HIGH MOLECULAR WEIGHT CHITOSAN, PROCESS FOR OBTAINING AND USES THEREOF

Abstract

The present disclosure relates to a method for obtaining a high molecular weight chitosan with a lower acetylation degree and its use in human or veterinarian medicine. More specifically, to the obtaining of this biomaterial by means of a simpler process, with reduced energy costs, when compared with conventional procedures.

Claims

1. A method for obtaining a high molecular weight chitosan, comprising: providing milled squid pen with a particle size between 63 and 125 m; and reacting NaOH with the milled squid pen particles selected in the previous step, for at least 1.5 hrs at 75 C., under stirring and in a N.sub.2 atmosphere.

2. The method of claim 1, wherein the step of providing milled squid pen comprises milling squid pen and selecting the milled squid pen with a particle size between 63 and 125 m.

3. The method of claim 2, wherein the amount of the selected milled squid pen particles is between 4-20 g

4. The method of claim 1, wherein the amount of NaOH is 200 mL.

5. The method of claim 1, wherein the NaOH is a solution of 50% (v/v) NaOH.

6. The method of claim 1, wherein the reaction time is between 1.5 and 3.5 hrs

7. The method of claim 1, further comprising: freezing the obtained chitosan at 80 C. and/or freeze-drying the obtained chitosan for 3 days.

8. The method of claim 1, further comprising previously washing the squid pen to eliminate impurities.

9. A chitosan comprising a molecular weight of 500-1200 kDa and an acetylation degree between 5-40%.

10. (canceled)

11. The chitosan of claim 9, wherein the acetylation degree is between 5-25%

12. The chitosan of claim 9, wherein the acetylation degree is between 5-15%

13. The chitosan of claim 9, wherein a protein concentration of the chitosan is up to 0.1 mg/ml.

14. (canceled)

15. The chitosan of claim 13, wherein the molecular weight is 1000 kDa-1200 kDA.

16. (canceled)

17. The chitosan of claim 9, wherein the chitosan is -chitosan.

18. (canceled)

19. (canceled)

20. (canceled)

21. (canceled)

22. (canceled)

23. A pharmaceutical composition comprising the chitosan of claim 9, and an active ingredient.

24. (canceled)

25. The composition of claim 23, wherein the composition comprises 0.1 to 50% of said chitosan.

26. The composition of claim 23, further comprising one or more additional polysaccharides, wherein the one or more additional saccharides are seaweed polysaccharides beta glucan, galactomannan, mucilage, cellulose, inulin, pullulan, dextrin, starch, glycosaminoglycans, or mixtures thereof.

27. The composition of claim 23, further comprising a protein, a growth factor, a digestive enzyme, a metabolic enzyme hormone, a drug, or mixtures thereof.

28. The composition of claim 27, wherein the protein is selected from the group consisting of: collagen, laminin, albumin, keratin, silk fibroin, fibronectin, and mixtures thereof.

29. The composition of claim 23, further comprising a cell culture media or a buffered media, wherein the cell culture media is a liquid, semi-solid, solid or gas cell culture media or a natural, synthetic or semi-synthetic cell culture media.

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

34. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0086] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of the present disclosure.

[0087] FIG. 1: Representative example of the FTIR spectra obtained for the studied chitosans. The characteristic bands related to the chitosan chemical structure are indicated.

[0088] FIG. 2: Representative example of the .sup.1H NMR spectra obtained for the studied chitosans. This spectrum corresponds to the chitosans obtained after the first reaction cycle. The characteristic peaks related to the chitosan chemical structure are indicated.

[0089] FIG. 3: Representative example of a SEC-MALLS chromatogram obtained for the chitosans of the present disclosure.

[0090] FIG. 4: Representative example of the .sup.1H NMR spectra obtained for the chitosans of the present disclosure. This spectrum corresponds to the chitosans from the second deacetylation cycle.

[0091] FIG. 5: Example of a membrane prepared by solvent-casting of a chitosan solution (0.5% in 2% acetic acid). A) Dried formulation and B) water re-hydrated formulation. C) Shows the scanning electron characterization of the dry membrane, while D) shows its energy dispersive X-ray spectrometry characterization.

[0092] FIG. 6: Comparison between solvent-casted chitosan solutions (0.1% in 2% acetic acid) three days after the casting. A) High molecular weight extracted chitosan of the present disclosure and B) medium molecular weight commercial chitosan.

[0093] FIG. 7: Comparison between solvent-casted chitosan (0.1% in 2% acetic acid) formulations. A) Membrane obtained using the high molecular weight extracted chitosan of the present disclosure and B) formless mass obtained using the medium molecular weight commercial chitosan.

[0094] FIG. 8: Example of a chitosan (0.5% in 2% acetic acid)-fucoidan hydrogel formed by ionic interaction. The colour of the hydrogel increases as it does the concentration of fucoidan: A) 2.5%, B) 5% and C) 10%.

