|Title of Invention||
A PROCESS FOR THE PREPARATION OF A BIOPOLYMER MATRIX
|Abstract||A process for the preparation of a biopolymer matrix comprl9tng subjecttng an aqueous solution of 5 to 20 g of alginate having two hydroxyl groups or a hydroxyl and an amino group In adjacent positions, to oxidation using 4.2 to 16.8 gms of oxidising agent, to obtain the algInate dlaldehyde (ADA), purifyIng the same followed by reaction of a 10 to 20% solution of ADA with an equal volume of a 20% solution of gelatin in the presence of a reagent at a pH of 7.4 to 9.4 to form the matrix of alginate dlaldehyde crosslinked gelatine.|
|Full Text||This invention relates to alginate dialdehyde (ADA) crosslinked gelatin and a process for the preparation thereof.
This invention further relates to alginate dialdehyde crosslinked gelatin as a wound dressing material.
Large scale bum wounds and other open wounds with skin loss often require treatment within temporary dressings. Their main functions are :
1. Reduce the evaporation of water from the wound bed and associated energy loss;
2. Prevention or minimisation of microbial invasion at the wound site;
3. Encouragement of vascularisation and tissue regeneration at the interface between the dressing and the wound surface.
Numerous wound dressing materials are available and are also being inves¬tigated. Dressings help to achieve haemostatic and thus control blood loss and protects the wound from microbial contamination by shielding the wound from the environment. Some dressings are designed to prevent evaporative fluid loss and thereby keep the wound bed in a moist condition which is beneficial for healing. Some wound dressings contain components which directly promote cell growth or migration or which attract or activate cells from the immune system which secrete growth-promoting substances. Dressings may also contain antimicrobial drugs, which help control infection of the wound.
Paraffm-embedded cotton gauze is widely used as wound dressing but has many limitations. "Hydrogel" dressings which are non-concessive dressings
made of hydrophilic polymers such as gelatin, polysaccharides, polydactyl-amide, polyhydroxyethyl m eth aery late, polyvinyl pyrrolidone, etc. are another type of dressing with a high capacity for the absorption of exudates. These dressings swell upon contact with wound fluid and can absorb several tines their own weight of exudates. Commercially available hydrogel dressings inchide intestine get (Smith and Nephew, UK) and Vision (CR Bard USA). A special type of hydrogels are the alginates, which are hydrophilic pofysaccharides extracted from seaweed. They are produced as thin non-woven tissues or as fibres. Upon contact the wound fluid, fluey turn into a gel which has a high absorptive capacity for wound fluid. Examples include Kaltostat (Brit-Cairn, UK) and Sorbsan (Steriseal, UK). Occlusive or semi-occhisive dressings prevent or retard the evaporation of water from the wound bed thereby keeping it moist. They are usually polymer Alms such as that fabricated from flexible potyurethane and contain a self-adhesive coating. Examples of such dressings are Opsite (Smith and Nephew, UK) and Tegaderm (3M, USA). Examples of semi-occhisive dressings are Omiderm (Latro Medical Systems, UK) and Elkin (Konin-klijke Utermohten, The Netherlands).
A more complex type of occhisive dressings is the hydrocoUoid (HCD) dressings consisting of hydrocoUoid particles (eg. gelatin, pectin, etc.) embedded in a hydrophobic matrix (eg. polyisobtttylene).HCD dressings have a h^ absorptive capacity, and are very useful for the treatment of wounds producing high amounts of exudates. Commercially available examples of these dressings include Dodder degree. (Connate, UK) and Tegasorb TM. (3M, USA).
Wound dressings is an area which is undergoing sweeping changes in its technology with the advent of genetic engineering and the emergence of many protein- and peptide-based therapeutic substances and cell culture technologies.
