Effects of solvent and concentration on the encapsulation of curcumin on Thai silk fibroin and gelatin hydrogels

By: William S. Bresnihan

A research report for the Chemical Engineering Department at Chulalongkorn University as part of the summer exchange program of Clemson University's Bioengineering

Department

Advisors: Dr. Siriporn Damrongsakkul

Dr. Sorada Kanokpanont

Dr. Juthamas Ratanavaraporn

Dr. Frank Alexis

Chulalongkorn University

Department of Chemical Engineering and Biomedical Engineering Program

Thailand

Clemson University

Department of Bioengineering

United States of America

2015

Abstract

Hydrogels are a growing peak of interest in medical applications especially in drug delivery. Hydrogels can be used as a carrier to load therapeutic drugs for diffusion into the body. This study examined the effects of silk fibroin and gelatin hydrogels when loaded with curcumin, an anti­cancer herbal compound, along with the effects elicited by both the concentration of curcumin in solution and the type of solvent (mixed ethanol and water) used to load curcumin onto the hydrogels. The loading and encapsulation efficiency for hydrogels, at each concentration of curcumin solution, and the type of solvent used were evaluated to determine the optimal parameters for application. From the results, no definitive conclusions could be drawn regarding the effects of solvent concentration or hydrogel composition on encapsulation or loading efficiencies of curcumin. The only concrete observation was that 4 mg/ml is the maximum concentration of curcumin that can be used in solution.

Introduction

For years, the use of hydrogels has been an area of acute interest for investigating drug delivery. Hydrogels are a material composed of polymeric constituents, primarily water.3 Their hydrophilic structure allows them to hold large amounts of water, among other water soluble chemicals, in their three­dimensional structure.3 They are optimal for biomaterial applications as their chemical composition is compatible with physiological conditions, particularly in the case of drug delivery. Hydrogels are desirable due to their biocompatibility, biodegradability, and their efficient drug release profiles. The drawbacks of some hydrogels however are the rate of degradation and the directly proportional rate of drug diffusion, whether they are too quick or too slow. .

Gelatin has been a popular choice for hydrogels because it is non­cytotoxic, biodegradable, easy to process, inexpensive, and efficient for drug delivery.1 Gelatin hydrogels do however degrade very quickly in a physiological environment due to its solubility in water.1 To combat this, certain ratios of silk fibroin (SF) have been introduced into gelatin based hydrogels to slow the rate of degradation.1 Silk is a natural protein that is biosynthesized from various creatures such as silkworms, spiders, and mites.2 Silk consists of two different proteins; fibroin, a core filament protein that gives silk its unique properties, and sericin, a glue like protein that binds strands of fibroin together to form a cacoon.2 Silk fibroin has been shown to demonstrate unique biomechanical properties. It is lightweight, strong, tough, and elastic. It also elicits a weak inflammatory reaction and has a slow biodegradation rate.1 In this study, silk

fibroin and gelatin hydrogels at the weight blending ratios of 50/50, and 0/100, are used as a carrier for the controlled release of curcumin.

The herbal compound curcumin is employed as this chemical has been shown to exemplify inhibiting effects on proliferating cancer cells1. Curcumin is a component of the herb Curcuma longa,1 classified as a hydrophobic molecule. It exhibits antioxidant, anti­inflammatory, antiviral, antibacterial, antifungal, and anticancer properties.1 Because of these characteristics, curcumin has elicited therapeutic interest for inflammation related diseases, as well as cancer and diabetes.1 However, the therapeutic value of curcumin is limited due to its water insolubility, rapid metabolism, and short half­life.1

The parameters to be investigated in this study include the ratios of solvent of ethanol to water (100:0, 90:10, and 80:20) that curcumin is dissolved in prior to administration on to the hydrogels. The solvent type is important; as curcumin dissolves more readily in ethanol due to its hydrophobicity, the amount of water in each type of solvent can drastically affect the encapsulation and the loading efficiency. The encapsulation efficiency is vital to analyze, as it shows how much of the compound is actually being absorbed or wasted in a given volume of solvent. The loading efficiency is important in a medical application sense, in that doctors administering the drug need to know exactly how much of the compound can be loaded into a given dosage.

