Growth factor release from amylopectin hydrogel based on copper coordination

Journal of Controlled Release 56 (1998) 135–148

Growth factor release from amylopectin hydrogel based on copper coordination Yasuhiko Tabata, Yasuhiro Matsui, Yoshito Ikada* Research Center for Biomedical Engineering, Kyoto University, 53 Kawahara-cho Shogoin, Sakyo-ku, Kyoto 606 -8507, Japan Received 12 December 1997; accepted 4 May 1998

Abstract This paper describes a biodegradable hydrogel matrix releasing basic fibroblast growth factor (bFGF) on the basis of protein metal coordination with the protein drug. The biodegradable hydrogel was prepared from amylopectin by its crosslinking with ethylene glycol diglycidyl ether, followed by introduction of diethylenetriaminepentaacetic acid (DTPA) residues for copper chelation. When bFGF was incorporated into the DTPA-introduced amylopectin hydrogel after chelation with Cu 21 , an insignificant amount of bFGF was released from the hydrogel in buffered solution, in contrast to that without Cu 21 chelation. An increased ionic strength in the solution did not affect the bFGF release, indicating the occurrence of coordinate bonding of bFGF to the DTPA-introduced hydrogel through Cu 21 chelation. An implantation study with 125 I-labeled amylopectin hydrogels demonstrated that they underwent degradation in the back subcutis of mice. Cu 21 chelation of hydrogels enabled bFGF to remain in the mouse back for a long time period, irrespective of DTPA introduction. However, DTPA residues were necessary to induce significant neovascularization by the Cu 21 -chelating hydrogels incorporating bFGF. The DTPA-introduced amylopectin prevented Cu 21 -induced deactivation of bFGF, again in marked contrast to DTPA-free amylopectin. It was concluded that biologically active bFGF could be incorporated to DTPAintroduced amylopectin through Cu 21 chelation in a stabilized state and was released as a result of hydrogel biodegradation, resulting in prolonged neovascularization.  1998 Elsevier Science B.V. All rights reserved. Keywords: bFGF; Amylopectin; Biodegradable hydrogel; Release; Metal coordination; Neovascularization

1. Introduction Since bioactive proteins are susceptible to proteolysis and denaturation after administration into the body, it is of prime necessity to contrive dosage forms for in vivo prolongation of the protein biological activity. One possible way for this purpose is to incorporate a protein drug into an appropriate matrix for achieving sustained release of the drug in *Corresponding author. Tel.: 181 75 7514115; Fax: 181 75 7514144; E-mail: [email protected]

the active state at the site of action over a long time period. Indeed, numerous studies on protein release by use of polymer matrices have been reported [1–4], but there is a major problem for the protein release technology. That is the loss of biological activity of the protein released. It has been demonstrated that this activity loss mainly results from denaturation and deactivation of the protein during the formulation process with a polymer matrix. When exposed to harsh environmental changes, such as heating and exposure to sonication and organic solutions, protein is generally denatured, losing its

0168-3659 / 98 / $ – see front matter  1998 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 98 )00081-9

136

Y. Tabata et al. / Journal of Controlled Release 56 (1998) 135 – 148

biological activity [5–7]. Therefore, a new formulation method under mild conditions for protein drugs with carriers is required to minimize protein deactivation. From this viewpoint, hydrogels may be a preferable candidate as protein release matrix because of their biosafety and inertness toward protein drugs [8]. However, sustained release of protein over a long time period will not be expected from hydrogels, since the release rate of protein from these matrix is generally diffusion controlled through aqueous channels in the hydrogels. Thus, for achieving effective protein release, a key strategy will be to immobilize the protein drug to polymer chains constituting the hydrogel through molecular interaction. In one trial, we have already prepared a biodegradable gelatin hydrogel incorporating basic fibroblast growth factor (bFGF) on the basis of polyion complexation between negatively charged gelatin and positively charged bFGF, and succeeded in releasing biologically active bFGF as a result of the hydrogel biodegradation [9–12]. In the present study, metal chelation is applied to immobilize bFGF into a biodegradable amylopectin hydrogel. For metal chelating, diethylenetriaminepentaacetic acid (DTPA) residues are introduced to amylopectin after crosslinking and Cu 21 is used as a chelating metal. The effect of Cu 21 chelation on the in vitro and in vivo profiles of bFGF release is investigated for the DTPA-introduced hydrogels. Neovascularization is induced by these hydrogels in the mouse subcutis to estimate retention of the bFGF activity in the hydrogel.