The major technological developments in the area of wound dressings have prmcqiaUy sought to obtain simple or composite ratings able to match most of the characteristics of an ideal skin replacement. Among dressings composed of natural polymers, alginates have found wide acceptability (vide infra). Non-woven calcium alginate dressings have been increasingly used in die management of partial and full diickness wounds. Calcium alginate is converted into a hydrophilic gel at the wound surface by an ion exchange reaction between the calcium in the alginate and sodium in the blood and wound exudates. The hydrophilic gel provides a moist environment which promotes healing and epidermal regeneration. Calchim alginate has also a role in blood coagulation. The calcium ions released in exchange for sodium ions promote hemostasia while the sodnim alginate serves as a matrix for the aggregation of platelets and erydirocytes. Gelatin is obtained by the controlled hydrolysis of the fibrous insohible protein collagen which is the major constituent of skm^ bones and connective tissue. Gelatin contains a high concentration of amino acids such as glycine, proline and hydroxyproline. Gelatin possess a high hemostatic effect. Gelatin-based wound dressings have been conventionally prepared by crosslinking gelatin using formaldehyde glutaraldehyde, carbodiimide, diisocyanate, etc. and recently a wound dressing based on gelatin crosslinked with dextran dialdehyde has been reported. (US Pat. No.6132759).
Although both alginate and gelatin have been investigated individually for many biomedical applications including wound dressings, published reports or patents on combining the two and thus achieving synergic beneficial effects of both for medical use has been rare. In the smgle known instance of gelatin-alginate composite aa a possible wound dressing material. A mixture of gelatin and sodium alginate in water is cast as a film and crosslinked using carbodiimide in 90% acetone-water mixture. However^ the method involves crosslinking both gelatin and alginate using a toxic compound such as carbodiimide.
Reports on the toxicity of alginate dressings prepared using calcium cross-linking method have caused concern on the use of these dressings. Calchim sodium alginate dressing (Kaltostat) is found to be cytotoxic to fibroblasts and keratinocytes in culture and high calcium concentration is known to inhibit the growth of cells in culture.
QMBCT^ Qf THE IWVfiWTIQW
It is therefore an object of this invention to propose an algmate dialdehyde crosslinked gelatin whose preparation does not involve any toxic compounds.
■S " I It is a further object of this invention to propose an alginate dialdehyde
V "V^ 1 ^ f V \ |crosslinked gelatin which is prepared by a rapid process.
Another object of this invention is to propose an ADA crosslinked gelatin
which he^s to prevent or check wound^infectiott, and helps in healing and
epithelializatlbn of the wound.
These and other objects of the invention will be apparent from the ensuing
BRIEF DESCRIPTION OF THE INVENTION
Thus, according to this invention is provided an alginate dialdehyde cross-linked gelatin as a wound dressing material.
According to the invention is further provided a porous for the preparatton of an alginate dialdehyde crosslinked gelatin.
In accordance with this invention, a polysaccharide is subjected to periodate oxidation to produce the dialdehyde. Periodate oxidation is a well-known reaction. Oxidation can be accomplished on any polysaccharide having geminal hydroxyl or one hydroxyl and one ammo group in adjacent positions using reagents such as sodinm or potassium periodate in aqueous mediam. The reaction produces dialdehyde residues in the polysaccharide. The extent of oxidation depends on the concentrirtion of the re^gents^substrate (alginate), time and temperature of the reaction and the molecular weight of the substrate. Ahhongh sodinm or potassnim periodate is the preferred reagent for such reactions, those skilled in the art would know that reagents such as periodic acid or lead tetra acetate in an organic solvent such as dimethyl sulphoxide may also be used. After oxidation, the dialdehyde derivative of alginate can be conveniently purified and separated from low molecular weight reaction components by using dialysis membranes, precipitation, ohrafiltration or gel permeation chromatography, followed by lyophilization.
The oxidation reaction also can be conducted in ethanol/water mixture, with the advantage that more amount of alginate can be used for the oxidation in comparison with the aqueous sohition thereby yielding siginificantly larger quantity of ADA in a single reaction. Alginates cannot be dissolved in water to prepare sohitions of high concentration since their viscosity is very high
which result in gels rather than solutions making the handling of the same difficult In ethanol/water mixture, it is demonstrated that a dispersion of alginate can undergo oxidation by periodate. Periodates are known not to oxidise ethanol, making the process simple and advantageous for the preparation of lan^e quantities of ADA.
