阅读说明：本技术 一种儿茶酚功能化壳聚糖/牡蛎肽温敏水凝胶的制备方法、产品及应用 (Preparation method, product and application of catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel ) 是由 胡章 张冬英 卢思彤 李思东 孔松芝 程瑜 廖铭能 于 2020-07-01 设计创作，主要内容包括：本发明公开了一种儿茶酚功能化壳聚糖/牡蛎肽温敏水凝胶的制备方法、产品及应用,所述制备方法包括儿茶酚功能化壳聚糖的制备以及儿茶酚功能化壳聚糖/牡蛎肽温敏水凝胶的制备；本发明制备的儿茶酚功能化壳聚糖/牡蛎肽温敏水凝胶无细胞毒性,生物相容性好,能够促进L929细胞迁移,溶血率小于5％,符合国家安全标准。(The invention discloses a preparation method, a product and an application of catechol functionalized chitosan/oyster peptide temperature-sensitive hydrogel, wherein the preparation method comprises the steps of preparing the catechol functionalized chitosan and preparing the catechol functionalized chitosan/oyster peptide temperature-sensitive hydrogel; the catechol functionalized chitosan/oyster peptide temperature-sensitive hydrogel prepared by the invention has no cytotoxicity and good biocompatibility, can promote migration of L929 cells, has a hemolysis rate of less than 5 percent, and meets the national safety standard.)

1. A preparation method of catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel is characterized by comprising the following steps of preparing catechol-functionalized chitosan solution by performing catechol functionalization on chitosan, adding oyster peptide into the catechol-functionalized chitosan solution, and then dripping beta-sodium glycerophosphate solution into the catechol-functionalized chitosan solution to be stirred and mixed to obtain the catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel.

2. The preparation method of the catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel according to claim 1, which comprises the following steps:

(1) preparation of catechol functionalized chitosan

Dropwise adding carbodiimide hydrochloride/N-hydroxysuccinimide solution into the chitosan solution/3, 4-dihydroxy benzene acrylic acid solution for reaction, dialyzing after the reaction is finished, and freeze-drying to obtain catechol functionalized chitosan;

(2) preparation of catechol-functionalized chitosan/oyster peptide/beta-sodium glycerophosphate temperature-sensitive hydrogel

Preparing catechol functionalized chitosan solution from the catechol functionalized chitosan prepared in the step (1), adding oyster peptide, and adding beta-sodium glycerophosphate solution under stirring to obtain the catechol functionalized chitosan/oyster peptide temperature-sensitive hydrogel.

3. The preparation method of the catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel according to claim 2, wherein the step (1) comprises the following steps:

dissolving chitosan in acetic acid to prepare a chitosan solution, and dissolving 3, 4-dihydroxyl phenylpropionic acid in water to obtain a 3, 4-dihydroxyl phenylpropionic acid solution; adding the 3, 4-dihydroxyl phenylpropionic acid solution into the chitosan solution to obtain a chitosan/3, 4-dihydroxyl phenylpropionic acid solution;

dissolving carbodiimide hydrochloride and N-hydroxysuccinimide in ethanol water solution to obtain carbodiimide hydrochloride/N-hydroxysuccinimide mixed solution;

and thirdly, dropwise adding the carbodiimide hydrochloride/N-hydroxysuccinimide mixed solution prepared in the second step into the solution prepared in the first step for reaction, and dialyzing, freezing and drying after the reaction is finished to obtain the catechol-functionalized chitosan.

4. The preparation method of the catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel according to claim 3,

in the first step, the mass fraction of chitosan in the chitosan solution is 2%, the concentration of 3, 4-dihydroxyphenylpropionic acid in the 3, 4-dihydroxyphenylpropionic acid solution is 1mol/L, and the mixing volume ratio of the 3, 4-dihydroxyphenylpropionic acid solution to the chitosan solution is 1: 10;

in the second step, the mixing molar ratio of the carbodiimide hydrochloride and the N-hydroxysuccinimide is 1:1, the volume ratio of the ethanol to the water in the ethanol water solution is 9: 1, and the concentration of the carbodiimide hydrochloride in the solution is 1 mol/L.

And step three, reacting for 10 hours, wherein the reaction pH value is 4.5-5.5, dialyzing in hydrochloric acid aqueous solution with the pH value of 5 for 3 days after the reaction is finished, dialyzing in distilled water for 4 hours, and freeze-drying to obtain the catechol functionalized chitosan.