2. Materials and methods

2.1. Materials Amylopectin, isolated from potato, and DTPA anhydride were purchased from Nacalai Tesque (Kyoto, Japan) and Dojindo, (Kumamoto, Japan), respectively. As a crosslinking agent, ethylene glycol diglycidyl ether (Denacol EX-810) was supplied from Nagase Chemical (Hyogo, Japan). An aqueous solution of human recombinant bFGF (10 mg / ml) was kindly supplied from Kaken Pharmaceutical (Tokyo, Japan). Cupric sulfate hydrate (CuSO 4 ? 5H 2 O), chloramine T, sodium pyrosulfite (SMS),

and tyramine were obtained from Nacalai Tesque. Na 125 I (740 MBq / ml in 0.1 N NaOH aqueous solution) and an anion-exchange resin, Dowex 1-8X, were purchased from NEN Research Products (DuPont, Wilmington, DE, USA) and Dow Chemicals (Midland, MI, USA) respectively. N,N9-carbonyl diimidazole (CDI) and other chemicals were purchased from Wako (Kyoto, Japan) and used without further purification.

2.2. Radioiodination of bFGF bFGF was radioiodinated according to a chloramine T method [13]. Briefly, 450 ml of the original bFGF solution was added into 100 ml of 0.5 M potassium phosphate-buffered (KPB) solution (pH 7.5). Then, 3.5 ml of Na 125 I solution and 100 ml of 0.05 M KPB solution (pH 7.2) containing 0.06 mg of chloramine T were added to the bFGF solution. After agitation at room temperature for 2 min, 100 ml of 0.01 M phosphate-buffered saline solution (PBS, pH 7.4) containing 0.12 mg of SMS was added to stop the radioiodination. The resulting mixture was passed through a column of Dowex resin to remove uncoupled, free 125 I molecules, followed by an addition of 650 ml of water to obtain an aqueous solution of 125 I-labeled bFGF. The bFGF concentration was quantitated by measuring the UV absorbance at 280 nm and adjusted to 3 mg / ml.

2.3. Preparation of DTPA-introduced amylopectin hydrogels Varied amounts of ethylene glycol diglycidyl ether were added to 27.8 ml of 10 wt% amylopectin aqueous solution in 0.2 N NaOH. The solution mixture was cast into a cell culture Petri dish (100 mm tissue culture dish, Code No. 3020100, Iwaki Glass, Tokyo, Japan), and left at 258C for 3 days until complete drying. Following punching out of the resulting sheet to form discs of 5 mm diameter, each disc was rinsed repeatedly with double-distilled water (DDW) until the pH of the DDW became neutral, dehydrated, and finally, swollen in dehydrated dimethyl sulfoxide (DMSO). The swollen hydrogel disc was placed in 2.0 mM DTPA anyhydride and 40 mM 4-dimethylaminopyridine solution in dehydrated DMSO to give a concentration of

Y. Tabata et al. / Journal of Controlled Release 56 (1998) 135 – 148

10 mg amylopectin / ml solution. The reaction mixture was allowed to gently stir at 408C for various time periods to introduce DTPA residues to the amylopectin hydrogel discs. The resulting hydrogels were washed several times with DDW and dried under vacuum. The dry weight of the hydrogel prepared was around 5.8 mg. The amount of DTPA residues introduced was quantitated by conductivity measurement. The water content of amylopectin hydrogels, defined as the weight ratio of water to wet hydrogels, was calculated from the hydrogel weight before and after swelling in DDW at 378C for 24 h.

