Process for extraction of fish collagen and formulations of 3D matrices of collagen for biomedical and therapeutic applications thereof

Abstract

Polyelectrolyte based bioactive super-absorbent material ionotropically crosslinked/neutralized involving polyvalent carboxylic acids, citrate, Kojic Acid, Alpha Arbutin along with fish scale collagen cross linked with other polyelectrolyte biopolymers preferably selected from chitosan, alginate or their combinations is used in this invention. The advancement is also directed to process of extraction of collagen of high purity from fresh water fish scale by salt and alkaline washing, crushing followed by continuous dialysis thereby minimizing the chance of collagen degradation. Also disclosed are different forms of collagen-chitosan composite biomaterial using citrate as the neutralizing buffer in combination with antimicrobial agent, antioxidant, skin plumper and melanin reducer wherein the different forms of collagen chitosan particularly sheet, flakes, powder, gel, particles, fiber, film, spray etc. reveal efficient wound healing properties. The advancement is thus directed to find wide application in various dermal wound healing, tissue engineering, 3D cell culture, cell expansion and cell delivery vehicle, mimicking the in vivo situation in dynamic condition, cosmetics and different other health care applications.

Claims

1. A process for extraction of collagen from fish scale consisting of: (i) washing, softening, and depigmenting the fish scale by washing the fish scale with a salt solution selected from NaCl or KCl, followed by depigmenting and further softening the fish scale by treating the washed and softened fish scale with an alkali selected from NaOH or KOH, for 24-48 hours with a shaking speed of 60-100 rpm; (ii) subjecting the fish scale obtained in step (i) to crushing and smashing at a temperature up to 20 C. to achieve disintegration through punching, grinding and tearing of the fish scale; (iii) treating the fish scale obtained step (ii) with 0.3 M to 0.5 M acetic acid at a temperature of 6 C. to 10 C. with stirring at a shaking speed of 60-100 rpm, thus providing dissolution of collagen directly from the treated fish scale to provide a collagen containing acetic acid solution; (iv) carrying out filtration of the collagen containing acetic acid solution obtained in step (iii) to provide a first solution of soluble collagen and a first residue; (v) subjecting the first solution of soluble collagen obtained in step (iv) to salt treatment to precipitate collagen from the first solution of soluble collagen, followed by filtration to generate a solution and a second residue; (vi) subjecting the second residue obtained in step (v) to acetic acid treatment to provide a second collagen containing solution; (vii) carrying out continuous dialysis of the second collagen containing solution obtained in step (vi) by introducing the second collagen containing solution into a reverse osmosis hollow fiber membrane in a dialyzer, wherein dialysate used for the continuous dialysis is water, and controlling inlet and outlet pressure of the second collagen containing solution in the reverse osmosis hollow fiber membrane, wherein the inlet pressure is maintained at 300 to 1000 mbar and the outlet pressure is maintained at 100 to 600 mbar, and wherein an inlet pressure of the dialysate is maintained at 1 to 200 mbar below the outlet pressure in the reverse osmosis hollow fiber membrane; and (viii) obtaining purified collagen from an outlet of the dialyzer, the purified collagen having a yield of greater than 22% based on a total fish scale weight basis, and purity of 92-95%.

2. The process as claimed in claim 1, wherein the first residue obtained in step (iv) has collagen content and is subjected to: i) crushing and smashing; ii) treating with acetic acid and pepsin; iii) carrying out filtration to generate a solution of soluble collagen and a residue; and iv) recycling the solution of soluble collagen to step (v) of claim 1, and the residue to step (ii) of claim 1.

3. The process as claimed in claim 1 step (vii), wherein the continuous dialysis is carried out until the second collagen containing solution has a pH of 5.5 to 6.5.

4. The process as claimed in claim 1 step (ii), wherein the fish scale is subjected to crushing and smashing at a temperature up to 10 C.