Cross-linking between gelatin and ADA is achieved by the formation of Schiff base Imk^ges between free amino groups present on die gelatin and the dialdehyde residues on ADA. This reaction is performed in aqueous medium and the speed and degree of cross-linking depend on a variety of parameters, such as the type of gehitin and its concentration, degree of dial¬dehyde substitution and molecular weight of the ADA, pH»buffer type and the presence of electrolytes in the reaction medium, etc.. The ADA k crosslinked with gelatin in the presence of a reagent selected from borax sohition, phosphate buffer and phosphate buffered saline. Two different grades of sodium alginate are employed in the embodiment of the invention. High (viscosity of 2% solution 14,000 cps at 25°C) and medhim viscosity (viscosity of 2% sohitton 3,500 cps at ZS^"C) sodium alginate from the brown algae Macrocystis pyrifera, are obtained from Sigma Chemical Co., St. Louis, MO, USA. However, those skilled m the art would understand that such viscosities are not a limiting factor for the invention, other viscosities could also be employed. This is particularly the case with biopolymers which are natural products and the viscosities of the material often depend on the biological source from which it is derived and can never be the same or identical. Gelatin employed m the invention is type A (Bloom 300) also obtained from Sigma, USA. Several types of gelatin exist, depending on the source of collagen used, and on the extraction and
production process employed. Type A is prepared by acid hydrolysis and type B is prepared by basic hydrolysis of the collagen. Both types of gelatin can be used for the preparation of the composite matrix, but_typ^e A is prderred since it is known to contain more amino groups and therefore is more efficient in the crosslinking reaction. Gelatins with a variety of Bloom numbers exists the Bloom number being a measure of the rigidity of the gel formed from the material. Although Ae present embodiment is usmg Bloom 300, other Bloom numbers are also suitable, but those skilled in the art would know that the lower the Bloom number, the weaker will be the mechanical strength of the fthns formed. Again, type A gelatins have higher Bloom numbers and therefore, this type is preferred in the invention. The composite matrix according to the invention may be prepared as membranes, sheets or foamr. Foams have the advantage that they will be able to absorb large amount of the wound exudates because of the macro-porous nature. The fabrication of such foams is simple and usually involves passing air, nitrogen or an inert gas such as helmm through the gelatin solution under agitation and then adding the ADA to crosslmk the same. Foams thus produced can be shaped as sheets, rods, plugs, pads, etc. and can be used in the hydrated, semi-hydrated or dry form suitable for application in the wound site. In accordance wiHk an efflbodiment, the composite matrix mi^ be prepared in situ in the would bed itself The in situ application will have several conform ability of the dressing on wounds without the dressing wrinkling or fluting in the area covering the wound. Most commercially available dressings in the form of membranes and sheets are problematic as far as the conformability is concerned and the in situ formation of the dressings will therefore be superior to such dressings. The composite matrix
may also be fabricated into micropartides or microspheres which could fmd applications as a controlled drug delivery system. The fabrication of such geometries of the composite matrix involves dispersing the gelatin sohition as fine droplets in a suitable dispersion medium such as vegetable oil or other hydroxycarbon oils such as paraffin stabilised by a suitable surfactant and crosslinking the particles by the introduction of ADA. Various techniques could be employed for generating the dispersion such as mechanical agitation, uttrasonicatton, vortex mixing, etc. depending on the size of the particles desired. Many drugs could be incorporated into the matrix during the fabrication of such particles. Such drug-loaded particles could be used for injection intramuscularly or subcutaneously for their slow release in the human body. They could also be used for coverng the wound bed since the large surface area of the particles would be helpful in absorbing wound exudates especially in chronic ulcers. Protems and poly¬peptides also could be incorporated into the matrix for their slow release up on implantation.
The alginate dialdehyde crosslinked gelatin m^ be provided with anti¬bacterial agents such as silver su^hadiazine or gen tarn ycin or other antibacterials which are beneficial in preventmg or checking wound mfection. Such antibacterials could be incorporated into the composite matrix either during the preparation of the matrix itself or could be mcorporated later after the formation of the matrix by sorption methods which usually involves socking the matrix in a sohition of the antibiotic (antibacterial) compound and allowing its sorption into the matrix. Such antibiotics would be released slowly from the matrix into the wound bed thereby help checking the bacterial colonization of the wound.
The compounds are not limited to antibiotics or antibacterials and thoie
skilled in the art would know that there are many other compounds that
helps wound healing and epithelialization such as many Growth Factors
currently available. Growth factors such as those belonging to the class of
the EGF» FGF, PDGF, TGF, VEGF» PD-ECGF or IGF families are suitable
for die said purpose.