2.4. Cu 21 chelation and bFGF incorporation to DTPA-introduced amylopectin hydrogels Each of dried, DTPA-introduced amylopectin hydrogel discs (58 mg, 10 discs) was added to 5 ml of aqueous solution containing 2.16 mg of cupric sulfate. Cu 21 chelation was allowed to proceed under gentle stirring at 408C for various time periods. After that, the hydrogel disc was washed three times with DDW, followed by vacuum drying. The Cu 21 concentration in the solution supernatant was determined by a chelate titration method to calculate Cu 21 chelating to 1 mg dry hydrogel. A Cu 21 -free aqueous solution was used to prepare DTPA-introduced hydrogels without Cu 21 chelation. For bFGF incorporation into the Cu 21 -chelating amylopectin hydrogels, 10 ml of the original bFGF solution containing 100 mg of bFGF was added to the dried hydrogel discs, followed by leaving at 48C overnight to obtain DTPA-introduced amylopectin hydrogels incorporating bFGF through a Cu 21 coordinate bond. Similarly, bFGF was incorporated to DTPA-introduced amylopectin hydrogels without Cu 21 chelation and DTPA-free amylopectin hydrogels with or without Cu 21 chelation.

2.5. Release of bFGF from DTPA-introduced amylopectin hydrogels with or without Cu 21 chelation For in vitro release experiments, bFGF was incorporated into dried DTPA-introduced amylopectin hydrogels with or without Cu 21 chelation by dropping the bFGF solution (10 ml) onto the dried hydrogels, followed by leaving at 48C overnight for

137

bFGF incorporation. The hydrogels incorporating bFGF were placed at 378C for various time periods in 1 ml of PBS (ionic strength, 0.17). To evaluate the effect of solution ionic strength on the bFGF release from the bFGF-incorporating hydrogels, the release test was performed for the initial 6 h in 1 ml of DDW (ionic strength, 0) and thereafter in 1 ml of 1.5 M NaCl aqueous solution (ionic strength, 1.5). Similarly, bFGF release was examined for DTPAfree amylopectin hydrogels with or without Cu 21 chelation. The sample solution was periodically exchanged with the fresh solution of the same type at the same volume. Each sample solution was subjected to high-performance liquid chromatography (HPLC) equipped with a heparin affinity column (Toyo Soda, Tokyo, Japan) to determine the concentration of released bFGF in the intact form. The affinity change of bFGF for heparin was evaluated based on the alternation of the retention time and shape of HPLC peaks. As comparison, aqueous solution of 10 mg / ml bFGF (10 ml) was mixed with 490 ml of DDW containing 309 mg of cupric sulfate or that without cupric sulfate. After standing at 48C overnight and an addition of 500 ml PBS, the solution mixture was subjected to HPLC measurement.

2.6. Estimation of in vivo degradation of DTPAintroduced amylopectin hydrogels with Cu 21 chelation DTPA-introduced amylopectin hydrogels were radioiodinated following introduction of tyramine residues into the hydrogels according to a CDI activation method [14]. Powdered CDI (3.48 mg) was added into 58 ml of dehydrated DMSO containing 580 mg of DTPA-introduced hydrogel (100 discs), followed by activation of hydroxyl groups at 408C for 12 h. The activated hydrogel was placed in 58 ml of 0.59 mg / ml tyramine solution in dehydrated DMSO and left at 408C for 48 h to prepare tyramine-introduced hydrogel discs, followed by drying under vacuum. The dried hydrogel discs were swollen with 30 ml of 0.5 M KPB containing Na 125 I (37.5 kBq) at room temperature for 12 h. Then, 10 ml of 0.05 M KPB solution (pH 7.2) containing 0.06 mg of chloramine T was added to every swollen hydrogel disc. After standing at 48C for 3 h, each of

138

Y. Tabata et al. / Journal of Controlled Release 56 (1998) 135 – 148

the discs was placed into 500 ml of 0.01 M PBS containing 1.25 mg of SMS to stop the radioiodination. The resulting hydrogel discs were rinsed several times with DDW until the radioactivity was not detected in the DDW any more, and then treated in the aqueous solution of cupric sulfate as described above. Following subcutaneous implantation of 125 Ilabeled DTPA-introduced amylopectin hydrogels with Cu 21 chelation into the back subcutis of ddY mice (6–7 weeks old, Shizuoka Animal Center, Shizuoka, Japan), the radioactivity of hydrogels and their surrounding tissue was measured at different time intervals to evaluate the time profile of in vivo hydrogel degradation (three mice per each sampling time). All the animal experiments were done according to the institutional guidance of Kyoto University on animal experimentation.

