Claims:We Claim:

1. A hydrogel for biomedical uses, said hydrogel comprising hyaluronic acid in chitosan gel, characterized in that the hyaluronic acid is obtained from microbial Streptococcus equi subsp.equi isolated from a horse nasal sample.

2. The hydrogel as claimed in claim 1, wherein the ratio of hyaluronic acid with chitosan is 1:1 to 1:5 w/w.

3. The hydrogel as claimed in claim 1, wherein the hyaluronic acid in chitosan gel is prepared by emulsified gel technique using sodium alginate solution emulsified with pure glycerol for hydration.

4. The hydrogel as claimed in claim 1, wherein the ratio of sodium alginate to chitosan gel is 1:1 to 1:5w/v.

5. A process for preparation of hydrogel for biomedical purpose comprising hyaluronic acid in chitosan gel, characterized in that the hyaluronic acid is obtained from microbial Streptococcus equi subsp.equi isolated from horse nasal sample, said process comprises of combining obtained hyaluronic acid with chitosan in a ratio of 1:1 to 1:5 w/w, to obtain hydrogel.
6. The process for preparation of hydrogel as claimed in claim 5, wherein the hyaluronic acid is obtained in following steps:
(i) isolating Streptococcus equi subsp.equi isolated from horse nasal sample;
(ii) preparing a culture for the isolated Streptococcus equi subsp.equi;
(iii) fermenting the culture;
(iv) centrifuging the fermented culture with ethanol;
(v) obtaining supernatant from the fermented culture;
(vi) obtaining hyaluronic acid from the suspension;
(vii) combining obtained hyaluronic acid of step (vi) with chitosan in a ratio of 1:1 to 1:5 w/w, to obtain hydrogel.

, Description:Field of Invention:
This invention relates to an organic hydrogel for biomedical applications comprising chitosan in hyaluronic acid. More particularly, the synthesis method does not involve harmful chemicals, and the hydrogel is non toxic, biodegradable, economical and shows better physical chemical properties. The invention relates to a hydrogel comprising chitosan in hyaluronic acid, wherein the hyaluronic acid is obtained from microbial Streptococcus equi subsp.equi isolated from horse nasal sample.

Background of the Invention:
Hyaluronic acid (HA) is a mucopolysaccharide exhibiting various biomedical properties and therefore a choice of interest in recent years with a scope of producing therapeutic and cosmetic agents. Hyaluronic acid also called as hyaluronan, is an anionic, nonsulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues and formed in the plasma membrane. Microbial source of hyaluronic acid has gained immense importance over avian source as it is viral free and non-allergic.
CN101676000A relates to a preparation method of a hyaluronic acid-chitosan biomembrane, wherein the biomembrane, which contains hyaluronic acid and carboxymethyl chitosan, is prepared by the crosslinking reaction of hyaluronic acid, carboxymethylchitosan and polyvinyl alcohol in the presence of a crosslinking agent.
The present invention relates to synthesis of organic hydrogel from bacterially derived HA. In general, chemical synthesis of hyaluronic acid is being carried out by two approaches: pre-glycosylation oxidation and post-glycosylation oxidation. Although chemical synthesis is an existing method to synthesize hyaluronic acid, stereo-selectivity and regio-selectivity of sugars is one of the key factors that involves cost-demanding production. Also, liberation of the target compounds and purification of the polyanionic target molecules is a roadblock to the large scale chemical synthesis of HA oligosaccharides.
To overcome such limitations prevailing in chemical synthesis, microbial synthesis of hyaluronic acid, specifically Streptococcus equi subsp.equi, has come as an alternative approach for synthesis of hydrogel.The hyaluronic acid prepared from S. equi subsp.equi obtained from horse nasal sample according to the process of present invention can be prepared on large scale suitable for commercialization.
The hydrogel prepared according to the invention is organic, and does not possess side effects and harsh chemicals.
Summary of Invention:
According to an embodiment of the invention, there is provided a hydrogel for biomedical uses, said hydrogel comprising hyaluronic acid in chitosan gel, characterized in that the hyaluronic acid is obtained from microbial Streptococcus equi subsp.equi isolated from horse nasal sample.
Yet according to another embodiment of the invention, the ratio of hyaluronic acid with chitosan is 1:1 to 1:5 w/w.
Yet according to another embodiment of the invention, the hyaluronic acid in chitosan gel is prepared by emulsified gel technique using sodium alginate solution emulsified with pure glycerol for hydration.
Yet according to another embodiment of the invention, the ratio of sodium alginate to chitosan gel is 1:1 to 1:5w/v.
Yet according to another embodiment of the invention, there is provided a process for preparation of hydrogel for biomedical purpose comprising hyaluronic acid in chitosan gel, characterized in that the hyaluronic acid is obtained from microbial Streptococcus equi subsp.equi isolated from horse nasal sample, said process comprises of combining obtained hyaluronic acid with chitosan in a ratio of 1:1 to 1:5 w/w, to obtain hydrogel.
Yet according to another embodiment of the invention, the hyaluronic acid is obtained in following steps:
(i) isolating Streptococcus equi subsp.equi isolated from horse nasal sample;
(ii) preparing a culture for the isolated Streptococcus equi subsp.equi;
(iii) fermenting the culture;
(iv) centrifuging the fermented culture with ethanol to obtain a supernatant;
(v) obtaining hyaluronic acid from the supernatant;
(vi) purifying the hyaluronic acid.
Brief description of drawings:
The manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.
Fig-1 shows SEM visualization of CHT-HA Hydrogel at different scale measurment.
Fig2 : In-vitro drug release of all five ratios of HA-CHT hydrogel at different pH range, (mean ± SD, n=3). (Fig2a=1:1 ratio;Fig2b=1:2 ratio;Fig2c=1:3 ratio;Fig2d=1:4 ratio;Fig2e=1:5ratio).
Detailed description of the Invention:
The invention provides a strain of Streptococcus equi subsp.equi from horse nasal sample as a potent strain for Hyaluronic acid (HA) production was identified after screening 297samples. Hyaluronic acid produced by a selected isolate was confirmed by HPLC-RID. Production of HA was also enhanced by optimizing the growth conditions by conventional and statistical approach. Modified Luria bertani broth was proven to be an effective growth medium for strain of Streptococcus equi subsp.equi. A high increase of HA yield upto 7.21 g/L was obtained. The novel strain was deposited at genebank. The complete data is available at:
https://www.ncbi.nlm.nih.gov/nuccore/MK156140.1?report=genbank&to=981