The invention will now be explained in greater det«l widi the help of tfie
following non-limiting examples:
Preparatioii of Alginate Dialddiyde (ADA):
Medium viscosity sodium alginate^ 20 gm or high viscosity sodium
alginate S g was dissolved in 500 mL and 450 mL distilled water
respectively by magnetic stirring in a beaker at
room tonpenture. Diffatm quantities of sodium n^^
sohition and.tbe contents were stined magnetically in the dark at 2S*C for 6 h. The sohitionwas then dialyzed using the 3500 M.W cut off dialysis tubing (Spectra/Por* M.Wcut off 3S00, Spectrum Laboratories, CA, USA) against distilled water (2.5 lit) with several changes of water until it was free from periodate (48 hours). Con^lete removal of periodate was ensured by testing the dialyzate for the absence of turbidity or precqiitate with an aqueous sohitbn of siNer nitrate. The solution was then frozen -TS^C, lyophilized and stored in a desiccator in the refrigerator at 4*"C. The extent of oxidation with respect to time was foUowed by iodometric titration of the residual periodate present in the reaction mucture at regular time intervals. A 5 mL aliquot of the reaction mixture was neutralized with an of 10 mL sodium bicarbonate sohition and iodine was Uberated by the addition of 20% potassium iodide sohition (2 mL). Liberated iodine was then titrated with standardized sodium thiosulphates sohition using starch as the indicator. Representative data are shown in Table 1.
Table 1. Oxidation of medium viscosity (mv) and high viscosity (hv) sodium alginate (6 h reaction)
Sodium Alginate (gram)
Sodium m-periodate (gram)
483 ± 1.6
Sodium alginate, medium viscosity 20 gm was dispersed in 100 mL distilled ethanoL Different molar concentrations of sodium m- periodate in distilled water 000 mL) was then introduced into the solution and was kept stirred in the dark at 25°C for 6 h. It was then dialyzed. lyophilized and stored as described in method A. Degree of oxidation was determined according to Zhao and Heindel (Zhao H . Heindel ND, Determination ofdegree of substitution of formyl
polyaldd^de dadran by the IqfdioxyliiiaDe liydroditoi^ 8:^400-402) in the following mannen
Approximate^ SO mg of lyophilized ADA was dissolved in 10 mL of 0.25 N faydroxylamine hydrochloride-methyl orange solution. The solution was allowed to stand for 2 hr and was then titrated against standard sodium l^dioxide solution until the red-to-yellow end point was achieved by matching the cotor of the san^ile sohition with that of a Uaidc one. Table 2 gives the representathw data:
Table 2. Oxidation of sodium alginate in ethanol/water mixture (6 h reaction)
Sodium alginate (gram) Sodium m-periodate (gram) Oxidation (%)
20 2 8.4211.4
20 5.86 20.4 ± 2
20 13 55 ±1
20 18.9 83.75 ± 0.2
Preparation of gcbtin-AOA geL
ADA of different percent oxidations was made to react with gelatin to form the crosslinked gel Gelation reactitm was carried out in the presence of 0.1M Borax (Sigma, USA) solution. One mL of ADA (10 and 20% sohition-obtained by the oxidation in ethanol^vater mixture- in O.IM Borax) was taken in a vial of 15 mL capacity (diameter 20 mm), to which ImL of gelatin (20% solution. Type A, Bloom 300) added, and stirred using a Teflon magnetic stir bar (dia 5 mm. length 10 mm). Gelling time was noted as the time required for the stir bar to stop. Concentration of alginate diakldiyde. Borax and gelatin was varied in order to study the variation in gelling time. All this gelling experiments were carried out at 3T*C. Representative data are shown in Table 3.
Tabic 3. GelUng time of gelatin using ADA of dififerent percentage oxidation
Oxidation (%) Gelling time (seconds)
20 (10% solution) 25±2
55 (20% solution) 21 ±1
84 (20% solution) 13 ±1
The gelling time was also determined by varying the concentration of gelatin, ADA and Borax for the 84% oxidized ADA and the representative data are shown in the foUowing tables.
Table 4. Variation of the concentration of ADA (84% oxidized-Method B) in 0.1 M Borax on the gelling time of 20% solution of gelatin (Bloom 300, type A).