5. The preparation method of the catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel according to claim 2, wherein in the step (2):

the addition amount of the oyster peptide is 1 g/L;

the solvent of the catechol-functionalized chitosan solution is water, and the mass fraction of the solvent is 2%; the solvent of the beta-sodium glycerophosphate solution is water, and the mass fraction of the water is 30%;

the mixing volume ratio of the catechol functionalized chitosan solution to the beta-sodium glycerophosphate solution is 7: 3 or 8: 2;

and after the beta-sodium glycerophosphate solution is dropwise added, stirring for 2min, and placing in a constant-temperature environment at 37 ℃ for constant-temperature placement to obtain the temperature-sensitive hydrogel.

6. The preparation method of the catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel according to claim 2, wherein the oyster peptide in the step (2) is prepared into chitosan/oyster peptide microspheres before being added, and the preparation method specifically comprises the following steps:

a. dissolving oyster peptide and chitosan in acetic acid solution to obtain oyster peptide/chitosan mixed solution, dropwise adding the oyster peptide/chitosan mixed solution into liquid paraffin containing an emulsifier under the condition of stirring, and stirring;

b. and (3) dripping a cross-linking agent solution, stirring, centrifuging, washing, and freeze-drying to obtain the chitosan/oyster peptide microspheres.

7. The preparation method of the catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel according to claim 6,

in the step a:

the mass fraction of acetic acid in the acetic acid solution is 1%;

in the oyster peptide/chitosan mixed solution, the mass fraction of chitosan is 1%, and the addition amount of oyster peptide is 0.4-1.4 mg/mL;

dripping the oyster peptide/chitosan mixed solution into liquid paraffin containing an emulsifier at 800rpm and 60 ℃, and stirring for 1.5 h; the emulsifier is a mixture of Tween 80 and Span-80, and the volume ratio of the oyster peptide/chitosan mixed solution to the liquid paraffin is 1: 10;

in the step b:

the mass fraction of the cross-linking agent solution is 25%, the addition amount of the cross-linking agent solution and the volume ratio of the liquid paraffin are 1: 50, the mixture is dripped out after 30min, the mixture is continuously stirred for 30min after the dripping is finished, the mixture is centrifugally collected, precipitates are repeatedly washed by petroleum ether and ethanol, and then the precipitates are frozen and dried to obtain the chitosan/oyster peptide microspheres;

the cross-linking agent is glutaraldehyde.

8. The preparation method of the catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel as claimed in claim 1, wherein β -sodium glycerophosphate solution and NaHSO are sequentially added dropwise after the oyster peptide is added₃Stirring and mixing the solution to obtain the catechol functionalized chitosan/oyster peptide temperature-sensitive hydrogel.

9. A catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel prepared by the method for preparing the catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel according to any one of claims 1 to 8.

10. The application of the catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel according to claim 9 in preparation of wound repair.

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to a preparation method, a product and application of catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel.

Background

Trauma is the most common disease in modern society, and has high morbidity and disability rate. To date, the health and quality of life of humans remains seriously threatened by bleeding from various causes, skin wounds and scar tissue formed by healing thereof. Therefore, the development of a medicament and a corresponding preparation capable of accelerating wound healing and reducing pathological scar formation is always the key and difficult point in the field of skin wound treatment, and has great social significance.

Hydrogels refer to a class of three-dimensional network polymers formed by physical or chemical crosslinking that can absorb a large amount of water and retain their three-dimensional structure. The main characteristics are as follows: (1) the polymer matrix forms a network structure through physical or chemical crosslinking, and the network is filled with a solvent which cannot flow freely and shows the semi-solid property of elasticity or viscoelasticity; (2) sensitive to temperature and external conditions; (3) has swelling property, syneresis property, thixotropy and adhesiveness; (4) has the advantages of easy spreading, comfortable feeling, no greasiness, easy removal, capability of absorbing tissue exudate, no interference with normal physiological action of skin, and certain water retention effect to promote transdermal absorption of medicine. The hydrogel prepared from natural organic materials does not cause immunological rejection in organisms, shows good biocompatibility, and simultaneously cells can normally adhere and grow in the hydrogel, and metabolites can be discharged through pores of the hydrogel. Compared with other artificially synthesized materials, the hydrogel structure prepared from natural organic materials is closer to living tissues, is similar to extracellular matrix parts in nature, can reduce friction on surrounding tissues, and obviously improves various biological properties of the material.

The temperature-sensitive hydrogel is one of the most studied hydrogels in a plurality of environment-sensitive hydrogels, and is mainly characterized in that sol-gel transformation can occur in response to temperature change. In recent years, the use of temperature-sensitive hydrogels as gel systems has been increasingly studied, and has received much attention in the biomedical field. Compared with the traditional hydrogel, the temperature sensitive hydrogel has obvious superiority when being applied to the fields of tissue engineering, drug sustained release and the like.