source 1..981
/organism="Streptococcus sp."
/mol_type="genomic DNA"
/strain="HNS35"
/isolation_source="Horse nasal swab"
/host="horse"
/db_xref="taxon:1306"
rRNA<1..>981
/product="16S ribosomal RNA"
ORIGIN
1 tgccccgtccacgagtgactaaatcacctgtagacttgcaccctcgctccgtaagtcgta
61 acaaggtaaccgtagagtttgatcctggctcaggacgaacgctggcggcgtgcctaatac
121 atgcaagtggaacgcacagatgatacgtagcttgctacaattatctgtgagtcgcgaacg
181 ggtgagtaacgcgtaggtaacctagcttatagcgggggataactattggaaacgatagct
241 aataccgcataaaagtggttgacccatgttaaccatttaaaaggagcaacagctccacta
301 tgagatggacctgcgttgtattagctagttggtagggtaaaggcctaccaaggcgacgat
361 acatagccgacctgagagggtgaacggccacactgggactgagacacggcccagactcct
421 acgggaggcagcagtagggaatcttcggcaatggggggaaccctgaccgagcaacgccgc
481 gtgagtgaagaaggttttcggatcgtaaagctctgttgttagagaagaacagtgatggga
541 gtggaaagtccatcatgtgacggtaactaaccagaaagggacggctaactacgtgccagc
601 agccgcggtaatacgtaggtcccgagcgttgtccggatttattgggcgtaaagcgagcgc
661 aggcggtttgataagtctgaagttaaaggcagtggcttaaccattgtatgctttggaaac
721 tgttaaacttgagtgcagaaggggagagtggaattccatgtgtagcggtgaaatgcgtag
781 atatatggaggaacaccggtggcgaaagcggctctctggtctgtaactgacgctgaggct
841 cgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgctgag
901 tgctaggtgttaggccctttccggggcttagtgccggagctaacgcattaagcattccgc
961 ctggagagcaatccaaccgg t
//

The production of hyaluronic acid in lab scale and in pilot scale were almost similar even after generations which stored for 3 years in -200C. The pilot scale production of hyaluronic acid was carried out in 5L fermenter under the optimized conditions also resulted in 7 fold increase. This shows that even the pilot scale making of the HA from the isolate showed a high yield. This is economic for manufacturing of the HA in high yields for manufacturing and commercialization.
The HA obtained is used to prepare biodegradable hydrogel with biodegradable chitosan polymer as entrapped in hyaluronic acid from S.equi subsp.equi. The synthesis method is completely organic in nature without using any toxic or hazardous chemical. The resultant organic hydrogel (HA-CHT-HGL) is characterized for theoperative preclinical delivery and alsoforits biocompatibility.
Streptococcus equi subsp.equi (MK156140) is identified as a potent strain of hyaluronic acid producer. Strain of Streptococcus equi subsp.equi showed better growth at a pH of 7.1. The growth was performed at a pH maintained at 7.1. The fermented growth medium was agitated at a speed of 180 rpm, temperature of 35ºC for a period of 26 hr were found to enhance the production of hyaluronic acid.
MATERIAL & METHODS
Preparation of Hyaluronic acid from Streptococcus equi subsp.equi
Streptococcus equi subsp.equi (MK156140) strain was isolated from a horse nasal swab and maintained in our laboratory. It was subcultured and maintained in nutrient media. The culture was incubated at 32ºCfor 24 h. Glycerol stock (50%) of the culture was maintained in a deep freezer (-20°C) for future experimental studies.
The inoculum was prepared in 250 ml Erlenmeyer flasks containing 50ml of LB media of pH 7.0. The media was autoclaved at 121ºC (15 lbs) for 20 min and inoculated with Streptococcus equi subsp.equi MK1561140. The inoculated flasks were kept on an orbital shaker (Scigenics Biotech) at 150 rpm for 24h, and the grown culture was used as inoculum. The inoculum size of 10-8 was used throughout the study for subsequent inoculation unless otherwise specified.
To extract the hyaluronic acid, the fermented broth was initially blended with a 10% volume of 5%(w/v) SDS for 10min and centrifuged at 5000xg/30 min. Hyaluronic acid was precipitated by mixing the supernatant with three volumes of ethanol and then centrifuged at 5000xg/10min. The sediment was redissolved with one volume of 1.5 M NaCl and 3 volume of ethanol and centrifuged again at 5000xg/10 min. Finally, the sediment was resuspended in distilled water to identify hyaluronic acid.
Isolates that are negative for catalase activity were inoculated into nutrient broth and incubated for 24 hrs at 37o C under shaken condition. The hyaluronic acid present in the capsule was extracted by adding 10% volume of 5% SDS and incubated for 10 min, followed by centrifugation at 5000 rpm for 30 min. Three volumes of ethanol were mixed with the supernatant and centrifuged at 3000 rpm for 10 min to precipitate the hyaluronic acid. The precipitated hyaluronic acid was determined by High Performance Liquid Chromatography with refractive index detector. Obtained Hyaluronic acid was estimated by CTAB method.
To estimate the hyaluronic acid, the fermented broth sample was diluted with equal volume of 0.1% of SDS and incubated for 10 minutes at room temperature to free the capsular bound hyaluronic acid. The mixture was then filtered through a 0.45µm syringe filter and used in a turbidimetric HA quantification assay. One ml of filtrate is taken in a clean test tube, to this 1ml of 0.1M acetic acid and 2ml of CTAB reagent (2.5% of CTAB dissolved in 0.5M NaOH) was added and incubated for 20 minutes at room temperature. Optical density was recorded at 600 nm using a UV-vis spectrophotometer