Concentration of ADA (wt%)in0.1 M Borax Gelling time (seconds)
5 23.5 ±0.5
15 14.5 ±2
20 13 ±1
25 15 ±1
Table 5. Variation of the concentration of giejatin (Bloom 300, type A) on the gelUng time (ADA 84% oxidized-Method B, 20% sohition in 0.1 M Borax):
Concentration of gelatin (wt%) (jelling time (seconds)
5 59 ±1
10 37 ±3
20 13 ±1
Tabk 6. Variation of the concentration of Borax on gelling time of 20% aohition of gelatin (Bloom 300. type A) with 20% sohition of ADA (84% oxidized-Method B):
Concentration of Borax
Gelling time (seconds)
46.5 ± 1.5
Rate of Swelling of the Geb in Phosphate Buffered Saline
One-half mL 10% and 20% ADA from medium viscosity sodium alginate O.IM Borax (percent oxidation 84.55 and 20-Method B) and one half mL gelatin solution (15% solution of Bloom 300. Type A in water) were mixed using a vortex mixer in a glass vial of 15 mL capacity
and allov^ to form gel of approximately 26 mm diameter and 20 mm thickness. It was then kept for 10 min at 37^: and 5mL of phosphate buffered saline (0.1 M. pH 7.4) was added to the gel and incubated the same at 37-C. At regular intervab of time, the weight of the gel was noted after removing PBS using Pasteur pipette. The percentage swelling was caknilated based on
initial weight of the gel and its swollen weight as:
Swelling (%) = Weight ofswoUen gel-Initial weight of gel x 100
Initial weight of gel
Representative data are shown in Table 7:
Table 7. Rate of swelling of gel in phosphate buffered saline with respect to time:
84 % oxidized 55% oxidized 20% oxidized
Time (Hours) ADA(20%) ADA(10%) ADA(20%) ADA(10%) ADA(10%)
0.5 28. 78 ± 2 16.01 ±2 15.62 ±1 14.80 ±1.2 30.28 ±0.25 1
1.5 46.90 ± 3 26.40 ± 4 26.50 ±2 25.05 ± 0.4 42.33 ±4 j
2.5 56.63 ± 4 31.80 ±4 33.14 ±2 34.40 ±2 47.60 ± 4
3.5 62.25 ± 2 37.08 ± 0.3 40.28 ± 4 39.27 ± 2 42.72 ± 3 j
20 82.05 ± 4 46.34 ± 3 64.1912 68.06 tl 159.80 ±0 5
22 91.90 ±1 50.71 ± 2 74.43 ±3 73.54 ± 4 71.30 rl
24 88.20 ± 3 47.8 0± 2 74.44 ± 3 72.56 ± 2.5 71.12±06
The gels thos prepared were then subjected to varions experiments to itndy the rate of evaporatioa of water from the gels aad the release patten of proteins from the geb. af i highlighted betow:
Rate of Evnponifion of Water from the Gcb:
Gels for the experiment were prepared as before by combining O.S mL 10% and 20% ADA from medium viscosity sodium alginate in O.IM Borax (percent oxidation 84, 55 and 20) and 0.5 mL gelatin solution (15% solution of Bloom 300, Type A in water) using a vortex mixer in a glass vial It was then kept open at 37"C in an mcuhator at 35 ± 2% relative humidity and periodically weighed in an analytical balance The percentage weight loss was calculated based on the initial weight of the gel. This is illustrated in the example shown in Table 8.
