WO2021045607A1

WO2021045607A1 - A sustained release hydrogel composition

Info

Publication number: WO2021045607A1
Application number: PCT/MY2020/050080
Authority: WO
Inventors: Ju Yen FU; Doryn Meam Yee Tan; Lay Hong CHUAH
Original assignee: Malaysian Palm Oil Board
Priority date: 2019-09-03
Filing date: 2020-09-03
Publication date: 2021-03-11
Also published as: KR20220075345A; MY201184A

Abstract

The present invention relates to a hydrogel composition for sustained release of tocotrienol comprising chitosan; gelatin; β-glycerophosphate; and noisome-encapsulated tocotrienol.

Description

A SUSTAINED RELEASE HYDROGEL COMPOSITION

FIELD OF INVENTION

The present invention relates to a hydrogel composition that provides controlled release of a therapeutic substance. Particularly, the hydrogel formed therefrom possesses thermo-responsive property and is suitable for subcutaneous injection.

BACKGROUND OF THE INVENTION

Chronic inflammation is a silent killer as they are stimulated by tissue damage that subsequently triggers vicious cycle. Numerous studies have been conducted in order to find suitable anti-inflammatory drug to treat inflammation. Synthetic drugs such as nonsteroidal anti-inflammatory drugs are however unable to offer prevention or enhance therapeutic responsiveness in patients. There are also findings showing that these drugs tend to cause side effects in the patients. Alternative anti-inflammatory medication is therefore highly desirable.

Tocotrienol is a unique form of vitamin E with unsaturated side chains. Its antioxidant activity is 50 times more potent than tocopherol. In addition, the anti-inflammatory effect of the tocotrienol leads to 3 major health benefits including protection against neurodegeneration, metabolic diseases and cancer. The main mechanism of tocotrienol in the inflammation pathway is through the downregulation of Nf-kB transcription factor.

The incorporation of tocotrienols in various food products is prevalent since 2010 as it has obtained “generally recognized as safe” status from the United States Food and Drug Administration (FDA). However, similar to most lipid compounds, the tocotrienol suffers from low bioavailability, ranging from 10-30% in animal study. Thus, an efficient delivery system is critically needed to enable successful introduction of the tocotrienol into the body and improves its efficacy in the body.

Hydrogel is a delivery system made up of entangled polymer networks that have the ability to trap large amount of water. Its highly porous structure permits loading of bioactive compounds into the gel matrix, acting as a depot system for controlled release system. While the bioactive compounds are gradually eluted from the hydrogel, a large amount of the compounds can be maintained at the surrounding tissues over a prolonged period of time. Chitosan and gelatin are among the common natural polymers for forming the hydrogels. The hydrogels made from these polymers are generally biocompatible, biodegradable and deformable.

Accordingly, the present invention provides a hydrogel composition and method for preparing a delivery system for the controlled release of tocotrienol.

SUMMARY OF INVENTION

The main aspect of the present invention is to provide a hydrogel composition for sustained release of tocotrienol, in which the hydrogel produced possesses thermo- responsive properties. The thermo-responsive hydrogel is able to convert from liquid state to solid gel at room temperature upon subcutaneous injection.

Another aspect of the present invention is to provide a hydrogel composition for sustained release of tocotrienol, in which the tocotrienol is encapsulated in niosome prior to mixing in the composition.

At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention is a hydrogel composition for sustained release of tocotrienol comprising chitosan; gelatin; b- glycerophosphate; and noisome-encapsulated tocotrienol.

In accordance with a preferred embodiment, the chitosan present in an amount of 2- 3% (w/v) of the composition.

In another preferred embodiment, the b-glycerophosphate present in an amount of 6- 12% (w/v) of the composition.

Preferably, the hydrogel composition further comprising 1-2% (w/v) of gelatin.

Preferably, the hydrogel composition further comprising 0.1-0.5% (w/v) of calcium chloride.

Advantageously, the particle size of the noisome-encapsulated tocotrienol is 170-190 nm.

It is also preferable that the noisome-encapsulated tocotrienol has a zeta potential of - 80 to -60 mV.

The present preferred embodiment of the invention consists of novel features and a combination of parts hereinafter fully described and illustrated in the accompanying drawings and particularly pointed out in the appended claims; it being understood that various changes in the details may be effected by those skilled in the arts but without departing from the scope of the invention or sacrificing any of the advantages of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction and operation and many of its advantages would be readily understood and appreciated.

Figure 1 shows graph plot of G’ (square) and G” (triangle) at 37 °C over 30 minutes of (a) blank hydrogel and (b) hydrogel comprising niosome-encapsulated tocotrienol.

Figure 2 shows graph plot of G’ (square) and G” (triangle) vs temperature of (a) blank hydrogel and (b) hydrogel comprising niosome-encapsulated tocotrienol.