[0095] FIG. 9: Comparison between the outcomes of chitosan (0.1% in 2% acetic acid)-fucoidan ionic interaction. A) Macro-hydrogels obtained using the high molecular weight extracted chitosan of the present disclosure and B) opaque solution obtained using the medium molecular weight commercial chitosan.

[0096] FIG. 10: Examples of membranes with a yellow colouring. The transparent chitosan membranes acquired this colour upon albumin loading.

DETAILED DESCRIPTION

[0097] The present disclosure refers to the physicochemical and structural characterization of high molecular weight -chitosan isolated from marine industry residues, more specifically from squid pens. It also refers to the application of this polymer and/or its derivatives in biomedicine.

[0098] In the present disclosure, Cht was extracted following a simpler procedure than those described in the literature. This extraction was performed in a shorter period of time and in a more eco-friendly manner when compared with conventional procedures, as less energy was utilized. More specifically, the received squid pens were gently washed with distilled water, to eliminate gross impurities. To achieve the greatest possible degree of reproducibility, the dried squid pens were milled (Ultra Centrifugal Mill ZM200, Retsch, Haan, Germany) and the obtained powder sieved (Analytical Sieve Shaker AS200, Retsch, Haan, Germany). Subsequently 5g of Cht powder with a particle size between 63 and 125 m were added to 200 mL of reaction medium (50% NaOH). The collection was left to react during 2 hrs at 75 C., under constant magnetic stirring. This process was performed under N.sub.2 atmosphere, in order to avoid the oxidation of the polysaccharide. Finally, the reaction product was abundantly washed with water until neutrality was reached.

[0099] In an embodiment, the obtained chitosan was frozen at 80 C. and freeze-dried for 3 days.

[0100] In an embodiment, the chemical structure of the obtained chitosan was characterized by FTIR spectroscopy. The FTIR spectrum was obtained using Shimadzu IRPrestige 21 spectromer (IRPrestige 21, Shimadzu, Europe). Samples were prepared as potassium bromide tablets at room temperature. The spectrum was collected by averaging 32 scans with a resolution of 4cm.sup.1, corresponding to the 4000-400 cm.sup.1 spectrum region. The obtained chitosan spectra displayed all the characteristic bands of chitosan. Indeed, all the studied samples lead to identical spectra. In this way, the spectrum of batch IV was selected as representative (FIG. 1). The peak at 3417 cm.sup.1 corresponds to OH and NH stretching vibrations, which is in concordance with the higher intensity of this peaks in the spectrum of chitosan with respect to that of chitin. The peak at 2879, related to CH stretching vibrations, is more intense in the spectrum of chitosan than in the chitin spectrum. This particular difference between the two spectra has been previously described in the literature. The peaks ranging from 1560 to 1690 cm.sup.1 are attributed to the NH bending vibration, while the band at 1695 cm.sup.1 is related to the NH absorption in NH.sub.2. Again, this is in concordance with the higher intensity of this peaks in the spectrum of chitosan with respect to that of chitin. This strong absorption peak at 1695 cm.sup.1 may be also assigned to the CO stretching vibration in the amide, due to partially acetylated amino groups. Finally, the peak at 1074 corresponds to the COC stretching vibrations.

[0101] In an embodiment, the acetylation degree was determined by nuclear magnetic resonance (NMR). The .sup.1H-NMR spectra of chitosan was obtained in a 2% DCI solution in D.sub.2O at 25 C., being recorded under the Burker Avance III spectral (Avance III HD 300 NMR-spectrometer, Bruker, Germany) conditions: resonance frequency of 400,13 MHz, with 1s pulse and 3.98 ms acquisition time. MestReNova Software 9.0 (Mestre-lab Research) was used for spectral processing. Chemical shifts are reported in ppm (). The NMR spectra confirmed that the product of the reaction was chitosan. The NMR spectra for all the studied samples were very similar. In this way, the spectrum of batch I was selected as representative (FIG. 2). The signal at 4.8 ppm coresponds to H1 GIcN, while the signal at 4.5 ppm corresponds to the H1 proton in GIcNA. The group of signals between 2.75 and 4.25 ppm correspond to the H2-H6 protons in the chitosan sugar. Meanwhile, the peak at 2 ppm corresponds to the 3 protons of the N-acetyl plus AcOH signals. The DA was determined from the relative integrals of this signal at 2 ppm, with respect to the combined H2-H6 protons. The NMR spectra also confirmed the good purification of the final product, as no signals from additional compounds were present in the different spectra.