Description

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

(1) FIG. 1: illustrates optical images of collagen sponge obtained after lyophilisation;

(2) FIG. 2: illustrates the flow diagram of collagen extraction from fresh water fish scale;

(3) FIG. 3: illustrates electrophoretic patterns of Type I collagen obtained from fish scale of Rahu, Catla, Mrigel and mixed species;

(4) FIG. 4: illustrates collagen based different forms; (a) sheet (b) film (c) sponge (d) particles (e) fibers and (f) beads;

(5) Fresh water fish origin of Indian subcontinent, East and South-east Asia (such as Golden Carp, Silver Carp, Grass carp, Labeo Bata, Lata, Catla, Rahu, Mrigel, Shol, Talapia, Nilontika, Hilsa, Bhetki, Balichura, Barali, Bata/Bangna, Bele, Rupchand, Cypinus, Chital, Foli/Chitol, Chuna, Common carp ((Channa gachua, Channa orientalis), Gozar (Channa marulius), Ilish (Tenualosa ilisha), Kalibaus, Nilotica (Oreochromis), Japanese Pnuti, Koi, Putitor mohashoul, Mohashoul, Shorpunti, Tapse, Mourala etc.) including fish scale are good source of collagen.

(6) Scales and other biological wastes of Fresh water fish of individual species (Rahu, Katla, Mrigel) and the mix scales of the above mentioned species (henceforth will be referred as mixed species) were collected from fish market of Contai, East Midnapur, West Bengal, India.

Example 1: Extraction of Collagen Type I from Fish Scale

(7) Fresh water fish scale of 1000 g was taken and washed with water thoroughly. It was again washed with 0.2-0.5M NaCl solution. After washing, the fish scale is kept in a container and soaked in 0.2-1.0 M preferably 0.5M NaOH solution for 24 to 48 h in a BOD incubator shaker with a shaking speed of 60-100 r.p.m and at temperature below 10 C.; preferably at 4 C.

(8) After soaking, alkali treated scale was washed with distilled water and smashed/crushed in a grinder/crusher while maintaining the temperature below 20 C. by adding ice/ice water intermittently.

(9) The smashed/crushed scale was kept in a container along with 0.5 M acetic acid solution (acid solution:scale=10-20:1 ratio by weight) for 7 days in a BOD incubator shaker at <10 C. and 60-100 r.p.m. After 7 days, the solution was filtered with nylon/cotton net of porosity 300-1000 m to recover the scale. The filtered solution (Part I) was collected in a container. The residue (Part II) is also collected in another container.

(10) In the solution (Part I), NaCl was added pinch by pinch and was gently shaken in a magnetic stirrer at r.p.m.<150. The dissolved collagen precipitated out form the solution continuously. Salt addition process was continued until the concentration of NaCl in the solution became 0.9 M. Then the precipitate was separated from the solution by a specially designed filter system where filtration was carried out in a single step using cloth filter (in the range of 50-500 m). After filtration, the precipitate was collected and the filtrate was discarded.

(11) The collected precipitate was re-dissolved in 0.3-0.5M acetic acid by gentle stirring. The dissolved solution was dialyzed in a hollow fiber membrane dialyzer (molecular cut off 3-80 kDa). The inlet pressure of the solution was kept at 300-1000 mbar and outlet pressure was at 100-600 mbar. The inlet pressure of water was kept at 0-200 mbar below the outlet pressure of the solution. The dialysis was continued until the pH of the solution reached in the range of 5.5 to 6.5. The solution obtained after dialysis was frozen at 20 C. for 12 h followed by lyophilizing at <30 C. for 24 to 48 h under vacuum. After freeze drying the purified collagen sponge was collected (FIG. 1).

(12) The collected residue (Part II) was smashed/crushed in ice-water mixture in a grinder/crusher and the ground/crushed scale was kept in a container. 0.5 M acetic acid (acid solution:scale=10-20:1 ratio by weight) and 0.1% pepsin were added in the bottle. The bottle containing scale and chemicals was kept under shaking condition for 3 days in a BOD incubator shaker at <10 C. at 60-100 r.p.m. After 3 days, solution and residue were collected separately. Same treatment, as given to part I solution, was applied to this solution and the same procedure was followed to obtain collagen from this solution. The residue was recycled and again smashed/crushed followed by acetic acid treatment and filtration to obtain part I and part II, as described above. The process of extraction collagen from fish scale is shown schematically in FIG. 2.