Figure 3 shows viscosity and flow behaviour of a freeze dried (a) blank hydrogel and (b) hydrogel comprising niosome-encapsulated tocotrienol at 25 °C.

Figure 4 shows results derived from an amplitude sweep test. G’ (square) and G” (triangle) were calculated from shear strain 0.001-100% for (a) blank hydrogel and (b) hydrogel comprising niosome-encapsulated tocotrienol.

Figure 5 shows SEM micrographs of hydrogel (b) at a magnification of a) 500x and b) 5,000x.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawings. However, it is to be understood that limiting the description to the preferred embodiments of the invention and to the drawings is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.

The present invention relates to a hydrogel composition for sustained release of tocotrienol comprising chitosan; gelatin; b-glycerophosphate; and noisome- encapsulated tocotrienol.

Chitosan is a linear polysaccharide composed of randomly distributed P-(l-4)-linked D-glucosamine and N-acetyl-D-glucosamine units. In one embodiment, the chitosan may be produced by deacetylation of chitin, a structural element in exoskeleton of crabs and shrimps. Preferably, the chitosan is present in an amount of 2-3% (w/v) of the hydrogel composition.

Polyelectrolyte such as gelatin is preferably added into the chitosan-based hydrogel to improve the gel strength. It is preferable that the hydrogel composition comprises 1- 2% (w/v) of gelatin. Negative charge moieties of the gelatin can bind with the chitosan via ionic interactions to form polyelectrolyte complexes. In addition, the hydrophilic gelatin also forms hydrogen bonding with water molecules and subsequently establishes an aqueous layer to prevent the aggregation of the chitosan chains at room temperature.

Ionic neutralization method using polyol phosphate salts such as b-glycerophosphate is preferably employed for forming in-situ thermo-responsive hydrogel. In accordance with a preferred embodiment, the b-glycerophosphate present in the hydrogel composition is within the range of about 6-12% (w/v).

When dissolved in water, negative charges from phosphate groups of the b- glycerophosphate neutralize the positive charges of the chitosan amino groups, forming ion complexes and reduce electrostatic repulsion of the chitosan polymer chains. At room temperature, the polyol portions of the b-glycerophosphate maintain solubility of the chitosan by forming hydration layers between the chitosan polymer chains owing to hydrogen bonding with surrounding water molecules. As temperature increases, proton transfer from the chitosan to the b-glycerophosphate occurs leading to reduced electrostatic linkages. Besides, increased temperature induces motion of water molecules, reducing hydrogen bonding between the b-glycerophosphate with the water molecules. When temperature rises above the transition temperature, the chitosan polymer chains in the solution precipitate in the form of gel and gelation occurs, as the hydration layers are too weak to prevent interactions between chitosan polymer chains.

Preferably, the hydrogel composition further comprising further comprising 0.1-0.5% (w/v) of calcium chloride. The calcium chloride is preferably added into the composition to prevent aggregation of the chitosan polymer chains at room temperature and maintain the solubility of freeze-dried hydrogel powder.

Pursuant to the preferred embodiment, the tocotrienol in the hydrogel composition is encapsulated in niosome. In a preferred embodiment, the average particle size of the niosome-encapsulated tocotrienol is about 170-190 nm. In another preferred embodiment, the zeta potential of the niosome-encapsulated tocotrienol is about -80 to -60 mV respectively. Encapsulation of tocotrienol in the nano-sized niosome can greatly improve the efficacy of the tocotrienol in anti-oxidant and anti-inflammatory activities.

Advantageously, a thermo-responsive hydrogel comprising the tocotrienol is produced from the composition aforementioned. In a preferred embodiment, the thermo- responsive hydrogel is preferably stored in the form of freeze-dried powder at about 4 °C in order to conserve its thermo-responsive property.

According to a preferred embodiment, the thermo-responsive hydrogel aforementioned can be administered to a subject via subcutaneous injection.

EXAMPLES

The present invention can be further understood through consideration of the following non-limiting Example.

Example 1 Preparation method

1. Niosome-encapsulated tocotrienol was prepared using the method as described in PI 2015700198.

2. Chitosan (Protasan HC1, medium molecular weight, 2.5 %) solution was prepared in deionized water at 4 °C for about 60 minutes.

3. b-glycerophosphate (10%) solution was prepared in deionized water at about 4 °C for about 60 minutes.

4. Gelatin (2%) solution was prepared in deionized water.

5. Calcium chloride (0.1%) solution was prepared in deionized water.

6. The gelation solution was added to chitosan solution and stirred for about 10 minutes at room temperature.

7. Next, the calcium chloride solution was added to the mixture and stirred for about 10 minutes at room temperature.

8. Subsequently, the b-glycerophosphate was added to the mixture and stirred for about 10 minutes at room temperature.

9. 2 mL of the niosome-encapsulated tocotrienol was added and stirred for about 30 minutes at room temperature.