Hydrogel Synthesis method
HA (1% w/v) and Chitosan (2% w/v) (chitosan is also referred herein as CHT) was thoroughly dissolved in 1% Aqueous acetic acid solution. The solution was stirred at 1000 rpm for 8 hours at room temperature for the complete dissolution of chitosan and production of homogenous HA-CHT viscous solution. The HA-CHT solution was stored at 4ºC for 24 h for the stabilization and for complete polymer hydration of chitosan. Sodium alginate (SA) solution (3% w/v) was prepared separately in distilled water and mixed with glycerol (2%), and stirred at 800 rpm for 5 h in room temperature to obtain the homogenous solution. 10 ml of SA solution was then added gradually into 40 ml HA-CHT solution, which was still in a state of stirring to obtain the hydrogel ratio of 1:4. The resulting hydrogel was stirred for another 2 hour at room temperature for the final homogenous viscous hydrogel loaded with HA. The obtained HA-CHT hydrogel was sterilized at 121ºC for 25 min. Thus, the hydrogels were prepared with five different ratios of SA and CHT (1:1, 1:2, 1:3, 1:4, 1:5) for further analysis.

Characterization Methods of HA-CHT Hydrogel
Organoleptic characteristics
The organoleptic characteristics of synthesized HA-CHT hydrogels were tested in terms of color, odor, texture, as well as the stiffness, grittiness, greasiness and tackiness by applied on skin for a couple of minutes.
Result: All five ratios of HA-CHT hydrogels showed odorless, buffy pale white appearance with smooth texture and soft feeling, less greasy on touch also lacking of any gritty particles, tackiness and irritant felling.

Table 2: Organoleptic characteristics of hydrogels
S. No. Studies
Hydrogel
(CHT:SA) ratio Color Odor Texture Feel

1 1:1 buffy pale white Odorless smooth Soft
2 1:2 buffy pale white Odorless smooth Soft
3 1:3 buffy pale white Odorless smooth Soft
4 1:4 buffy pale white Odorless smooth Soft
5 1:5 buffy pale white Odorless smooth Soft

Homogeneity test
A small quantity of HA-CHT hydrogels were pressed between the thumb and index finger and the consistency of the gel was noticed (whether homogeneous or not).
Results: All five ratios of HA-CHT hydrogels showed decent homogeneity and consistency without any aggregation of polymers and ingredients. Moreover, the samples did not show any grittiness or inconsistency in appearance and feeling, which showed a better stable and homogenous hydrogel.

Table 3: Homogeneity of hydrogels

S. No. Hydrogel (CHT:SA) ratio Homogeneity
1 1:1 ***
2 1:2 ***
3 1:3 ***
4 1:4 ***
5 1:5 ***
Keys: *** = High Homogeneity, ** = Moderate Homogeneity, * = Low Homogeneity

Spreadability Analysis
The spreadability of HA-CHT Hydrogel samples were measured by spreading Hydrogels individually on a pre-marked (circle of 2 cm2 diameter) clean glass plate, covered with another glass slide, 2gmof weight was kept on the upper glass plate for 5 min. The spreadability of hydrogels were measured by determining the diameter of spreaded hydrogels on a glass plate. The formulation analysis was triplicated and the measurement was repeated thrice with average value along with SD.
Results: All five ratio HA-CHT hydrogels showed acceptable range of spreadability. The spreadability was optimum without formation of any sign of lumps or bulges exhibiting ideal transdermal or topical formulation with decent stability.
Table3: Spreadability analysis of CHT-HA Hydrogel (mean ± SD, n=3).
S. No. Hydrogel (CHT:SA) ratio Spreadability (cm)
1 1:1 2.50 ± 0.14
2 1:2 2.90. ± 0.09
3 1:3 3.45 ± 0.24
4 1:4 3.95 ± 0.61
5 1:5 4.55 ± 0.83

Scanning Electron Microscopy (SEM)
The structure, morphology and orientation of HA-CHT hydrogels were assessed by scanning electron microscopy (SEM), Nova Nano SEM 450, Germany. Prior to the SEM evaluation, the hydrogels were lyophilized by using freeze dry lyophilizer (REMI, New Delhi, India). The dried hydrogel was placed on SEM stub by employing double sided adhesive tape at 50mA for 5-10 minutes via sputter (KYKY SBC-12, Beijing, China). A scanning electron microscope aided with secondary electron detector was employed to obtain digital images of the developed hydrogel system.
Results: The Scanning Electron microscopy (SEM) of all five synthesized ratiosof HA-CHT hydrogels showed smooth morphology without formation of any lumps and surface texture of hydrogel, shown in Figure 1. The phase separation or other clusters are showing better stability and amalgam of polymers and entrapment of HA.