(13) Washing in NaCl removes the odour and colour/pigment from the scale and therefore, high purity collagen isolation is possible by minimizing the filtration which is significantly time consuming. The removal of pigment subsequently helps to reuse the hollow fiber membrane for more number of times.

(14) NaOH treatment makes the scale softer with significant swelling characteristics to facilitate grinding/crushing without significant increase in the temperature and protein degradation.

(15) This step of grinding/crushing promotes exposure of collagen into acidic environment during acetic acid treatment, which further facilitates easy dissolution of collagen without demineralization of scale.

(16) The hollow fiber membrane was used for dialysis and purification which could be advantageously reused several times (4-10 times) and collagen production rate increased significantly.

(17) Thus, in this method, the minerals were not attempted to be demineralized by EDTA treatment as demineralization by chelating is time consuming and costly affair, which also eliminates the possibility of presence of remnant EDTA (a chelating agent for Ca.sup.+2, Fe.sup.+2, Fe.sup.+3 causing demineralization) in the isolated collagen.

Example 2: Characterization of the Extracted Collagen Obtained from Ex 1

(18) i) SDS-PAGE:

(19) Electrophoretic patterns of Type I collagen from fish scale of Rahu, Catla, Mrigel and mixed species is shown in FIG. 3. In all the cases (i.e. Rahu, Catla, Mrigel and mix sources) the mobility of O-chains was obtained as expected, based on the molecular weight markers.

(20) The estimated molecular weight for a chain of these collagens, using globular proteins as standards was approximately 120-150 kDa. As in type I collagen from calf skin which was compared with the isolated collagens comprised of at least two different -chains (1 and 2) with different mobility, which indicated that the collagens from these two species are of type I collagen.

(21) ii) FTIR Analysis:

(22) In the FTIR spectra of fish scale collagen of mixed species, the amide I band found is in the range from 1600 cm.sup.1 to 1700 cm.sup.1. This is mainly associated with stretching vibrations of the carbonyl groups (CO bond) along the polypeptide backbone, which is a sensitive marker of the peptide secondary structure. The ratio of absorption intensity between 1240 cm.sup.1 (amide III) and 1463 cm.sup.1 (amide II) band is approximately equal to 1.0, which confirms the triple helical structure of collagen.

(23) iii) Amino Acid Analysis by HPLC:

(24) The amino acid composition of the collagen isolated from mixed species of fresh water fish scale was expressed as amino acid residues per 1000 total amino acid residues and is shown in Table 1. As collagen is triple helical in nature with characteristic amino acid repeat, (Gly-Pro-Hyp), glycine (Gly) was the most abundant with the amount of 340 of the total amino acid present in collagen extracted from mixed species. The total amount of imino acids per 1000 total amino acid residues, proline (Pro) and hydroxyproline (Hyp), are 120 and 80, respectively, which is likely to have effect on the stability of the collagen fibers and influences the denaturation temperature as well. Absence of cysteine in collagen emphasizes presence of type I collagen. The hydroxyproline content in the collagen isolated from scale of Katla, Rahu, Bata, Mrigel etc. (fresh water fish origin) had marginal variation which was significantly higher than the marine source and thus significantly higher thermal stability.

(25) TABLE-US-00001 TABLE 1 Amino acid composition of ASC from scale of Mixed species (expressed as residues per 1000 total amino acid residues) Amino acid Residues/1000 residues Aspartic acid/Asparagine 47 Cysteine 0 Glycine 340 Hydroxyproline 80 Glutamic acid/Glutamine 77 Alanine 115 Proline 120 Lysine 26 Valine 22 Methionine 14 Isoleucine 10 Leucine 23 Serine 35 Threonine 21 Tyrosine 3 Phenylalanine 13 Tryptophan 0 Histidine 4 Arginine 50

Example 3: Yield Percentage of Different Fish Scales

(26) Using this procedure, the mixture of scale from different spices yielded collagen up to 22% which is substantially higher than the most of the reported values. The collagen yield was up to 5% of the scales by EDTA treatment and acetic acid dissolution as discussed in the F. Pati et al without crushing of the scales.