Disclosure of Invention

Based on the content, the invention provides a preparation method, a product and application of the catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel; the modified chitosan/active peptide composite material is prepared by taking oyster peptide, catechol-functionalized chitosan and other marine bioactive substances as main raw materials, has excellent application effects in the aspects of skin wound repair, hemostasis, antibiosis and the like, is a natural nontoxic biological composite material, and provides technical guidance and theoretical basis for realizing high-value utilization of marine biological resources.

In order to achieve the purpose, one of the technical schemes of the invention is a preparation method of catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel, which comprises the following steps: preparing the chitosan into a catechol functionalized chitosan solution after the chitosan is functionalized by catechol, then adding oyster peptide into the chitosan solution, and then dripping a beta-sodium glycerophosphate solution into the chitosan solution and stirring and mixing the mixture to obtain the chitosan.

Preferably, the method comprises the following steps:

(1) preparation of catechol functionalized chitosan

Dropwise adding carbodiimide hydrochloride/N-hydroxysuccinimide (DEC/NHS) solution into chitosan solution/3, 4-dihydroxyphenylpropionic acid (CS/HCA) solution for reaction, dialyzing, and freeze-drying to obtain catechol-functionalized chitosan (CS-C);

(2) preparation of catechol functionalized chitosan/oyster peptide (CS-C/OP/beta-GP) temperature-sensitive hydrogel

Preparing a catechol functional chitosan solution from the catechol functional chitosan (CS-C) prepared in the step (1), adding Oyster Peptide (OP), dropwise adding a beta-sodium glycerophosphate (beta-GP) solution into the prepared catechol functional chitosan solution under the stirring condition to obtain the catechol functional chitosan/oyster peptide (CS-C/OP/beta-GP) temperature-sensitive hydrogel;

preferably, the step (1) comprises the steps of:

dissolving chitosan in acetic acid to prepare a chitosan solution, and dissolving 3, 4-dihydroxyphenylpropionic acid (HCA) in water to obtain a 3, 4-dihydroxyphenylpropionic acid solution; adding the 3, 4-dihydroxyphenylpropionic acid solution into the chitosan solution to obtain a chitosan/3, 4-dihydroxyphenylpropionic acid solution (CS/HCA);

dissolving carbodiimide hydrochloride and N-hydroxysuccinimide in ethanol water solution to obtain carbodiimide hydrochloride/N-hydroxysuccinimide mixed solution (EDC/NHS);

and thirdly, dropwise adding the carbodiimide hydrochloride/N-hydroxysuccinimide mixed solution prepared in the second step into the solution obtained in the first step for reaction, and dialyzing, freezing and drying after the reaction is finished to obtain the catechol functionalized chitosan (CS-C).

Preferably, in the step (i), the mass fraction of chitosan in the chitosan solution is 2%, the concentration of 3, 4-dihydroxyphenylpropionic acid in the 3, 4-dihydroxyphenylpropionic acid solution is 1mol/L, and the mixing volume ratio of the 3, 4-dihydroxyphenylpropionic acid solution to the chitosan solution is 1: 10;

in the second step, the mixing molar ratio of the carbodiimide hydrochloride and the N-hydroxysuccinimide is 1:1, the volume ratio of the ethanol to the water in the ethanol water solution is 9: 1, and the concentration of the carbodiimide hydrochloride in the solution is 1 mol/L;

and step three, reacting for 10 hours, wherein the reaction pH value is 4.5-5.5, dialyzing in hydrochloric acid aqueous solution with the pH value of 5 for 3 days after the reaction is finished, dialyzing in distilled water for 4 hours, and freeze-drying to obtain the catechol functionalized chitosan.

Preferably, in the step (2):

the addition amount of the oyster peptide is 1 g/L;

the solvent of the catechol-functionalized chitosan solution is water, and the mass fraction of the solvent is 2%; the solvent of the beta-sodium glycerophosphate solution is water, and the mass fraction of the water is 30%;

the mixing volume ratio of the catechol functionalized chitosan solution to the beta-sodium glycerophosphate solution is 7: 3 or 8: 2;

and after the beta-sodium glycerophosphate solution is dropwise added, stirring for 2min, and placing in a constant-temperature environment at 37 ℃ for constant-temperature placement to obtain the temperature-sensitive hydrogel.