Tabk 8. Rate of eviqwiatiim of water fom the gds at 37*C and at 35 ± 2 % rekthv hmn^
Time (Hours) Weight Loss (%)
84% oxidized 55% oxidized 20% oxidized
ADA(20%) ADA(10%) ADA(20%) ADA(10%) ADA(10%)
0.5 1.14 ±0.08 2.08 ±0.4 1.21 ±0.1 1.56 ±0.3 1.83±0.2
1 2J6±0.2 3.67 ±0.5 2.35 ±0.2 2.70 ±0.4 3.55±0.4
2 3.81 ±0.4 5.40 ±0.6 3.67 ±0.3 3.90 ±0.5 7.51±0.7
3 5.34 ±0.6 7.10 ±0.7 5.05 ±0.5 5.45 ± 0.4 7.51 ± 1
5 8.57 ±0.8 11.26 ±0.7 8.00 ±0.7 8.50 ± 0.6 12.07 ±0.9
19.5 26.84 ±1.6 34.24 ±2.3 25.62 ±1.7 23.60 ±2 34.87 ±1.8
22.5 29.63 ± 1.9 37.84 ±2.5 28.23 ±1.8 28.65 ± 2 42.12 ±2.0
23.5 31.33 ±2 41.0511.9 29.86 ±2.1 30.12 ±2.3 44.55 ± 3 1
27.5 - 38.57 ±2.5 48.27± 2.8 36.81 ±2.3 36.75 ±2.5 55.0112.5 1
43.75 57.46 ±3 73.97 ±3.5 55.48 ±3.1 55.78 ±3.2 79.8113
46.75 60.20 ±3 75.45 ±5.8 58.16 ±3.3 58.31 ±3 82.2613
64.75 64.08 ±3.3 81.32 ±6.4 61.97 ±3.8 63.16 ±4 85.4915.5
87 82.74 ±1 87.02 ±0.4 81.86 ±2.2 84.30 ±3 88.05 ±3J
102 • 83.64 ±0.3 87.20 ±0.3 84.7210.03 89.17 ±0.6 87.77 ±0.1
132 83.72 ±0.3 87.30 ±0.3 84.83 ±0.06 89.20 ±0.7 87.8 ±0.5
162 83.87 ±0.4 87.29 ±0.3 84.97 ±0.02 89.34 ±0.6 87.96 ±0.5
196 83.96 ±0.3 87.40 ±0.3 85.08 ±0.04 89.42 ±0.7 88.09 ±0.6
Gelling experiments in the absence of borax.
ADA (55% oxidized - Medium viscosir.) prepared by method B was tried to crosslink with gelatin (Type A, Bloom 300) in the absence of Borax. Gelling lime was noted in the
presence of buffers fike phosphate buflered saline (0. IM, pH7.4) and phosphate buffer (O.IM. pH 7.4). Representative data for the gellii^ time is illustrated in the example shown in Table 9.
Table 9. Variation of type of buffer on gelling time of 20% solution of gelatin (Bloom 300, type A) with 20% solution of ADA (55% oxidized-method B):
Buffers Gelling time (seconds)
Phosphate buffered saline 153 ±10
Phosphate buffer 121115
In all other buffers, gelling time was prolonged (more than 2 hours). ADA (60% oxidized) prepared by method A was also used to crosslink with gelatin. Gelling time obtained was more than 15 minutes. The reason for this high gelling time was examined and it was found that there is decrease in molecular weight for ADA prepared by method A. Molecular weights of ADA determined at different degree of oxidation by the two methods are shown in Table 10.
Table 10. Molecular weight of ADA having different percentage oxidation (Medium viscosity)
Compound Percentage oxidation Molecular weight (MO (Dakons)
Sodium Alginate 0 2.31,400
ADA (Method B) 8.42 ±1.4 1,37,943
ADA (Method B) 20.4 ±2 74,896
ADA (Method B) 55.0 ± 1 60,002
ADA (Method B) 83.7 ±0.2 30,800
ADA (Method A) 60±6 20,048
Controlled Release of FTTC Albumm from the GcL
To study the release profile of high molecular weight proteins and how the method of preparation, viscosity and percentage oxidation affect the release profile. diflFerent types of gels were loaded with HTC-Iabeled bovine serum albumin (1% loading) and cumulative release was foUowed. ADA prepared from medium viscosity (BM65, AM55) and high viscosity (BH61, AH77) by both methods was used for this experiment. A and B denote alginate dialdehyde prepared by method A and B respectively and M and H stand for medium and high viscosity alginate and number denotes percentage oxidation.
Briefly, 0.15 mL of ADA (20% solution in 0.1M borax) was taken in a capped test mbe, to which was added 0.15 mL of gelatin(15% solution) containing HTC- albumin (1% loading) and aUowed to form geL It was then kept at 37«C for 10 minutes. 10 mL of PBS (pH 7.4) was introduced to it and incubated at 37»C. At regular intervals, ImL aliquots were withdrawn and absorbance of released FITC-albumin was read at 496 nm in a UV-Visible spectrophotometer. Cumulative release was then cakulated. AU the experiments were done in tripUcate. Representative data are illustrated in the example shown in Table 11.