Example 4: Collagen Based Wound Dressing

(27) Collagen extracted by the Example 1 is used to prepare different forms of products for wound dressing by combining with a polysaccharide such as chitosan, alginate or their combination followed by citrate buffer neutralization. The basic steps comprise:

(28) i) Chitosan-collagen solution at different weight ratio (20:0.25-10 wt % on total weight basis) was prepared individually or as a mix by adding 0.1-1 M acetic acid to the final concentration of the solution 0.5-5 wt % depending on the molecular weight of chitosan used.
ii) The solution was further neutralized to a different extent by drop wise addition of 5 wt % citrate buffer (in the range of 1-10 wt %) at varied pH but higher than 3.13 (preferably at pH 7.4) under continuous stirring by mechanical/magnetic stirrer (200-300 r.p.m.) to reduce the charge density of chitosan gradually.
iii) When pH of the solution reached to 7.0 the appearance became white and the solution was completely neutralized.
iv) The neutralized solution was then cast in a tray (preferably in Teflon coated tray) and stored in freezer at 20 C. for 12 h followed by lyophilisation at <30 C. under vacuum of 110.sup.1-510.sup.2 mbar or below until the freeze solution dried substantially.
v) The dried mix was crushed and sieved by nylon/cotton mesh of varied mesh sizes.
vi) The different forms (particles, powder, flake, bead etc.) obtained after sieving were stored in an air tight bag under sterilized condition at room temperature or below for final applications.

(29) These downstream products like collagen based sheet, film, sponge, particles, fibres and beads are shown in FIG. 4.

Example 5a: Preparation of Chitosan-Collagen Powder, Particles, Flakes and Beads

(30) To form beads, citrate buffer (in the range of 1-10 wt %) at pH in the range of 4.76 to 10 (preferable at pH 7.4) is added to the solution of Example 4 following under continuous stirring by mechanical means (200-500 r.p.m.) to reduce the charge density of chitosan and form beads. When pH of the solution was closed to 7.0 the appearance of neutralized beads turned into white and looked like precipitate. The beads were collected, washed and dried to a different extent and by different means. The neutralized beads were stored in freezer at 20 C. for 12 h followed by lyophilisation at <30 C. under vacuum of 110.sup.1-510.sup.2 mbar or below until the beads dried substantially or completely. The drying of beads was also attempted under vacuum (110.sup.1-510.sup.2 mbar or below) or in a temperature controlled oven (at 40-80 C.). Finally the beads were either crushed followed by sieving or directly sieving in nylon/cotton mesh of varied mesh sizes and depending upon the mesh size different forms like powder, particles, flakes and beads were obtained.

Example 5b: Preparation of Chitosan-Collagen Sheet

(31) The differentially neutralized chitosan/chitosan-collagen solution obtained in Example 4 was then cast in a tray (preferably in Teflon coated tray) and stored in freezer at 20 C. for 12 h.

(32) 2.5 wt % collagen solution was prepared separately by adding collagen in 0.3 M acetic acid solution (250 mg collagen in 10 ml acetic acid solution). This solution was neutralized by adjusting the pH with 5 wt % citrate buffer.

(33) The collagen/neutralized collagen solution was poured on the top of the frozen mix followed by similar freezing and lyophilisation (at <30 C. under vacuum of 110.sup.1-510.sup.2 mbar or below) into sponge. The sponge was pressed into different extent to form sheet of required thickness. Opposite to collagen layer of the sheet, a non-adhesive mesh (nylon or any other similar materials) was fixed either by pressing or by using cellulose based bio-adhesive in order to provide support to the sheet, form a non-sticky barrier and retain the shape of the sheet

(34) Optionally, 1-5 wt % alginate solution was prepared with distilled water by shaking in any mechanical means. 5 wt % citrate buffer was added (ratio of alginate solution to citrate buffer was 5-10:1 by volume) drop wise to the solution by continuous mechanical/magnetic stirring (100-300 rpm) to increase charge density of the solution.

