Characteristics of methacrylated gelatin hydrogel incorporating proteins

Hyung Seok Kang, Jin-Bae Jeong, Jong-Duk Kim

Dept. of Chemical Eng. Korea Advanced Institute of Science and Technology

단백질을 포집한 기능성 젤라틴 하이드로젤의 특성

강형석, 정진배, 김종득

화학공학과, 한국과학기술원

Introduction

Many studies about gelatin which is hydrolysis product of skin structural protein have been conducted to use carries of protein and peptide drugs.

So far, gelatin has been formulated into microparticles or nanoparticles by w/o emulsion or spray drying and various kinds of membranes[1]. Biocompatibility and biodegradability of gelatin as hydrogel induce studies to apply the material as promising protein carries. Also, its amphoteric characteristics made it possible to modify some residues into functional groups to avoid activity loss of proteins. Most of studies used crosslinking of lysine residues to formulate protein carries, as a result, the method induced activity loss. Other studies used sorption method to load proteins into crosslinked gelatin carries that resulted in low amount of loading of proteins and fast release rate[2-4].

In this study, amine groups in gelatin backbone are substituted to double bonds and functionalized gelatin is formulated into hydrogels with proteins[5]. In order to simulate swelling behavior in gastric and intestinal condition, swelling of hydrogels are monitored in acidic medium with pepsin and basic medium with trypsin.

Theory

Gelatin is functionalized by reacting with glycidyl methacrylate to add double bonds to side group of gelatin. Proteins can be effectively incorporated into hydrogel formulation of methacrylated gelatin without reducing activity loss due to reaction between adjacent double bonds around protein molecules. The rate of protein release can be controlled by modulating degree of substitution of amine groups and initiator concentration.

Experimental

Functionalization of gelatin

Gelatin(Sigma, type B, 225 bloom) was methacrylated by reacting with glycidyl methacrylate in phosphate buffer(pH 7.4, 100mM) for 24 hours. Amount of lysine residues in gelatin is quantitated and equimolar amount of glycidyl methacrylated was added to gelatin solution. The reaction was ceased by adding glycine solution. After 24 hours, the solution was extensively dialyzed with distilled water for 3 days. The dialyzed solution was finally lyophilized.

Hydrogel preparation

Hydrogels were prepared by free radical initiation of methacrylated gelatin by ammonium persulfate(Aldrich) and N,N,N',N'-tetramethylenediamine(Aldrich) with proteins in buffer solution. Lysozyme(Sigma), bovine serum albumin(Sigma) and lipase(Sigma) were used as model proteins. The reaction is conducted in 37℃ for 1 hour and washed several times with distilled water. Table I shows codes and composition of reaction mixture.

Table I. Codes and composition of hydrogel formulation

	MGL	Lipase	TEMED	APS	Buffer
MGL-L1	400mg	100mg	25 □l	1.25mg	2.4ml
MGL-L2	400mg	100mg	25 □l	2.50mg	2.4ml
MGL-L4	400mg	100mg	25 □l	5.00mg	2.4ml

Protein release and swelling behavior

The release study of proteins from hydrogels was conducted in phosphate buffer(pH 7.4 10mM). Amount of released protein was quantitated by Bradford method(Bio-Rad) at 595 nm. Swelling ratio of hydrogels was monitored by weighing swollen hydrogels. Gastric medium is simulated to 10mM buffer solution of pH 2 with pepsin(Sigma, 2260U), and intestinal medium is simulated to 10mM buffer solution of pH 7,4 with trypsin(Sigma, 1300U).

Result and Discussion

The natural gelatin was functionalized by introducing double bonds to side groups. Most of lysine groups were substituted to double bonds by TNBS method. Functionalized gelatin(MGL) was formulated into cylindrical hydrogel with proteins by  conducting reaction in 1 ml disposable syringe.

Figure 1 shows swelling behavior of series of hydrogel with lipase as a function of initiator concentration. As increasing initiator concentration, the swelling ratio decreased because average molecular weight of cross-links decrease. Table 2 shows initial swelling rate and equilibrium swelling ratio calculated by second order swelling kinetics that is effective in rapid swelling hydrogel in water. By increasing initiator concentration, equilibrium swelling ratio not only decreased, but initial swelling rate was reduced due to same reason.

Table 2. Calculation of initial swelling rate and equilibrium swelling ratio

	MGL-L1	MGL-L2	MGL-L4
Initial swelling rate	4.84	4.09	3.99
Equilibrium swelling ratio (swollen wt/dried wt)	24.4	19.6	15.4

Figure 2 and 3. show swelling behavior of MGL-L series in gastric  simulation medium. In medium of high concentration of pepsin, hydrogels were rapidly degraded while hydrogels showed transient maximum swelling with time in medium of low concentration of pepsin. Crosslinking density brought about little effect on swelling ratio in gastric medium. Regardless of pepsin concentration, hydrogels were surface-eroded. This phenomena may be caused by the fact that amino groups of lysine residues were almost substituted to non-charged groups and amphoteric gelatin was converted into anionic functionalized gelatin as a result. The fact means that MGL became deswollen in acidic medium and diffusion of pepsin enzyme into hydrogel became difficult, therefore, pepsin enzyme can mainly hydrolyze hydrogel surface.

Figure 4 shows swelling behavior of MGL-L series in intestinal simulation medium. Relatively lower concentration of trypsin compared to pepsin could effectively degrade hydrogels. In basic medium, carboxylic acids of MGL are ionized so that hydrogels become rapidly swollen and trypsin enzyme can easily diffuse through hydrogel pores and hydrolyze hydrogels faster than pepsin enzyme do. Therefore, hydrogels in intestinal medium showed bulk erosion. Figure 5 shows swelling ratio of hydrogels after 1 hour as a function of trypsin concentration. Crosslinking density caused little effect on swelling ratio as shown figure.

In conclusion, various proteins were incorporated into methacrylated gelatin hydrogels. The swelling ratio could be controlled by initiator concentration so that protein release rate could also be controlled. In gastric medium,  hydrogels showed surface erosion due to deswelling of anionic hydrogel in acidic condition, but hydrogels showed bulk erosion in intestinal medium due to rapid swelling. The crosslinking density can control swelling ratio and protein release rate, and but erosion in both medium does not depend on crosslinking density.

References

1. X. S. Wu et al, 23rd Annual Meeting of the Society for Biomaterials, 199(1997)

2. S. Downes et al, J. Materials Sci.: Materials in Medicine, 5, 819-823(1994)

3. Y. Ikada et al, Advanced Drug Delivery Reviews, 31, 287-301(1998)

4. K.P.Rao, J. Biomater. Sci. Polymer Edn, 7,623-645(1995)

5. K. Park, Biomaterials, 9, 435-441(1988)

그림 2 Swelling behavior of MGL-L hydrogel series (pepsin concentration : 1 mg/ml, incubation in 10ml pH 2 buffer)

그림 1 Swelling behavior of MGL-L hydrogels as a function of initiator concentration

그림 4 Swelling behavior of MGL-L hydrogel series (trypsin concentration : 0.01 mg/ml, incubation in 10ml pH 7.4 buffer)

그림 3 Swelling behavior of MGL-L hydrogel series (pepsin concentration : 0.1 mg/ml, incubation in 10ml pH 2 buffer)