pH Analysis
The pH of the HA-CHT hydrogels were measured by using digital pH meter (HI-TECH WATER TECH. New Delhi, India). The formulation analysis was triplicated and the measurement was repeated thrice with average value along with SD.
Results: The pH analysis of all five ratios of HA-CHT hydrogels were showed slightly acidic pH. This is due to the ratio of SA (sodium alginate) in CHT hydrogel, as SA exhibited acidic characteristics in solution. The slightly acidic characteristics of synthesized hydrogel mimic the skin pH and showed significant transdermal property in clinical platform.
Table 4: pH analysis of HA-CHT Hydrogels (mean ± SD, n=3)
S. No. Hydrogel (CHT:HA) ratio pH
1 1:1 5.0 ± 0.23
2 1:2 5.4. ± 0.78
3 1:3 5.9 ± 0.20
4 1:4 6.3 ± 0.48
5 1:5 6.6 ± 0.83
Gelation Temperature and Time
Gelation temperature of HA-CHT hydrogels were screened by placing hydrogel in a water bath with 10 ml vial, increasing the temperature slowly from 15 to 40 °C for 10 min at a rate of 0.5 °C min–1. The vials were inspected for gelation by slanting them at 90? angle to check the flow upon tilting. Gelation time is defined as the time taken by the hydrogel formulations to completely change from sol to gel at an optimum temperature. The formulation analysis was triplicated and the measurement was repeated thrice with average value along with SD.
Results: The gelling time showed by all five ratios of HA-CHT hydrogels are varied due to the concentration of SA. The SA has thickening properties, increasing the concentration of SA with chitosan significantly reduce the gelling time. Optimum gelling time and concentration played crucial role in effective transdermal delivery of hydrogel. The gelling time of all five batches of Hydrogel are described in below table.
Table 5: Gelation time of HA-CHT Hydrogels (mean ± SD, n=3)
S. No. Hydrogel (CHT:SA) ratio Gelling time (sec)
1 1:1 55.1 ± 1.34
2 1:2 48.3 ± 2.48
3 1:3 42.9 ± 2.99
4 1:4 39.5 ± 1.76
5 1:5 36.7 ± 2.56

Rheological Profile
The rheological characteristics of HA-CHT hydrogels were evaluated using Rheometer (Brookfield) at 25oC. The analysis were triplicated and the measurement was repeated thrice with average value along with SD.
Results: The all five ratio of HA-CHT hydrogels were showed increased range of viscosity due to different concentrations of SA. The rheogram of hydrogels described the positive correlations between the SA concentration and viscosity, the viscosity of hydrogels were significantly increased with increasing polymer ratio and increasing in shear rate of hydrogel. The viscosity is demonstrated in below table (table 5).
Table 5: Rheology of HA-CHT Hydrogels (mean ± SD, n=3)
S. No. Hydrogel (CHT:SA) ratio Viscosity (Cps)
1 1:1 2798 ? 2.20
2 1:2 3033 ? 2.93
3 1:3 3275 ± 3.19
4 1:4 3569 ± 1.09
5 1:5 3821 ± 2.54

Swelling Analysis
The degree of swelling was calculated by finding out weight of swollen hydrogels. The swelling behavior of the various batch of HA-CHT hydrogels were studied at different pH conditions. The swelling potential was studied at 3 different pH as acidic, neutral and basic (pH 4, 7 and 9 respectively). The frozen HA-CHT hydrogel were pelletized using a hydraulic pelletiser. The dry weight of the pellet was noted (Wo). The pellet was then placed in corresponding pH solution for 5 minutes at room temperature, then removed from the solution and surface adsorbed solution was removed using filter paper and wet weight was recorded (Ww). The swelling ratio was calculated by the following formula. The swelling at each pH was studied in triplicate
Swelling ratio = -Wo/Wo
Results: Swelling pattern of hydrogels were evaluated to measure the swelling efficiency at pH at 4, 7, and 9. All five batch ratios of HA-CHT hydrogels showed significant swelling at acidic pH 4 compared to neutral and alkaline solutions. The high-water uptake due to presence of CHT and SA molecule, shows fine tendency to absorb water and swelled. The acidic swelling demonstrated the enhanced ionic attraction between ionized hydrogel groups and hydrated counter ions. The swelling ratio or swelling index is demonstrated in following table.
Table 6: Swelling Index of HA-CHT hydrogel (mean ± SD, n=3).
S. No. Hydrogel (CHT:HA) ratio Swelling ratio (%)
pH 4 pH 7 pH 9
1 1:1 75 24 10
2 1:2 81 30 14
3 1:3 87 33 18
4 1:4 90 40 25
5 1:5 96 55 30
Entrapment Efficiency (EE)
To measure the EE of HA in chitosan hydrogel, the hydrogels were centrifuged at 10000 rpm for 5 minutes by using digital centrifuge (REMI, NE Delhi, India). The collected supernatant was carefully diluted with PBS (pH5) and the drug content was resolute spectrophotometrically by using UV spectrophotometer (SCHIMADZU, Japan) at 280 nm, HA is used as blank. The EE of HA can be calculated by employing the following formula:
EE = weight of drug in hydrogel / initial weight of drug taken X 100
The formulation analysis were triplicated and the measurement was repeated thrice with average value along with SD was reported.
Results: The all five ratios of HA-CHT hydrogels showed significant drug loading efficiency (>60 % regard as significant). The drug concentration and the time of incubation plays key role in EE. Optimum polymers ratio leads to the enhanced drug loading efficiency. Moreover the concentration of SA which played important role in EE. Along with the concentration of drug and incubation time the stirring speed and stirring time also plays vital role in the EE. The synthesized hydrogel showed decent EE which also confirms absence of leakage and agglutination of HA and polymer in surface of prepared gel. The EE of different ratios of hydrogel are given in following table.
Table 7: Entrapment Efficiency (EE) of HA-CHT hydrogel (mean ± SD, n=3).
S. No. Hydrogel (CHT:HA) ratio EE (%)
1 1:1 60.91 ? 1.64
2 1:2 66.22 ? 4.25
3 1:3 71.21 ? 3.29
4 1:4 75.98 ? 2.85
5 1:5 79.36 ? 1.24