It is hypothesized that a solvent ratio of 80:20 for ethanol to water will exhibit the highest encapsulation and loading efficiency.

Materials

Chemicals:

Thai silk cocoons raced “Nangnoi Srisaket 1” from Nakornratchasrima Province, Thailand

Type A gelatin, Nitta gelatin, Japan

Curcumin

sodium carbonate lithium bromide de­ionized water curcumin

gluteraldehyde

glycine

ethanol (99.9%) Dimethyl Sulfoxide Protease type XIV

Lab equipment:

oven microplate­reader centrifuge

freeze­dryer Methods

Preparation of Silk Fibroin Solution

The first step in the prepared approach is to degum the silk fibroin. 40 grams of yellow Bombyx Mori silk cocoons were boiled in 0.02M sodium carbonate to remove sericin. Afterwards the degummed silk was rinsed extensively with deionized water to remove any excess sodium carbonate. After air drying, 8 grams of the SF was dissolved in 24 grams of 9.3 M lithium bromide solution at 60°C. A dialysis was then performed using cellulose tubes (MWCO = 12 kDa) to remove the lithium bromide. After dialysis, the SF solution was centrifuged at 9000 rpm to remove residual impurities. The supernatant of the centrifuged SF solution was then collected. The concentration of the SF aqueous solution was calculated to be 7.68%. The SF solution was then stored in a refrigerator at 4°C. It can be stored at this temperature for 2­3 weeks before gelation occurs.

Fabrication of Hydrogels

The next step is the preparation of the hydrogels. Two SF/G hydrogels with the weight ratios of SF/G 50/50/ and 0/100 were prepared. For the SF/G 50/50 hydrogel, a 5% weight gelatin solution was needed. To do so, 5 grams of type A gelatin was mixed with 60 ml of DI water. A 5% weight (wt) SF solution was prepared from the stock SF solution prepared earlier in the procedure. Afterwards, 24 ml of the 5% wt SF solution was then mixed with 24 ml of 5% gelatin solution. These steps were necessary to insure that the ratio of gelatin to SF was 50/50. Next the gluteraldehyde (GA) solution was prepared for the cross­linking of the gelatin and SF. For the GA solution, 1.152 ml of GA was mixed with 34.848 ml of DI water. When both solutions were homogeneously mixed, 12 ml of GA solution was added to the SF/G solution and stirred slowly. Lastly, 20 ml each of the resulting solution was poured into 3 teflon molds and covered with 3 layers of aluminum foil. Holes were poked at random in the aluminum foil to allow for vaporization during the freeze­drying process. For the G100 hydrogel samples, 48 ml of

the respective aqueous solution was mixed with 12 ml of the GA solution, and stirred slowly for 1 minute. Afterwards, 3 teflon molds were each filled with 20 ml of the mixed solution and covered with 3 layers of aluminum foil. Holes were perforated in the foil as previously mentioned.

The solutions were then placed in a freezer at ­20°C for 24 hours, then transferred to a ­40°C freezer for 24 hours, followed by lyophilization (freeze­drying) for 72 hours. Afterwards, the obtained hydrogel samples were washed with a 0.1 M solution of glycine to remove the excess cross­linking agent gluteraldehyde. The samples are freeze­dried again for 72 hours. After the final freeze­drying session, the hydrogels can then be cut into cubes with dimensions of 5 mm by 5 mm by 2 mm. Each cube should weigh approximately 5 mg. 30 cubes of each type of hydrogel were prepared in the aforementioned dimensions.

Preparation of Standard Curve for Curcumin

To prepare the standard curve for curcumin, 10 ml of a standard solution were prepared from a 1000 mg/ml stock solution for concentrations of 2, 4, 6, 8, 10, and 12 μg/ml. The concentrations of curcumin in different solvents were separated to avoid confusion, and afterwards 1 ml of each solution at each different concentration was placed in a cell plate, wrapped in aluminum foil, perforated with holes, and allowed to vaporize in a chemical hood for 48 hours. After the allotted time, 1 ml of dimethyl sulfoxide (DMSO) was pumped into each cell plate well to get leftover curcumin back into solution. 100 µl of each concentration of curcumin were pipetted into a 96 well plate and labeled accordingly. 4 samples of each concentration were used to ensure accuracy. The samples were then run through a micro­plate reader to measure the absorbance spectra of each type of solution. The absorbance of each well plate for each ratio solvent at each concentration was recorded at 430 nm. Blank cell plates with pure DMSO were also evaluated to isolate the absorbance spectra of curcumin and the solvent alone.