Preferably, the oyster peptides in the step (2) are prepared into chitosan/oyster peptide microspheres (M-OP) before being added, and the method specifically comprises the following steps:

a. dissolving oyster peptide and chitosan in acetic acid solution to obtain oyster peptide/chitosan mixed solution, dropwise adding the oyster peptide/chitosan mixed solution into liquid paraffin containing an emulsifier under the condition of stirring, and stirring;

b. and (3) dripping a cross-linking agent solution, stirring, centrifuging, washing, and freeze-drying to obtain the chitosan/oyster peptide microspheres.

Preferably, in the step a:

the mass fraction of acetic acid in the acetic acid solution is 1%;

in the oyster peptide/chitosan mixed solution, the mass fraction of chitosan is 1%, and the addition amount of oyster peptide is 0.4-1.4 mg/mL;

dripping the oyster peptide/chitosan mixed solution into liquid paraffin containing an emulsifier at 800rpm and 60 ℃, and stirring for 1.5 h; the emulsifier is a mixture of Tween 80 and Span-80, and the volume ratio of the oyster peptide/chitosan mixed solution to the liquid paraffin is 1: 10;

in the step b:

the mass fraction of the cross-linking agent solution is 25%, the addition amount of the cross-linking agent solution and the volume ratio of the liquid paraffin are 1: 50, the mixture is dripped out after 30min, the mixture is continuously stirred for 30min after the dripping is finished, the mixture is centrifugally collected, precipitates are repeatedly washed by petroleum ether and ethanol, and then the precipitates are frozen and dried to obtain the chitosan/oyster peptide microspheres;

the cross-linking agent is glutaraldehyde.

Preferably, the preparation of the catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel comprises the following steps of preparing a catechol-functionalized chitosan solution by performing catechol functionalization on chitosan, adding oyster peptide into the catechol-functionalized chitosan solution, and then sequentially dripping β -sodium glycerophosphate solution and NaHSO₃Stirring and mixing the solution to obtain the catechol-functionalized chitosan/oyster peptide temperature-sensitive hydrogel.

Preferably, NaHSO is added₃Under the condition of the solution, the solvent of the β -sodium glycerophosphate solution is water, the mass fraction of the water is 10 percent, and the NaHSO₃The mass fraction of the solution is 3 percent, after β -sodium glycerophosphate solution is dripped, equal volume of NaHSO is dripped₃And (3) solution.

The invention also provides the catechol functionalized chitosan/oyster peptide temperature-sensitive hydrogel prepared by the preparation method of the catechol functionalized chitosan/oyster peptide temperature-sensitive hydrogel.

The invention also provides application of the catechol functionalized chitosan/oyster peptide temperature-sensitive hydrogel in preparation of a wound repair medicament.

Compared with the prior art, the invention has the following beneficial effects:

(1) CS is grafted and modified by HCA, a new hydrophilic group is introduced, the water solubility of the CS is improved to more than 5g/100mL, the application range of CS-C is greatly expanded, and the results of antioxidant performance analysis show that the CS-C can well eliminate DPPH free radicals and hydroxyl free radicals, and the results of cell activity, apoptosis and erythrocyte hemolysis rate detection show that the CS-C has good biocompatibility.

(2) CS-C and beta-GP are mixed according to a certain proportion to prepare the CS-C/beta-GP temperature-sensitive hydrogel, the gel temperature of the temperature-sensitive hydrogel is 37 ℃, and the gel time is 12-18 min. The hydrogel has strong adhesion property, can be adhered to wound tissues for a long time, avoids secondary infection caused by falling off, and is beneficial to wound repair. The microstructure of the freeze-dried hydrogel presents a porous network structure, which is beneficial to free passage of water and small molecular drugs, healing of wounds, drug release and the like.

(3)NaHSO₃The introduction of the (3) improves the temperature sensitivity of the hydrogel, so that the hydrogel can be quickly gelated only by β -GP with lower concentration at physiological temperature, overcomes the potential toxicity possibly caused by β -GP with high concentration, and simultaneously, NaHSO₃The introduction of (2) shortens the gel time to 3-7 min.

(4) The oyster peptide is small molecular oligopeptide with the molecular weight of 200-800Da and prepared by taking oyster meat as a raw material through processing such as enzymolysis, separation, refining, drying and the like, and consists of 2-6 amino acids; the oyster peptide can be quickly absorbed by human body, not only contains rich protein, vitamins, trace elements and taurine with proper proportion, but also contains various special nutrient components of marine life, and the oligopeptide has higher bioactivity function.

(5) The M-OP prepared by the invention is in a complete spherical shape, the particle size is 1-10 mu M, the encapsulation rate is 72.8%, the drug loading rate is 11.9%, the in vitro release behavior is good, and the cumulative release rate reaches 70.8%.