In-Vitro Drug Release Studies
In vitro drug release studies of HA-CHT Hydrogel were performed by the dialysis bag using a shaking incubator (REMI, New Delhi, India) at 100 rpm. Saline phosphate buffer with pH4, 5, 6 and 7 were employed as dissolution medium so as to mimic the skin environment because the human skin pH value between 5-5.5. Each dialysis bag (pore size: 12kD; Sigma Chemical Co., St Louis, MO) were loaded with about 5 ml of hydrogel previously filtered through the Sephadex Column G-50. The volume and temperature of dissolution medium were 50 ml, and 37+2oC, respectively. At predetermined time interval 5 ml of sample was withdrawn, replaced with same volume of fresh media, filtered and measured for drug (HA) content at UV spectrometric range 280 nm of HA against blank using UV–Visible spectrophotometer. Mean results from triplicate measurements along with standard deviation were reported.
Results: The drug release of all five ratios of hydrogel at different pH displayed the biphasic behaviour with initial burst in early 1-6 hours followed by sustained release. The drug release is mainly depending on the degree of de-acetylation of CHT in which the HA is entrapped. The early bursting of hydrogel is due to the hydrophilic nature of CHT molecules and solubility of SA in water showing tendency to absorb large amount of water leading to the early degradation. Higher the degree of de-acetylation showed reduced degradation or erosion. The drug release results obtained from all five hydrogel ratios at pH 4 shows 65-75% drug release within 24 h. Whereas at pH 5 and 6 it shows 75-90% release in 24 hours due to sustain degradation of chitosan in slightly acidic environment. At pH 7, 25-35% release was noticed in same time interval, low release at neutral pH environment revealing biosafety margin on normal melanin cell. Fig2 shows graphs for In-vitro drug release of all five ratios of HA-CHT hydrogel at different pH range, (mean ± SD, n=3). (Fig2a=1:1 ratio; Fig2b=1:2 ratio; Fig2c=1:3 ratio; Fig2d=1:4 ratio; Fig2e=1:5ratio). Over all the drug release pattern showed significant HA release from HA in slightly acidic environment making synthesized hydrogel a better nanocandidate for transdermal delivery.

Stability Studies
The stability of the HA-CHT hydrogel during storage is asignificant prerequisite for its successful application and is evaluated on the light of structural integrity of hydrogel and percentage residual drug content. All five ratios of hydrogels were stored in amber colored glass bottles with the drug at different temperatures like 4?1°C; 25?2°C and 40?2°Cfor 45 days and analysed the homogeneity, change in surface morphology and drug content as per ICH guidelines.

Results:
The stability studies demonstrated no significant change in the homogeneity and surface morphology of all five ratios of hydrogel, exhibiting stable nature at varied temperature. Slight changes in homogeneity and surface morphology was noticed in the higher concentration of SA ratio in increased temperature, due to the heavy thickness and high temperature, but the deviation was insignificant. As the temperature elevated and time duration of storage increases, swelling of hydrogel was found, but leakage, cracking, dryness or grittiness was absent in all five ratio of hydrogel.
The all five batches of hydrogel were also evaluated for drug content measurement after stored at 4±1°C, 25±2°C and 40±2°C for a period of 45 days. It was observed that the hydrogels were quite stable at 4±1°C and 25±2°C showing absence of leakage of drug (HA). Whereas, the less hydrogels residual drug content was noticed after storage at 40±2°C for 45 days and showed sign of leakage for gel. In the consequences, the direct effect of high temperature on all five ratiosof hydrogelexhibited slight reduction in drug content,andlimited sign of leakage of drugs and greasiness on appearance. Over all the percent drug content of all ratiosof hydrogel stored at varied temperature range over 45 days showed significant drug entrapment was >70%.
Table 8:Stability analysis of CHT-HA Hydrogel at different temperature and time(mean ± SD, n=3).
S. No. Hydrogel (CHT:HA) ratio Studies Initial observation
(0 day) Final Observation
(45 days) on storage at different temperature
Temperature? 4?1°C 25?2°C 40?2°C 4?1°C 25?2°C 40?2°C
1. 1:1 Homogeneity *** *** *** *** *** **
Surface morphology - - - - - +
2. 1:2 Homogeneity *** *** *** *** *** **
Surface morphology - - - - - +
3. 1:3 Homogeneity *** *** *** *** *** **
Surface morphology - - - - - +
4. 1:4 Homogeneity *** *** *** *** ** **
Surface morphology - - - - + +
5. 1:5 Homogeneity *** *** *** *** ** **
Surface morphology - - - - + +
Keys: - = No change, + = Slight change, ++= significant change, *** = High Homogeneity, ** = Moderate Homogeneity, * = Low Homogeneity