Loading of Curcumin onto Hydrogels

After the cubes of each type of hydrogels are cut in the appropriate dimensions, the drug loading process can begin. Three separate concentrations of curcumin solutions, 1, 2, and 4 mg/ml, were prepared for each ratio of solvent. For the 4 mg/ml solution using the solvent ratio of 80:20 for ethanol to water, it was clear that the solution had reached its super saturation point and was therefore discarded for any future use in the experiment. After the preparation of curcumin solutions was completed,

3 samples of both G100 and SF/G 50/50 were placed into different microvials corresponding to the 3 different ratio solvents. The samples were then organized according to concentration, hydrogel composition, and solvent ratio. After the completion of this step, 500 µl of each type of curcumin concentration solution and solvent type were placed into the microvials. Afterwards, the microvials were wrapped in aluminum foil to prevent curcumin from reacting to any light and placed in the ­4°C freezer overnight to allow for vaporization.

Figure 1. G100 hydrogels in the respective curcumin solution concentrations.

Figure 2. SF/G : 50/50 hydrogels in each respective curcumin solution concentration.

Protease digestion of curcumin loaded hydrogels _

Following the overnight vaporization step, all the hydrogels were taken out of the microvials containing the curcumin solutions and placed into new microvials. The hydrogels were then minced at random to increase the surface area so that the enzyme protease type XIV could elicit a stronger and quicker reaction. Next the protease solution was created at a concentration of 2 mg/ml by dissolving 120 mg of protease in 60 ml of phosphate buffer saline (PBS) with a pH of 7.4 while heating and stirring. Three blank hydrogel samples of both G100 and SF/G 50/50 that contained no curcumin were prepared as well to see how the protease alone would react with each type of gel.

Lastly, 1 ml of the 2 mg/ml protease solution was pipetted into each microvial containing a hydrogel. The samples were then wrapped in aluminum foil to protect them from light, and incubated overnight at 37°C, as this is the physiological temperature. Protease drives the breakdown of peptide chains, and so is necessary to break down the hydrogel into smaller fragments so it can more easily dissolve in solution.

After a night in the protease solution, the hydrogels were transferred to a freeze­dryer set at ­80°C and allowed to sit for 48 hours. The different minced hydrogels, following dissolution via protease, in each concentration and solvent can be seen in Figures 1 and 2.

Dissolution of hydrogel fragments with Dimethyl Sulfoxide

One ml of DMSO was added to each microvial containing a hydrogel sample following the removal of the samples from the freeze­dryer. This step was necessary to draw curcumin out of the hydrogel fragments and into solution. Each sample was then centrifuged at approximately 10,000 rpm for 1 minute. Afterwards, 20 µl of the each curcumin solution along with 180 ml of DMSO were pipetted into separate wells on a 96 well cell­plate. The plate was then run through the microplate­reader to assess the optical density of each sample at 430 nm.

Upon obtaining the optical density data from the microplate­reader, the equations generated from the standard curves of curcumin for each type of solvent were used to calculate the concentration of curcumin that was absorbed by each type of hydrogel in each respective solvent. After these concentrations were calculated, both the encapsulation efficiency and loading efficiency were calculated, according to the equations bellows, then averaged to get an idea of how the different efficiencies compared to one another for each type of hydrogel dissolved in each solvent.

Encapsulation Efficiency = (weight of curcumin encapsulated in hydrogel) μg(total weight of curcumin dissolved in solution) μg

Loading Efficiency = (weight of hydrogel sample) μg(weight of curcumin encapsulated in hydrogel) μg

Results and Discussion

The parameters to be investigated during this experiment were as follows: comparing the encapsulation and loading efficiencies for G100 versus SF/G 50/50 hydrogels, observing the effects of loading hydrogels with a 100%, 90% and an 80% ethanol solvent on encapsulation and loading efficiencies, and to analyze the changes in encapsulation and loading efficiency as the concentration of curcumin in solution was increased.