2.7. Estimation of in vivo bFGF release from DTPA-introduced amylopectin hydrogels incorporating bFGF with or without Cu 21 chelation DTPA-introduced amylopectin hydrogels incorporating 125 I-labeled bFGF with or without Cu 21 chelation were implanted into the back subcutis of mice (six mice per group). DTPA-free amylopectin hydrogels incorporating 125 I-labeled bFGF with or without Cu 21 chelation and aqueous solution of 125 I-labeled bFGF were used as controls. Twenty ml of 125 I-labeled bFGF solution was mixed with 10 ml of aqueous solution containing 309 mg of cupric sulfate that was equivalent to the amount of Cu 21 chelated to the hydrogel, followed by leaving at 48C overnight. After an addition of 70 ml of PBS, the solution mixture was subcutaneously injected into the mouse back. At different time intervals, blood samples were taken out directly from the heart by syringe aspiration to measure their radioactivity on a gamma counter (ARC-301B, Aloka, Tokyo, Japan). The mouse skin containing the implanted hydrogel was cut into a piece of 335 cm 2 and the corresponding facia site was thoroughly wiped off with a filter paper to absorb 125 I-labeled bFGF. The radioactivity of the residual hydrogel and the skin piece plus the filter paper was measured on the gamma counter and their ratios to the initial hydrogel radioactivity were expressed as the percentage of remaining activity in and around the hydrogels, respectively.

2.8. In vivo assessment of neovascularization induced by DTPA-introduced amylopectin hydrogels incorporating bFGF with or without Cu 21 chelation DTPA-introduced amylopectin hydrogel discs incorporating bFGF with or without Cu 21 chelation were implanted into the back subcutis of mice. Aqueous solution of bFGF was also subcutaneously injected into the mouse back. Cu 21 -chelating or Cu 21 -free hydrogels incorporating bFGF or every amylopectin hydrogel without bFGF was used as controls. The mice were sacrificed at 1, 3, 5, 7, 10, 14, and 20 days after bFGF treatment to evaluate neovascularization in the mouse back subcutis. The bFGF dose was 100 mg / mouse and six mice were used for each experimental group. The neovascularization of bFGF was estimated by determining the weight of tissue hemoglobin as a marker of vascularization [9].

2.9. Statistical analysis All the data were analyzed by Students’ t-test and statistical significance was accepted at P,0.01. Results were expressed as the means6standard error.

2.10. Chromatographic evaluation of bFGF Ten ml of aqueous solution of 10 mg / ml bFGF was mixed with 490 ml of DDW containing 309 mg of cupric sulfate, 5.8 mg of amylopectin and 309 mg of cupric sulfate or 5.8 mg of DTPA-introduced amylopectin and 309 mg of cupric sulfate. Following leaving it at 48C overnight and an addition of 500 ml PBS, each solution mixture was subjected to HPLC measurement to compare the heparin affinity of sample bFGF with that of intact bFGF.

3. Results

3.1. Preparation of DTPA-introduced amylopectin hydrogels Fig. 1 shows the effect of the concentration of ethylene glycol diglycidyl ether on the water content of crosslinked amylopectin hydrogels. Hydrogels with water contents higher than 93 wt% could be

Y. Tabata et al. / Journal of Controlled Release 56 (1998) 135 – 148

139

tioned. No change in the water content of hydrogel was observed before and after DTPA introduction. Cu 21 chelation to DTPA-introduced and DTPAfree amylopectin hydrogels is shown in Fig. 3 as a function of time. Cu 21 ions were chelated to both the hydrogels with time up to 60 min, followed by saturation, although Cu 21 chelation to the DTPAintroduced hydrogel was higher than that to the DTPA-free hydrogel. Since the DTPA density in the hydrogel used was 0.1 mmol / mg dry hydrogel, the molar ratio of Cu 21 to DTPA residues was around 0.7 / 1 at the saturation of chelation.

Fig. 1. The effect of concentration of crosslinking agent on the water content of amylopectin hydrogels (NaOH concentration: 0.2 N, reaction temperature: 258C).