Table 9 a-e:Stability analysis by percent residual drug content of all five ratios of HA-CHThydrogelon storage at different temperature, (mean ± SD, n=3).
S. No. Time (days) % Residual drug content on storage at temperature (a) CHTSA ratio in hydrogel (1:1)
4?1°C 25?2°C 40?2°C
1 0 100 100 100
2 15 97.2+ 1.32 %$12120.22th.09 95.7 ± 2.37 90.7 ± 1.37
3 30 93.3+ 1.93 90.3 ± 1.36 82.7 ± 1.56
4 45 90.0 ±2.18 87.2 ± 1.92 75.3 ± 1.90

S. No. Time (days) % Residual drug content on storage at temperature (a) CHT:SA ratio in hydrogel (1:2)
4?1°C 25?2°C 40?2°C
1 0 100 100 100
2 15 96.4 + 2.36 %$12120.22th.09 94.2 ± 3.29 89.3 ± 3.49
3 30 94.1 + 2.36 89.7 ± 3.37 81.4 ± 1.74
4 45 90.1 ±3.27 88.4 ± 2.56 75.1 ± 2.46

S. No. Time (days) % Residual drug content on storage at temperature (a) CHT:SA ratio in hydrogel (1:3)
4?1°C 25?2°C 40?2°C
1 0 100 100 100
2 15 95.1+ 4.24 %$12120.22th.09 94.8 ± 1.98 89.5 ± 2.38
3 30 92.7+ 2.36 90.3 ± 3.29 81.0 ± 1.84
4 45 89.7 ±1.89 88.5 ± 2.37 74.2 ± 3.62

S. No. Time (days) % Residual drug content on storage at temperature (a) CHT:SA ratio in hydrogel (1:4)
4?1°C 25?2°C 40?2°C
1 0 100 100 100
2 15 91.2 + 1.02 %$12120.22th.09 93.0 ± 2.37 90.9 ± 4.29
3 30 90.1 + 1.45 86.4 ± 1.36 80.3 ± 2.46
4 45 88.0 ±3.29 81.2 ± 1.92 73.9 ± 2.19

S. No. Time (days) % Residual drug content on storage at temperature (a) CHT: SA ratio in hydrogel (1:5)
4?1°C 25?2°C 40?2°C
1 0 100 100 100
2 15 90.2+ 1.89 %$12120.22th.09 90.4 ± 1.98 88.5 ± 3.27
3 30 88.4+ 3.62 86.0 ± 3.89 79.1 ± 2.30
4 45 85.7 ± 1.74 83.4± 2.22 72.5 ± 3.27

Turbidity Analysis
The turbidity of the hydrogel played vital role in estimation of dimension, morphology, and drug release rate. For the turbidity analysis, one ml of the HA loaded hydrogel samples was diluted with 2 ml of distilled water and stored at room temperature for varied time of storage. Then, the turbidity of the sample was measured in a UV spectrophotometer of the Ultrospec 2000 produced by the UK in quartz tubes at a wavelength of UV range of HA.
Results: The turbidity of all five ratios of hydrogel sample was inacceptable limit of stability. The hydrogel in initial storage (1-10 days) showed less turbidity compared to extended storage duration. As the storage duration increases the turbidity of hydrogel was also increased due to elevated swelling and aggregation of CHT and SA polymer which shows tendency to absorb large amount of water and get swelled. The turbidity increases with increasing swelling phenomenon. Therefore, the turbidity of hydrogel was increased over a month, due to the high absorption of water in gel and colloids formation. The increasing turbidity does not affect the morphology and stability of hydrogel, lacking of drug leakage, dryness and caking of gel does not create over a period of storage.

Table 10: Turbidity analysis of HA-CHT hydrogel (mean ± SD, n=3).
S. No. Hydrogel (CHT:HA) ratio Turbidity (%) Days
1 5 10 15 20 25 30
1. 1:1 Turbidity (%) 1.0 10.1 30.9 45.1 65.4 79.6 88.1
2 1:2 Turbidity (%) 2.3 11.3 33.1 47.9 66.7 80.1 89.5
3 1:3 Turbidity (%) 3.6 14.5 35.3 45.1 67.8 81.7 90.4
4 1:4 Turbidity (%) 4.0 15.3 36.0 45.1 69.3 83.5 91.7
5 1:5 Turbidity (%) 5.5 17.6 38.7 45.1 71.2 85.1 92.0