Figure 3. The encapsulation efficiency of curcumin in gelatin (G100) hydrogels at various curcumin concentration (1, 2, and 4 mg/ml) and solvent type (ethanol:water = 100:0 (E100), 90:10 (E90) and 80:20(E80)).

Figure 4. The encapsulation efficiency of curcumin in silk fibroin and geltain (SF/G 50/50) hydrogels at various curcumin concentrations (1 mg/ml, 2 mg/ml, and 4 mg/ml) and solvent type (ethanol:water = 100:0 (E100), 90:10 (E90), and 80:20 (E80)).

Figure 3 shows the encapsulation efficiency for the G100 hydrogels. The only trend that can be observed from Figure 3 is that a curcumin concentration of 4 mg/ml results in a higher encapsulation efficiency. Figure 4 shows the encapsulation efficiency of SF/G 50/50 hydrogels, and the same trend can be seen ,e.g. a 4 mg/ml curcumin solution concentration results in a higher encapsulation efficiency. This trend can be seen for both types of hydrogel across each type of solvent. However, the standard deviation represented by the error bars on the graph is very high, so this is difficult to state definitively. The solvent type did not have a noticeable effect on the encapsulation efficiency. G100 seems to have a higher encapsulation efficiency at a curcumin solution concentration of 4 mg/ml, but again the standard deviation across each sample is too large to say definitely that G100 is superior in that regard.

Figure 5. Loading efficiency of curcumin in gelatin (G100) hydrogels in various curcumin concentration (1 mg/ml, 2 mg/ml, and 4 mg/ml) and solvent type (ethanol:water = 100:0 (E100), 90:10 (E90), and 80:20 (E80)).

Figure 6. Loading efficiency of silk fibroin and gelatin (SF/G 50/50) hydrogels at various curcumin concentrations (1 mg/ml, 2 mg/ml, and 4 mg/ml) and solvent type (ethanol:water = 100:0 (E100), 90:10 (E90), and 80:20 (E80)).

Figure 5 depicts the loading efficiency observed for the G100 hydrogel samples. The only noticeable trend is that the 4 mg/ml curcumin solution concentration samples exhibit a higher loading efficiency. Figure 6 shows the loading efficiency for SF/G 50/50 hydrogels, and the same trend can be observed in that a higher curcumin concentration results in a higher loading efficiency. However, the room for error is too high to draw any definitive conclusions. No obvious trend is seen across each solvent type. As was the case with the encapsulation efficiency, G100 seems to have a higher loading efficiency at a curcumin concentration of 4 mg/ml, but the standard deviation prevents any solid conclusions from being drawn.

Conclusion and Future Studies

Per the original parameters of investigation in this experiment, no definitive conclusions can be drawn, other than the fact that 4 mg/ml is the maximum concentration that can be used for curcumin in solution (as it reaches it saturation point in both the E100 and E90 solvents. In the E80 solvent, too much water is present and as a result prevents the curcumin from fully dissolving. The range of error was too high to draw any conclusions about whether or not one hydrogel was superior to the other, which solvent would be best to use to dissolve curcumin in and subsequently load onto the hydrogels, or which concentration of curcumin is optimal for encapsulation and loading efficiencies. The high source of error comes from a high standard deviation in

the optical density between samples, which in turn results from the few number of samples used per set of hydrogels and each respective solvent type.

To minimize these effects, in the future more samples should be evaluated to decrease the standard deviation between each data set. Also, in future experiments, it could be helpful to optimize the volume of curcumin solution loaded onto each hydrogel as it could have a significant impact on both the encapsulation and loading efficiencies.

References

1. Ratanavaraporn J, Kanokpanont S, Damrongsakkul S. J of Materials Sci: Mater Med, 2014, 25 p 401­410

2. Kaewprasit K, Promboon A, Kanonkpanont S, Damrongsakkul S. J of Biomedical Materials: Applied Biomaterials Part B 2014,102B, p 1639­1647

3. Enas M. Ahmed. 2013. J of Advanced Research, 2015, Volume 2, p 105­121

Acknowledgements

This research experience was supported by both Clemson and Chulalongkorn University, and I wish to thank Dr. Siriporn Damrongsakkul, Dr. Juthamas Ratanavaraporn, Dr. Sorada Kanokpanont, and Dr. Frank Alexis.