(6) The composite temperature-sensitive hydrogel CS-C/OP/beta-GP and the composite temperature-sensitive hydrogel CS-C/M-OP/beta-GP have porous structures, uniform pore sizes, water absorption rate of more than 550 percent, capability of keeping a moist microenvironment, no cytotoxicity, good biocompatibility, capability of promoting migration of L929 cells, hemolysis rate of less than 5 percent and accordance with the national safety standard.

(7) Experiments such as whole blood coagulation index, in-vitro coagulation time, platelet adhesion, erythrocyte adsorption and the like show that the CS-C/OP temperature-sensitive hydrogel can promote blood coagulation; the mouse liver hemostasis and tail-cutting hemostasis model shows that the CS-C/OP hydrogel can accelerate hemostasis and has the same hemostasis effect as the commercially available gelatin sponge; the CS-C/OP temperature-sensitive hydrogel can keep the moist environment of the wound surface wound, accelerate the healing speed of the wound, shorten the healing time, relieve the aggregation of various inflammatory cells of the wound surface wound, accelerate the generation of collagen fibers and new blood vessels to promote the synthesis of total protein in granulation tissues and the repair of the wound, and simultaneously proves the effect of accelerating the healing of the wound surface and the expression quantity of Ki-67 and VEGF.

Drawings

FIG. 1 is a SEM analysis chart of the freeze-dried hydrogels CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP prepared in example 1 of the present invention;

FIG. 2 is a water absorption performance graph of the lyophilized hydrogel CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP prepared in example 1 of the present invention;

FIG. 3 is a graph showing the effect of different concentrations of CS-C/β -GP on L929 cell viability prepared in example 1 of the present invention;

FIG. 4 is a graph showing the effect of different concentrations of CS-C/OP/β -GP on L929 cell viability prepared in example 1 of the present invention;

FIG. 5 is a graph showing the effect of different concentrations of CS-C/M-OP/β -GP on L929 cell viability prepared in example 1 of the present invention;

FIG. 6 shows the microscopic image of the live/cell staining of the lyophilized hydrogels CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP prepared in example 1 of the present invention;

FIG. 7 shows the effect of CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP on apoptosis of L929 in the lyophilized hydrogel prepared in example 1 of the present invention;

FIG. 8 is a model of L929 cell scratch test in cell migration test for the lyophilized hydrogels CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP prepared in example 1;

FIG. 9 is a graph showing migration of cells after the freeze-dried hydrogels CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP samples prepared in example 1 are processed for scratch models for 12h, 24h, 36h and 48 h;

FIG. 10 is a graph showing the hemolysis rate of CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP samples of the lyophilized hydrogel prepared in example 1;

FIG. 11 is a microscopic photograph of erythrocytes from samples CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP of the lyophilized hydrogel prepared in example 1;

FIG. 12 is a graph showing the results of hemolysis experiments on samples CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP of the lyophilized hydrogel prepared in example 1;

FIG. 13 is a photograph of the healing of the skin wound of mice dressed with samples of the lyophilized hydrogels CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP prepared in example 1;

FIG. 14 shows the percentage of wound healing in mice coated with samples of lyophilized hydrogels CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP prepared in example 1;

FIG. 15 shows the total protein concentration of the wound in each experimental group of the lyophilized hydrogel CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP sample dressings prepared in example 1;

FIG. 16 shows the TNF- α and IL-6 expression profiles of wounds in each experimental group when samples of the lyophilized hydrogels CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP prepared in example 1 are applied;

FIG. 17 shows the samples of lyophilized hydrogel CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP prepared in example 1, dressing H & E staining to evaluate pathological changes in skin in each experimental group;

FIG. 18 shows Masson staining results of various groups of samples of the lyophilized hydrogel CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP sample dressings prepared in example 1;

FIG. 19 is the mean optical density of collagen content in the treated wound surface of each group of sample dressings from the samples CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP of the lyophilized hydrogel prepared in example 1;

FIG. 20 shows that the lyophilized hydrogel samples prepared in example 1, CS-C/β -GP, CS-C/OP/β -GP and CS-C/M-OP/β -GP, the skin wound surface VEGF (A) and Ki-67(B) of each group were applied by immunohistochemical staining;

FIG. 21 is the mean optical density of VEGF (A) and Ki-67(B) expression in the wound surface treated with each set of sample dressings from samples CS-C/β -GP, CS-C/OP/β -GP, and CS-C/M-OP/β -GP of the lyophilized hydrogel prepared in example 1;

fig. 22 is a schematic diagram of hydrogel bonding strength test in effect verification 3.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.