(35) The separately prepared neutralized chitosan (by the method of Example 4) solution was mixed with alginate-citrate buffer to form gel. The gel was cast in a tray (preferably in Teflon coated tray) and subsequently frozen in a freezer at 20 C. for 12 h. Then the neutralized collagen solution was poured on the top of frozen mix in the tray and lyophilized at <30 C. under vacuum of 110.sup.1-510.sup.2 mbar or below. Sponge was formed after complete lyophilization.

(36) The sponge was pressed into different extent to form sheet of required thickness. Opposite to collagen layer of the sheet, a non-adhesive mesh (nylon or any other similar materials) was fixed either by pressing or by using cellulose based bio-adhesive in order to provide support to the sheet, form a non-sticky barrier and retain the shape of the sheet. The sheet was stored in an air tight bag under sterilized condition at room temperature or below for further applications.

Example 5c: Preparation of Chitosan-Collagen Film

(37) The neutralized solution of Example 4 was cast in a tray (preferably in metallic (like stainless steel)/glass/polymer based tray) and dried under sterilized condition preferably under air flow until the solution dried completely to form the film. The film was carefully removed and stored under sterilized sealed bag at room temperature or below for further applications.

Example 5d: Preparation of Chitosan-Collagen Fiber

(38) Chitosan-collagen solution obtained from Step 1 of Example 4 was filtered and the filtrate was wet spun in polycarboxylic acid bath at varied pH with pKa values higher than at least two carboxylated group ionization (above pKa.sub.2). The filtrate was also wet spun in a citric acid bath with pH between 4.76 and 10 preferably at 7.4. The fibers were washed, dried and kept in air sealed bag to store at below 30 C. preferably at 15 C. under sterilized condition for final applications.

Example 5e: Preparation of Chitosan-Collagen Spray

(39) The chitosan-collagen powder prepared in 5a was suspended in aqueous/dilute acetic acid medium and could be sprayed on the wound bed prior to covering by occlusive dressing. The collagen-citrate based solution/suspension could also be sprays on the assaulted area.

Example 5f: Preparation of Chitosan-Collagen Spray-Gel

(40) Chitosan-collagen mix (20:1-10 wt % on total weight basis) solution using 0.2 mol % acetic acid with pH below 6 along with citric acid (1-10 wt %) solution at pH above 5 were promoted for in-situ gelation during spraying using a special spraying device which facilitated mixing of both the solution into gel at the specific site of interest.

Example 5g: Preparation of Collagen Based Transparent Film, Curved Contact Lenses, Delivery Vehicles

(41) Blending collagen with other polymer like Polyvinyl alcohol, silk or chitosan (20:1-10 wt % on total weight basis) followed by co-valent/ionotropic crosslinking. Covalent or ionic crosslinking using unsaturated acid/aldehyde/amine etc. also favours in situ iodination during crosslinking using iodine as blend. This iodinated crosslinked polymer dressing system is efficient in antimicrobial activity due to slow release of iodine into the wound site.

Example 6: Characterization of Chitosan-Collagen Based Wound Dressing

(42) 1) Swelling Study:

(43) Collagen, chitosan-collagen, and chitosan-alginate-collagen samples (in example 5a to 5g) showed a steady state of swelling in 48 h of soaking period without any disintegration or dissolution in the medium and preserved their physical integrity. The sponge and sheet samples showed a rapid swelling in first 10-24 h because of rapid diffusion of water molecules. Then it gradually attained equilibrium in about 36 h and maintained equilibrium swelling state. The sheet showed higher swelling than the other forms and after 24 h it reached to 6000%.

(44) ii) Degradation Study:

(45) The enzymatic degradation of raw collagen and collagen based different samples (in example 5a to 5g) were investigated by monitoring the residual mass percent as a function of exposure time to collagenase solution. The collagen samples degraded rapidly compared to the chitosan-collagen samples. Within 24 h. the residual mass reduced to 18.4% of the initial mass for sponge/sheet (example 5b).