3.2. Release profiles of bFGF from DTPAintroduced amylopectin hydrogels incorporating bFGF with or without Cu 21 chelation

prepared by altering the concentration of the crosslinking agent used for the hydrogel preparation. The amylopectin hydrogel with the water content of 93 wt% was used throughout this study unless otherwise specified. Fig. 2 shows the DTPA introduction to the amylopectin hydrogel as a function of reaction time. The amount of DTPA residues introduced increased with time up to 6 h and thereafter levelled off. The amylopectin hydrogel with DTPA introduced at a density of 0.144 mmol / mg dry hydrogel was used for the following experiments unless otherwise men-

Fig. 4 shows the in vitro profiles of bFGF release from DTPA-introduced amylopectin hydrogels incorporating bFGF with Cu 21 chelation or other hydrogels in different aqueous solutions. The DTPA-introduced hydrogel with Cu 21 chelation exhibited approximately 10% of bFGF release during the initial 1 h, but thereafter no further release, whereas release of the intact bFGF was practically not detected from the DTPA-free hydrogel with Cu 21 chelation. A large initial release of intact bFGF was observed from Cu 21 -free hydrogels, irrespective of DTPA introduction (Fig. 4A). An increase in the solution

Fig. 2. The time course of DTPA introduction into the amylopectin hydrogel with a water content of 93 wt% (DTPA concentration: 2.0 mM, reaction temperature: 408C).

Fig. 3. The time course of Cu 21 chelation to DTPA-introduced (s) and DTPA-free (d) amylopectin hydrogels in DDW at 408C. The introduction extent of DTPA residues is 0.1 mmol / mg dry hydrogel.

140

Y. Tabata et al. / Journal of Controlled Release 56 (1998) 135 – 148

Fig. 4. The time profiles of bFGF desorption from amylopectin hydrogels incorporating bFGF with (opens marks) and without Cu 21 chelation (closed marks) in PBS at 378C: (s,d) DTPA-introduced and (n,m) DTPA-free hydrogels incorporating bFGF. (B) The effect of solution ionic strength on the in vitro profiles of bFGF desorption from DTPA-introduced amylopectin hydrogels incorporating bFGF with (s) and without Cu 21 chelation (d) at 378C. The release test solution was changed from DDW (ionic strength, 0) to 1.5 M NaCl aqueous solution (ionic strength, 1.5) after 6 h of release test.

ionic strength from 0 to 1.5 induced a temporary rapid release of bFGF from the DTPA-introduced hydrogel without Cu 21 chelation, in marked contrast to the Cu 21 -chelating DTPA-introduced hydrogel (Fig. 4B). HPLC chromatograms are shown in Fig. 5 for bFGF released from DTPA-introduced or DTPA-free amylopectin hydrogels incorporating bFGF with Cu 21 chelation. The sample solution for the DTPAintroduced hydrogel, though very small, exhibited a peak at the retention time where the intact bFGF was detected. However, for the DTPA-free hydrogel, the peaks were split, similar to those of bFGF in the presence of Cu 21 ions, while no intact bFGF peak was observed.

3.3. In vivo profiles of hydrogel degradation and bFGF release The time profile of hydrogel radioactivity is shown in Fig. 6 after subcutaneous implantation of 125 Ilabeled DTPA-introduced amylopectin hydrogels with Cu 21 chelation into the mouse back. The radioactivity decreased with implantation time, clearly indicating in vivo degradation of the hydrogels.

The hydrogel appearance in the mouse subcutis changed with implantation time: the shape of the hydrogel collapsed for the initial 4 days and thereafter a viscous solution remained in the implanted site. Since the solution viscosity was higher than that of DTPA-introduced amylopectin with Cu 21 chelation, this indicates that the hydrogel still remained without being completely degraded. A similar time profile of the hydrogel biodegradation was observed, irrespective of bFGF incorporation and Cu 21 chelation (data not shown). Fig. 7 shows the decrement patterns of bFGF radioactivity after subcutaneous implantation of DTPA-introduced amylopectin hydrogels incorporating 125 I-labeled bFGF with or without Cu 21 chelation. The radioactivity of bFGF injected in the solution form rapidly decreased, similar to that of Cu 21 -free hydrogels, irrespective of DTPA introduction. The radioactivity was retained at a larger amount and for a longer time period for DTPAintroduced or DTPA-free amylopectin hydrogels incorporating 125 I-labeled bFGF with Cu 21 chelation than other hydrogels and bFGF solution. A low level of radioactivity was detected in tissues surrounding the implanted hydrogels, but any radioactivity of

Y. Tabata et al. / Journal of Controlled Release 56 (1998) 135 – 148

141

Fig. 5. Heparin HPLC chromatograms of bFGF released from DTPA-introduced (A) and DTPA-free (B) amylopectin hydrogels with Cu 21 chelation during the initial 30 min in PBS at 378C, (C) 100 mg of intact bFGF, and (D) 100 mg of bFGF in the presence of Cu 21 ions.

bFGF was not detectable in the blood over the time period studied. Simple mixing of bFGF with Cu 21 ions prolonged its in vivo retention.