13. Biocompatibility Analysis
(a) Hemolysis Assay
The hemolysis study was carried out for HA-CHT Hydrogel employing fresh human blood. 1.5mL acid citrate dextrose (ACD) was added to 10 mL fresh blood. 100µL of HA-CHT hydrogel sample of concentration ranging from 0.1 to 1 mg/mL were added to 1 mL of blood sample and incubated for 2h with shaking in an incubator chamber at 37°C. The incubated sample was centrifuged at 5000 rpm for 5 min to obtain plasma. The plasma was then mixed with 1 µl of 0.01% NaHCO3 solution. The sample was then scanned at 264 nm. The plasma hemoglobin was assessed by employing the equation:
Plasma Hb = (2A 264 x 76.25).
(b) Coagulation Assay by Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) analysis
The plasma coagulating effect of HA-CHT hydrogels was determined by plasma coagulation assay. The plasma coagulation was assayed via two tests, i.e., prothrombin time (PT) and activated partial thromboplastin time (APTT). Fresh blood was collected in to ACD containing tubes. It was centrifuged at 4000 rpm at 25° C for 15 minutes to obtain platelet-poor plasma (PPP). The 1mg/mL concentration of HA-CHT hydrogels ratio was used for other tests. About 900µL of PPP was treated with 0.09 µL of HA-CHT sample and kept at 37 °C for 20 minutes. After the incubation period, the prothrombin time (PT) and activated partial thromboplastin time (APTT) were determined using a coagulation analyzer and reagent kits (CK Prest and Fibriprest from DiagnosticaStago, France). The experiment was done in triplicate using saline treated PPP as negative control.
Results: The hemolysis effect of all five HA-CHT hydrogel ratios displayed a negligible hemolysis effects, it showed hemolysis below 5%. It is considered as safe for clinical purpose as per ISO/TR 7406 ethical course of action (ref). As the ratio of SA increases slight increment in hemolysis is also observed due to the thinking effect of gel. Overall the effect of hemolysis is way under the limit if biologically safe margin for any transdermal preparation.
The PT and APTT analysis demonstrated the biocompatibility of transdermal gel by mean of coagulation evaluation. The coagulation time displayed by the assessment of PT and APTT by all five hydrogel samples showed normal coagulation interval on treatment with blood. The time limit for coagulation is under 45 sec in all cases which shows normal time for biological coagulation phenomenon. The PT and APTT assessment of all batches of Hydrogel demonstrates the biosafety of the hydrogel in clinical use. The hemolysis and coagulation assessment is demonstrated in below table.
Table 11: Haemolysis and Coagulation analysis of HA-CHT hydrogel (mean ± SD, n=3).
S. No. Hydrogel CHT-HA ratio Hemolysis (%) Prothrombin Time (PT) (sec) Activated Partial Thromboplastin Time (APTT) (sec)
1 1:1 1.61 ± 0.85 20.3± 1.35 44.9± 2.13
2 1:2 2.36 ± 0.36 18.1± 2.55 41.9± 1.38
3 1:3 3.55 ± 0.59 16.8± 3.29 37.2± 3.11
4 1:4 4.01 ± 1.34 14.2± 1.38 32.1± 1.09
5 1:5 4.89 ± 0.17 12.5± 2.18 27.9± 1.35

HA from Streptococcus equi subsp.equi (MK156140) isolated from horse nasal sample showed a promising result in producing hyaluronic acid under laboratory conditions and therefore taken for upscaling manufacturing processes, considering the economic ways for synthesis of HA for preparation.