10. Final mixture was freeze-dried for about 24 hours.

11. Prior to use, the freeze-dried powder was reconstituted with 3 mL of deionized water.

12. Hydrogel is formed upon incubation at about 37 °C. Example 2

Determination of gelation time

Gelation time of the hydrogel was determined using inverted tube method. Briefly, about 3 mL of reconstituted hydrogels were added to 10 mL test tubes. Two types of hydrogels were prepared for the analysis, in which the (a) first hydrogel was a blank hydrogel and the (b) second hydrogel was a hydrogel comprising the niosome- encapsulated tocotrienol.

The test tubes were immersed in water bath which has been pre-warmed at about 37 °C. At every 30 seconds interval, the test tubes were inverted to observe flowability of the hydrogel samples. The gelation time of the hydrogels after addition of the niosome-encapsulated tocotrienol were recorded at about 2 minutes.

Rheological measurement of the gelation time was also measured using oscillatory time sweep test at 37 °C. The rheology measurements were conducted on a modular compact rheometer MCR 302 (Anton Paar, Austria), equipped with spindle CP50-1. As shown in the Figure 1, the G’ value of the hydrogel (b) increased sharply between about 1.5 to 2 min of incubation at 37 °C. The results correlated well with inverted tube method.

Example 3

Determination of sol-gel temperature

Rheology measurements were conducted on a modular compact rheometer MCR 302 (Anton Paar, Austria), equipped with spindle CP50-1. Sol-gel temperature of hydrogel (a) and hydrogel (b) were determined using temperature sweep test. The loss modulus (G”) and storage modulus (G’) of each sample were measured at the temperature of 15-45 °C, at heating rate of 2 °C/min. The sol-gel temperature was defined as the temperature where the samples have equal value of G” and G’. As shown in Figure 2, the hydrogel (a) remained stable without much changes in the G’ and G” values despite the increase in temperature. On the other hand, as temperature increased above 37.5 °C, the G’ value of the hydrogel (b) increased significantly indicating conversion to gel state.

Example 4

Determination of gel strength

Rheology measurements were conducted on modular compact rheometer MCR 302 (Anton Paar, Austria), equipped with spindle CP50-1. To determine the flow behavior and viscosity of freeze-dried hydrogels (a) and (b), steady shear sweep test was performed at room temperature (25 °C). The sheer rate was set at a range of 0.001 to 1000 s ¹. As shown in Figure 3, both the hydrogels exhibit shear-thinning behavior at 25 °C, in which their viscosity decreased dramatically above shear rate of 0.1 s ¹.

Amplitude sweep test was conducted to check the linear-viscoelastic (LVE) region of the fully formed hydrogels, describing the rigidity of the hydrogels at rest. LVE region is defined as range of shear strain in which the hydrogels exhibited constant G’. As depicted in Figure 4, the hydrogel (a) showed linear behavior of G’ up to 10% strain while LVE region of hydrogel (b) ranged from 0.001 to 5 % strain. Hence, it was concluded that addition of niosomes decreased the rigidity of the hydrogel.

Example 5 Morphology analysis

Surface morphology of the hydrogels was examined using field emission scanning electron microscope (FESEM). The hydrogel solution was incubated at 37 °C for 1 hour followed by 24 hours of freeze-drying. The samples were examined at 10,000 kV under FESEM (FEI Quata 400F with X-max detector). As shown in Figure 5, the hydrogel (b) generally exhibited asymmetrical porous microstructures. Although the invention has been described and illustrated in detail, it is to be understood that the same is by the way of illustration and example, and is not to be taken by way of limitation. The scope of the present invention is to be limited only by the terms of the appended claims.

Claims

1. A hydrogel composition for sustained release of tocotrienol comprising chitosan; b-glycerophosphate; and noisome-encapsulated tocotrienol.

2. The hydrogel composition according to claim 1, wherein the chitosan present in an amount of 2-3% (w/v) of the composition.

3. The hydrogel composition according to any one of claims 1 to 2, wherein the b-glycerophosphate present in an amount of 6-12% (w/v) of the composition.

4. The hydrogel composition according to any one of claims 1 to 3 further comprising 1-2% (w/v) of gelatin.

5. The hydrogel composition according to any one of claims 1 to 4 further comprising 0.1-0.5% (w/v) of calcium chloride.

6. The hydrogel composition according to any one of claims 1 to 5, wherein the particle size of the noisome-encapsulated tocotrienol is 170-190 nm.

7. The hydrogel composition according to any one of claims 1 to 6, wherein the noisome-encapsulated tocotrienol has a zeta potential of -80 to -60 mV.

8. A hydrogel produced from the composition according to any one of claims 1 to 7 exhibits thermo-responsive property.

9. The hydrogel produced from the composition according to claim 8 is suitable for subcutaneous injection.