Table 11. Cumulative release of FITC albumin from gels:
Cumulative release (%)
BH61 19.90 ±4
21.40 ±2 21.40 ±2 21.47 ±2 22.34 ±2 24.32 ± 3 24.32 ± 2 24.47 ± 2 28.10±2 45.70 ±2 69.80 ±4 73.60 ±4 86.20 ±3
AH77 18.30 f 2 17.20 ±3 22.00 ±4 22.30 ±2 26.99 ± 2 28.40 ±2 30.30 ±3 39.30 ± 3 49.60 ± 3
55.30 ±3 77.90 ± 5
98.20 ±2 98.20 ± 2
28.00 ± 1 28.80 ± 2 28.82 ± 1 29.03 ±1 31.60 ±2 31.80 ±1 33.11 ±3 35.70 ±3 43.26 ±2 75.20 ±3
94.60 ±1 74.70 ± 1
AM5S 14.06 1
19.60 ±3 24.1614 22.63 ±3 23.95 ± 2 28.30 ± 3 30.46 ± 4 33.2314 34.55 ± 4 37.03 ± 3 50 52 ±3 66.51 ± 1 (-•8 30 t 4
ControUed Release of Silver sulphadiazine from Gels.
About 0.15 mL of (10 and 20% solution)ADA (medium viscosity) prepared by method B (percentage oxidation 84, 50 and 20%) was taken in capped test tube. Gelatin (0.15 ml of 15 % solution. Type A, Bloom 300) containing silver sulphadiazine (USP) was added to it and allowed to form gel. The gel was kept for 10 minutes at 37°C. 10 mL of PBS (pH 7.4) was introduced to this and incubated at ST"C. At regular intervals, one mL aliquot solution was withdrawn and absorbance of the released silver sulphadiazine was read at 256nm in a UV- Visible spectrophotometer. Cumulative release was then cakulated. All the experiments were done in
1. A process for the preparation of a iopoiymer matrix comprising subjecting an aqueous solution of 6 to 20 g of alginate repaving two hydroxyl groups or a hydroxyl and an amino group in adjacent pensions, to oxidation using 4.2 to 16.8 gms of oxidizing agent, to obtain the alginate dialdehyde (ADA), purifying the same followed by reaction of a 10 to 20% solution of ADA with an equal volume of a 20% sirloin of gelatin In the presence of a reagent at a pH of 7.4 to 9.4 to form the matrix of alginate dialdehyde crossiiniced gelatine.
2. The process as claimed in claim 1. wherein the oxidising agent used is selected from sodium periodate solution potassium periodate solution, periodic acid and lead acetate in an organic solvent such as diethyl suiphoxide.
3. The process as claimed in claim 1, wherein the ADA is purified by using dialysis membranes, precipitation, uttrafittratton and gel permeation chromatography followed by lyophilisation.
4. The process as claimed in claim 1, wherein ADA is prepared in a solvent such as water ethanol/water mixture.
5. The process as claimed in claim 1, wherein the alginate employed is of medium (viscosity of 2% solution, 3S00 cps at 25^} or high viscosity (viscosity of 2% solution, 14000 cps at 250C )
6. The process as claimed in claim 1, wherein said reagent used for crosslinking is selected from borax solution, phosphate buffer and phosphate buffered safine.
7. The process as claimed in claim 1, wherein said gelatin used is Type A, obtained by acid hydrolysis of collagen and type B. obtained by basic hydrolysis of collagen.
8. A process for the preparation of a blopolymer matrix substanllalty as
|Indian Patent Application Number||921/MAS/2002|
|PG Journal Number||13/2008|
|Date of Filing||11-Dec-2002|
|Name of Patentee||SREE CHITRA TIRUNAL INSTITUTE FOR MEDICAL SCIENCES & TECHNOLOGY|
|Applicant Address||AN INDIAN INSTITUTE OF BIOMEDICAL TECHNOLOGY WING, POOJAPPURA, THIRUVANANTHAPURAM 695 012,|
|PCT International Classification Number||A61L 15/28|
|PCT International Application Number||N/A|
|PCT International Filing date|