[0102] In an embodiment, chitosan was analysed by size exclusion chromatography-multiangle laser-light scattering. The SEC-MALLS method allows the determination of molecular weight and polydispersity. SEC-MALLS measurements were performed with a Viscotek TDA 305 (Malvern, United Kingdom) with refractometer, right angle light scattering and viscometer detectors on a set of four columns: pre-column Suprema 5 m 850 S/N 3111265, Suprema 30 5 m 8300 S/N 3112751, Suprema 1000 5 m 8300 S/N 3112851 PL and Aquagel-OH MIXED 8 m 7.5300 S/N 8M-AOHMIX-46-51, with refractive index detection (RI-Detector 8110, Bischoff). A 0.15M NH.sub.4OAc/0.2M AcOH buffer (pH=4.5) was used as eluent, at a rate of 1 mL/min. The chitosan samples were dissolved in this same buffer. The elution times and the RI detector signal were calibrated with a commercial calibration polysaccharide from Varian, Pullulan with Mp 47.1 kDa (Mw 48.8 kDa; Mn 45.5 kDa) and narrow polydispersity (1.07). Values of dn/dC were taken from the literature. Table 1 shows the values of different obtained parameters, among them, the Mw was 1,003.9481.20 kDa. This is a higher molecular weight than that obtained following conventional procedures. The obtained SEC-MALLS chromatograms showed a single and symmetric peak, suggesting that there is a homogenous polysaccharide population (see a representative example in FIG. 3).

[0103] In an embodiment, the DA was further reduced, by submitting the product from the first reaction cycle to a new reaction cycle. For this purpose, all the content resultant from the previously described reaction cycle was mixed with 200 mL of reaction medium (50% NaOH). The system was left to react during 2 hrs at 75 C., under constant magnetic stirring. This process was performed under N.sub.2 atmosphere. The reaction product was abundantly washed with water until neutrality was reached. The new DA was 5.66%0.15 (see the spectrum in FIG. 4) and the reaction efficiency 80.34%1.61.

TABLE-US-00001 TABLE 1 Values of RI area, Peak RV, Mn, Mw and Mw/Mn for the studied chitosans, obtained after SEC-MALLS characterization. A 0.15M NH4OAc/0.2M AcOH buffer (pH = 4.5) was used both as dissolution buffer and as eluent. RI area Peak RV Mn Mw Sample* (mvmL) (mL) (KDa) (KDa) Mw/Mn Batch I 285.672 16.609 567,234 1,066,341 1.655 46.097 0.319 47,602 391,000 0.407 Batch II 348.972 17.971 573,434 1,040,581 1.818 40.835 1.567 72,615 141,855 0.116 Batch III 354.408 18.093 573,056 1,023,845 1.800 39.654 1.520 58,818 65,760 0.169 Batch IV 371.726 17.939 589,945 .sup.884,992 1.500 46.981 1.011 45,279 76,501 0.051 *Sample concentration: 2 mg/mL

[0104] In an embodiment, the previous reaction was afterwards scaled. Accordingly, it was performed parting from 15 g of squid pens powder. The amounts of reagents were proportionally adjusted. The second reaction cycle was also performed. All the obtained results were very similar to those obtained before the scaling.

[0105] In an embodiment, membranes for tissue engineering or drug delivery applications were prepared by using the obtained chitosan. The membranes were prepared by solvent-casting. More specifically, 0.5% and 1% chitosan where dissolved in 2% acetic acid and casted over plastic Petri dishes. The solvent was left to evaporate at room temperature in an appropriate chamber. The resulting membranes were neutralized (0.1 M sodium hydroxide during 10 minutes). Water was utilized to eliminate sodium hydroxide residues.

[0106] In an embodiment, with long-term storage in mind, the prepared membranes were oven-dryed (see the example in FIG. 5A). The water contact angle was 80.43, which is in concordance with the literature. These membranes were characterized by scanning electron microscopy, showing a flat surface (see the example in FIG. 5C). Energy dispersive X-ray spectrometry characterization confirmed the presence of C, O and N in the membranes (see the example in FIG. 5D).

[0107] In an embodiment, thinking in application/administration after long-term storage, the prepared membranes were re-constituted by re-hydration with distilled water (see the example in FIG. 5B).

[0108] In an embodiment, membranes were obtained using lower chitosan concentrations (0.1%). The resultant formulations were compared to that obtained with medium molecular weight commercial chitosan, with a similar AD to that of the chitosan from the present invention. The procedure was the same as in the previous paragraph. Clear differences were observed between formulations. In the case of the present invention chitosan the membranes were formed faster (three days faster) (see FIG. 6). The authors relate this faster solvent evaporation to the high molecular weight of their chitosan, which provokes a specific arrangement of the chitosan chains, thus diminishing the entrapment of the water molecules. In addition, the formulation obtained with the present invention chitosan maintains indeed the form of a membrane (see the example in FIG. 7A), while the commercial chitosan gives rise to a formless mass (see the example in FIG. 7B).

