formulations were performed in albino rats for establishing its targeting potential in colon. 5ASA suspension was comprised in the study for the comparison of results. The results that was found indicate that maximum (84.27±2.32%) drug concentration 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% drug concentration was seen in small intestine while the colon was found to be still devoid of drug. The relatively sharp was found to be decreased in the drug concentration in stomach at time intervals (2–4 hrs) could be attributed to drug absorption through the stomach, systemic distribution and intestinal drug transit. After 6 hrs, maximum percentage of drug concentration was seen in the colon 9.43±2.10% (Fig. 6). In case of nanocomposites (5ASA-GG-MMT NC's), was enterically protected with MMT, no drug concentration was regained from small intestine even after 4 hrs (Fig. 6). However, the drug concentration was reobtained only in small intestine after 5 hrs from 5ASA-GG-MMT NC's. The formulation could have entered into ileum after 5 hrs and due to which 3.42±0.02%, drug concentration was regained from small intestine after 5 hrs. In colon, abundant microflora and extended transit time was exploited for targeted drug release and prolonged drug exposure. After entry of nanocomposites in colon, the release of drug was found to be continuously increased. These results were in well agreement with those we had observed during in vitro drug release studies. The peak drug concentration in colon observed with 5ASA-GG-MMT was 70.09±6.34%. There were significant differences in the drug concentration after 12 hrs from 5ASA-GG-MMT NC's in comparison to 5ASA-GG However, the drug concentration was found to be continuously declined and after 20 hrs, 27.23±0.56%, drug concentration was regained in colon (Fig. 6). Thus, higher drug concentration was found with these colon-specific nanocomposites at all time points. The drug concentration was found to be still detectable in the colon after 24 hrs in the case of colon-specific nanocomposites. The drug concentration was recovered in colon and it was found 14.46±2.22% from 5ASA-GG-MMT NC's after 24 hrs. This high concentration in the colon might be attributed to protection of the core 5ASA-GG from the environment of the upper part of GI tract by the MMT, that is why preventing drug release in the stomach and small intestine. 5ASA-GG NP & 5ASA-GG-MMT NC Therapeutic Activity In response to examine the therapeutic activity, the explanatory result of 5ASA-GG NP's & 5ASA-GG-MMT NC's systems were studied on a pre-existent inflammatory model in rats. After induction of experimental inflammation, there was a substantial increment in clinical outcome index in response to intestinal inflammation within 24hrs. All the 5ASA containing formulations shown reduction in the inflammation, differences became statistically significant on day 6. Similar results were obtained in response to administration of oral solution of 5ASA at 30 mg/kg. Results of clinical activity index, and colon weight/length ratios were estimated to be lower in contrast to the inflammatory control (Fig. 7). All 5ASA loaded formulations were shown a myeloperoxidase activity considerably different from the untreated group which revealed similar therapeutic 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 reducing the 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 presented the same level of therapeutic effect compared to 5ASA-GG NP's (Fig.7 ). CONCLUTIONS In this study an oral controlled drug delivery system for 5ASA loaded GG nanoparticles and 5ASA–GG-MMT nanocomposites was developed by w/o/w double emulsion solvent evaporation technique. About nanoparticle 62.12% entrapment efficiency and 79.23% release and nanocomposites 82.75% entrapment efficiency and 95.45% release were achieved for the highly hydrophilic drug, 5ASA. The presence of drug within the 5ASA–GG nanoparticles and 5ASA–GG-MMT nanocomposites also confirmed by DSC data. The observed morphology and structure of the 2µm-5µm spherical 5ASA–GG nanoparticles and 5ASA–GG-MMT nanocomposites (confirms by SEM data). The drug release profile of GG was found to be pH dependent, the presence of MMT platelets within the 5ASA–GG formulations results in controlled and higher % release of drug. Results of therapeutic activity shown that 5ASA–GG-MMT nanocomposites performed better in reducing the inflammation in contrast to 5ASA-GG nanoparticles and 5ASA suspension. Therefore, it can be said that the synthesized formulations have high potential as a controlled drug delivery system for 5ASA. Organ distribution studies were found to be revealed that the drug concentration was observed higher in the colon tissue, with low systemic exposure to the drug. However, proposed system was found to be reduced the side effects of the drug caused by its absorption from the upper part of the GI tract. The new colonic delivery system significantly was found to improve the efficacy of 5ASA in the healing of induced colitis in rats. The described system was provided therefore this may be very useful for clinical treatment of human colonic inflammatory bowel disease.