Pilot Scale production of HA:
The selected isolate Streptococcus equi subsp.equi (MK156140) was inoculated into 500 ml modified LB broth and incubated under shaken condition at 350C until the cell count reached to 108 CFU. The actively growing culture was transferred into 5 L modified LB broth in 10 L fermentor () and incubated for 24 – 26 hours at 350C and pH of about 7.0. And various other parameters such as agitation speed 180 rpm, pressure 0.5 kg/cm2 and aeration 2.0 vvm was maintained uniform throughout the fermentation process. The hyaluronic acid produced by the isolate estimated at regular interval of time (2 hours) by CTAB method using UV- vis spectrophotometer (Labman, LMSP- UV1000B).
Extraction and purification of HA:
Hyaluronic acid present in the capsule of S. equi subsp.equi was extracted and precipitated with alcohol. The hyaluronic acid was extracted by blending the culture broth with 10% volume of 5% SDS for 10 min and centrifuged. The hyaluronic acid was precipitated by adding 3 volume of ethanol to one volume of supernatant and further centrifuged. The viscosity of the hyaluronic acid were reduced by adding one volume of NaCl and two volume of ethanol and centrifuged.
Purification:
The crude hyaluronic acid was treated with isopropyl alcohol (IPA) (1:3v/v) to reduce the viscosity. The protein present in the crude extract was removed by adding 0.1% trichloroacetic acid (TCA) until the pH of the solution reaches 2.0. The extract was further purified by passing through the activated charcoal bed (2cm thickness). The collected filtrate was further centrifuged at 7000 rpm for 30 min at 4ºC and the supernatant was filtered through 0.45µm filters. The hyaluronic acid present in the filtrate was precipitated with the addition of IPA and dried under vacuum.
Results:
Media supplemented with sucrose as a carbon source and beef and yeast extract as nitrogen source, along with various physical parameters such as agitation speed 180 rpm, aeration rate 2.0 vvm, temperature 35ºC, pH 7.0 and pressure 0.5 kg/cm2 were found to enhance the yield of hyaluronic acid 0.9 g/L to 7.8 g/L within 24 h of incubation period under pilot scale.
Similar such study was carried out by kim et al.,(2006) with S. zooepidemicus. Who reported the production of HA under optimized and large scale production as 5.4 g/L which is comparatively lesser than the production level (7.8 g/L) of S. equi subsp.equi.
The quality of the product is one of the important criteria for any product to receive its commercial importance. The quality of the hyaluronic acid obtained after the microbial fermentation was enhanced with the help of various techniques, one such among them is by treating with activated charcoal which is found to be economical and effective method of purifying bacterial hyaluronic acid.
Many techniques in lab scale have been employed in purifying or enhancing the quality of the hyaluronic acid. Among them, activated charcoal was found to be economical and an alternative method for the purification of bacterial hyaluronic acid and it is been employed at pilot scale. In the present study 4.18 g/L, with (52.30%) of recovery rate of HA was observed at pilot scale (5L).
There are very few reports available with respect to pilot scale fermentation for hyaluronic acid production from Streptococcus species. Among which Im et al., (2009), reported 6.94g/L crude hyaluronic acid from Streptococcus sp. ID9102 and kim et al., (1996) reported 6-7g/L hyaluronic acid from S.equi subsp.equi KFCC 10830 which is lesser findings than the case in the present invention.
It is been stated that the production rate is closely associated to biomass of the organism. Recovery of the product i.e., hyaluronic acid is been a demanding process in industrial scale much work yet to be carried out to increase the same. In our study we were able to get 4 fold increase in the yield and 52.30% recovery of the hyaluronic acid from Streptococcus equi subsp.equi (MK156140).
Table 1: Comparison of hyaluronic yield between large scale and pilot scale for 2 consecutive generations of S.equi subsp.equi
Generation I yield g/L Generation II yield g/L
Lab scale 7.16 g/L 7.18 g/L
Pilot scale 7.8 g/L 7.7 g/L
In the view of industrial production of any product it is necessary to know the viability of the organism in terms of production rate of the desired product, hence we performed the experiment for the production of hyaluronic acid in lab scale and pilot scale as well and did not find any major difference in the production rate. This may be the promising result to prove that the organism isolated by us is able to produce hyaluronic acid in industrial scale without any much decrease in the yield rate.
Conclusion: The present study focused on the production of hyaluronic acid in pilot scale fermentor (5L) from Streptococcus equi subsp.equi MK156140. The parameters which have been studied under lab scale have been employed in pilot scale and obtained the crude hyaluronic acid extract 7.8 g/L. The resultant HA was purified by passing through the bed of activated charcoal in order to remove the impurities resulting in enhancing the hyaluronic acid content by 4.18 g/L (52.30% recovery). Based on the production rate of HA from S.equi subsp.equi both in lab scale and pilot scale we conclude that this isolate can be used in industrial production as it has promising functions in both therapeutics and cosmetics field.
The synthesized CHT-HA hydrogel very decent homogeneity with whitish pale buffy appearance devoid of any grittiness, tackiness and irritation on touch. The SEM analysis showed balance surface morphology and texture with negligible sign of clumps formation and agglutination at different measurement scale bar. The spreadability analysis showed significant spreading potential when evaluated with different optimized batches of hydrogel resulting in decent gel delivery. The rheology evaluation possess decent viscosity limit satisfying the clinical delivery of gel formulation. The gelling time and temperature profile showed that the hydrogel is stable and exhibits significant gelling time in varied temperature range. The swelling index showed that the developed hydrogel possess slightly acidic characteristics as the swelling is profound slightly acidic pH microenvironment. The turbidity analysis showed that the hydrogel is physiochemical stable in longer storage duration as the turbidity is decent and low during longer storage time. This showed the decent stability of hydrogel and make a significant transdermal gel for delivery of HA.
The in-vitro drug release pattern of hydrogel showed the lightly acidic characteristics of as the maximum HA was released at pH between 5-6 showing skin surface dependent drug release potential and comply with the skin surface pH. This pH responsive drug release pattern also signifies that the developed hydrogel possess targeted drug delivery system which does not bother the normal skin cell as the normal skin pH is 7.4.
The stability studies again signifies and validates the preliminary in vitro results of hydrogel The stability studies showed that the hydrogel is physically and thermally stable at varied temperature range on longer storage of conditions without deviating the homogeneity, morphology and texture of hydrogel. The surface morphology, spreading potential of hydrogel does not deviate on longer storage along with slight decline in drug content was found which regard as insignificant loss as the limit does not exceeds below 70%. The stability analysis showed and confirmed the physiochemical and thermal stability of developed hydrogel.
The blood compatibility parameters including hemolysis and coagulation assays showed that the developed hydrogel is biocompatible and biologically safe on preclinical application. The hemolysis if hydrogel was way under the limit for the operative biological safe margin as per the guidelines ISO/TR 7406 ethical course of action, which allows maximum 5 % hemolysis. The coagulation profile suggested that the hydrogel does not varies the normal coagulation time delivery which again confirmed the biological safe delivery of developed hydrogel.
Overall the preliminary characterization parameters of synthesized organic hydrogel showed the effective synthesis of organic gel formulation in preclinical and clinical platform which shows enhance stability and biological safe formulation. This also elaborate that the hydrogel is eligible for the furtherer advanced clinical and preclinical evaluation for the better production of HA loaded chitosan SA biodegradable gel for future biomedical applications which is not only highly effective but biofriendly and ecofriendly, pH and thermosensitive, nontoxic, non-tedious drug therapy, highly economic, having negligence adverse effects, easily available and producible at remote area and above all having patient compliance advantage.

Various modifications to these embodiments are apparent to those skilled in the art from the description and the accompanying drawings. The principles associated with the various embodiments described herein may be applied to other embodiments. Therefore, the description is not intended to be limited to the embodiments shown along with the accompanying drawings but is to be provided broadest scope of consistent with the principles and the novel and inventive features disclosed or suggested herein. Accordingly, the invention is anticipated to hold on to all other such alternatives, modifications, and variations that fall within the spirit and scope of the present invention and appended claims.