[0109] In an embodiment, hydrogels with potential for tissue engineering and drug delivery were prepared by using the obtained chitosan. These hydrogels were formed by electrostatic interaction with other polysaccharides i.e. chondroitin sulphate, fucoidan, gellan gum or alginate. More specifically, chitosan (concentrations 0.5 and 1% in 2% acetic acid) was mixed by mechanical agitation with the previously mentioned polymers (concentrations 2.5, 5 and 10% in water) at different rations. The gelation of the different formulations occurred immediately, and the hydrogels were neutralized (0.1 M sodium hydroxide during 10 minutes). Water was utilized to eliminate sodium hydroxide residues (see the example in FIG. 8).

[0110] In an embodiment, membranes were obtained using lower chitosan concentrations (0.1%). The resultant formulations were compared to that obtained with medium molecular weight commercial chitosan, with a similar AD to that of the chitosan from the present invention. The procedure was the same as in the previous paragraph. Clear differences were observed between formulations. The interaction between the present invention chitosan and the tested polysaccharide gave indeed rise to macro-hydrogels (see the example in FIG. 9A), while in the case of the commercial chitosan only solutions were observed (see the example in FIG. 9B).

[0111] In an embodiment, the amount of protein present in the extracted chitosan obtainable by the extraction method describe in the present disclosure was compared with that present in a previously purified commercial chitosan.

[0112] To assess the deproteinization efficiency of our methodology 10 mg of chitosan (with MW=1,00381 kDa and a DA=22.8% (0.7)) were immersed in 3 mL of milliQ water. The system was left under mechanical stirring for 48 hours. After this time a Micro BCA Protein Assay Kit (23235, ThermoFisher) was used for protein determination, following the instructions given by the supplier.

[0113] The same process was repeated with commercial chitosan (MW=190-310 kDa and an DA=18%, information provided by the supplier), after its purification in our lab.

[0114] These studies were performed in triplicate (n=3) and the results indicated as averageSD.

TABLE-US-00002 TABLE II Protein content determined by microBCA analysis. Chitosan Proteins (mg/mL) Batch I 0.029 0.011 Batch II 0.052 0.002 Batch III 0.075 0.004 Batch IV 0.058 0.023 Commercial 0.101 0.02 (MW, DA)

[0115] The results, presented in Table II, indicate a lower protein content in the extracted chitosan obtainable by the extraction method described in the present disclosure than in the commercial chitosan.

[0116] In an embodiment, it was proved the ability of the obtained chitosan to form membranes at low concentration. This property allows the use of the membranes of the present disclosure in medicine/biomedicine, namely tissue engineering. This interest grows if these membranes are able to act as drug delivery vehicles of bioactive molecules. Albumin was used as a model molecule to evaluate this ability.

[0117] Aliquots (50 l) of 1, 2.5 and 5% albumin solution (PBS pH=7.4) were placed over 1 cm.sup.2 membrane samples. The solution was left to penetrate the membrane for 1 hour at 37 C., point at which total solvent evaporation was observed. The supports where the membranes were placed during the loading step were washed with PBS (pH=7.4). These washing solutions were analyzed using a Standard UV-VIS Photospectrometry (UV-1601, Shimadzu, Australia), and the amount of albumin loaded within the membrane calculated utilizing Eq. 1:

% EE=[(iDfD)/iD]*100,Eq. 1:

where iD is the incorporated drug and fD is the free drug.

[0118] In an embodiment, the loaded membranes were immersed in 3.5 mL of PBS solution (pH=7.4) and placed at 37 C. under mechanical stirring. After 0.5, 1 and 5 hours the release mediums were analysed using a Standard UV-VIS Spectrophotometry (UV-1601, Shimadzu, Australia). The release studies were not accumulative. The concentration of released drug was calculated with the help of an albumin calibration curve.

[0119] These studies were performed in triplicate (n=3) and the results indicated as averageSD.

[0120] In an embodiment, all the drug included within the loading solutions was effectively incorporated by the membranes. Indeed, after the loading the membranes (originally transparent) acquired the same colour as the albumin powder (yellow). This effect can be appreciated in FIG. 10.

[0121] The release of this protein was also very efficient. Indeed, all the albumin was released after half an hour of study. This is probably due to the high hydrophilicity of albumin. In addition, the experiment was really reproducible, with all the UV spectra overlapping. This indicates a very homogeneous and reproducible arrangement of the chitosan chains during the formation of the membranes.

[0122] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0123] Where singular forms of elements or features are used in the specification of the claims, the plural form is also included, and vice versa, if not specifically excluded. For example, the term a polysaccharide or the polysaccharide also includes the plural forms polysaccharides or the polysaccharides, and vice versa. In the claims articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0124] Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

[0125] Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0126] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0127] In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims.

[0128] The above-described embodiments are combinable.

[0129] The following claims further set out particular embodiments of the disclosure.