(46) vi) In Vivo Animal Study (Full-Thickness Cutaneous Wound Models)

(47) Thirty-six rats (male wistar weighing220 g) divided into two groups were studied for wound healing under animal ethical clearance using standard protocol. A full-thickness square defect (2 cm2 cm) was created in the upper back area of each rat and then implanted with or without a chitosan-collagen film/sheet. Wounds were wrapped with dressing for protection. Wound closure was measured every other day after wounding. Animals were euthanized after 14 days and tissues were harvested for analysis. All the tissue samples were cut into a full-thickness manner from the wounded sites, and then were used for histological analyses. Photographic images were also captured to understand the healing process. The entire studies were carried out under ethical clearance.

(48) Measurement of Wound Healing:

(49) The wound healing process after treatment with a collagen-chitosan sheet and film and its comparison with the healing process of the control is shown in FIG. 7. During the first day post-operation, the collagen-chitosan sheet and film efficiently absorbed wound exudates and tightly attached to the wound surface.

(50) After 5 days of post-operation, scabs were observed in both groups which after 10 days post-operation, were falling off the wounds, and the wounds were mostly filled with restored skin. It is notable that the wounds of the collagen-chitosan sheet and film group were greatly reduced in size as compared to the control which is more evident after 15 days. Complete wound closure was observed in collagen-chitosan sheet treated group after 18 days. The restored skin treated with a collagen-chitosan sheet and film was similar to normal skin, while an elongated scar was still observed in the skin after healing of a control group due to excessive contraction.

(51) The defects treated with collagen-chitosan sheet demonstrated superior healing and the skin defect was reconstructed with a good cosmetic outcome. The wound area decreased at an accelerated rate for the wounds treated with collagen-chitosan sheet and collagen-chitosan film at the end of 14.sup.th day compared to the control. The difference between the control group and the collagen-chitosan sheet/film group showed significant healing compared to the control after 14 days of treatment (p<0.05). From this animal study, the positive role of collagen and citric acid in accelerating wound healing is evident.

(52) vii) Tensile Strength of Regenerated Skin:

(53) Assessment of regenerated skin tissues was done by measuring the mechanical properties of the regenerated tissues. Analysis of the all biomechanical parameters (mainly ultimate tensile strength and young's modulus) demonstrates that biomechanical property of collagen-chitosan sheet and collagen-chitosan film treated group was better compared to the control group.

(54) viii) Histological and Immunofluorescence Studies

(55) Histological analyses were performed to assess the regenerated tissues guided by collagen-chitosan based samples (in example 5a to 5g) in rats. H&E staining of wound sections after 14 days post-surgery revealed distinct differences in maturity of the epidermis/dermis and quality of granulation tissue between collagen-chitosan sheet/film-group and the control group. Both collagen-chitosan sheet and collagen-chitosan film-group demonstrated accelerated maturation and a well formed epidermis with compact orthokeratosis, while control group displayed more inflammatory granulation tissue and partially re-epithelialized epidermis with overlying serum crust. Collagen-chitosan film treated wounds demonstrated pale but more mature collagen bundles than the controls. In contrast, collagen-chitosan sheet treated wounds displayed well-organized compact collagen bundles that were oriented parallel to the epidermis. Furthermore, trichrome semi-quantitative analysis revealed significantly increased collagen intensity in collagen-chitosan sheet treated wounds compared to all other wounds (p<0.05).

(56) Type IV collagen antibody staining which stains the blood vessels was used to demonstrate the vascularization of each experimental group after 2 weeks of implantation. Higher number of blood vessel/density were observed in the collagen-chitosan sheet/film implanted group than in the control group (A large number of micro-vessels were observed in the collagen-chitosan sheet and film implanted group, and the micro-vessel density was much higher compared with the control group. This finding could be attributed to the presence of bioactive cues in the collagen-chitosan samples along with its porosity improved the activation and facilitated the endothelial cells to form new blood vessels in the wound bed.