3.4. Neovascularization induced by DTPAintroduced amylopectin hydrogels incorporating bFGF with or without Cu 21 chelation

Fig. 6. The time course of the remaining radioactivity of 125 Ilabeled DTPA-introduced amylopectin hydrogels with Cu 21 chelation after implantation into the back subcutis of mice.

Fig. 8 shows light microphotographs of mouse tissues 6 days after implantation of DTPA-introduced amylopectin hydrogels incorporating bFGF with Cu 21 chelation or other agents. Vascularization was remarkable around the implantation site of the DTPA-introduced hydrogel incorporating bFGF with Cu 21 chelation. This is in marked contrast to that without Cu 21 chelation. Injection of free bFGF was not effective in inducing vascularization at all and the tissue appearance of the injected site was similar to that of PBS-injected, control mice. bFGF-free hydrogels exhibited no vascularization, irrespective of Cu 21 chelation and DTPA introduction.

142

Y. Tabata et al. / Journal of Controlled Release 56 (1998) 135 – 148

Fig. 7. (A) The time course of the remaining radioactivity of amylopectin hydrogels incorporating 125 I-labeled bFGF with (open marks) and without Cu 21 chelation (closed marks) after implantation into the back subcutis of mice: (s,d) DTPA-introduced and (n,m) DTPA-free hydrogels incorporating 125 I-labeled bFGF. (B) The time course of the remaining radioactivity of PBS containing 125 I-labeled bFGF with (s) and without (d) Cu 21 chelation after injection into the back subcutis of mice.

Vascularization induced by DTPA-introduced amylopectin hydrogels incorporating bFGF with Cu 21 chelation or other agents is shown as a function of time in Fig. 9. Injection of PBS solution containing bFGF did not increase the weight of hemoglobin at the injection site over the time range studied. The level of tissue hemoglobin was similar to that of bFGF-free PBS-injected or untreated mice. The DTPA-introduced amylopectin hydrogel alone did not induce vascularization at all, irrespective of Cu 21 chelation. The DTPA-free hydrogel with or without Cu 21 chelation did not enable bFGF to induce its vascularization activity, while neither bFGF-free hydrogels with nor without Cu 21 chelation were effective. On the contrary, both of the DTPA-introduced amylopectin hydrogels incorporating bFGF with and without Cu 21 chelation induced significant neovascularization. For the former hydrogel, the tissue hemoglobin increased within 3 days after hydrogel implantation and the increased level remained up to 8 days after implantation, followed by return to the initial level of hemoglobin at day 14, whereas the latter hydrogel significantly enhanced neovascularization but only during the initial 4 days.

3.5. Heparin affinity of bFGF Fig. 10 shows the heparin HPLC chromatograms

of bFGF in the presence of DTPA-introduced or DTPA-free amylopectin chelated with Cu 21 ions. In the chromatograph of bFGF1Cu 21 ions1DTPA-free amylopectin, the HPLC peak of intact bFGF disappeared and new peaks appeared at longer retention times, similar to the chromatograph of the mixed Cu 21 and bFGF solution. On the contrary, the presence of DTPA-introduced amylopectin reduced the chromatographic change of bFGF even though the solution contained Cu 21 ions. The peak of the intact bFGF was preserved although other peaks were observed.

4. Discussion Amylopectin is a water-soluble component of starch that has been extensively used for pharmaceutical, medical, and food applications. To prepare a biodegradable hydrogel through chemical crosslinking of this biosafe material, a diepoxy compound was selected among many agents in the present study to crosslink polysaccharide [15], because an experiment for cytotoxicity revealed that this agent was less toxic than other chemical crosslinking agents, such as glutaraldehyde and water-soluble carbodiimide [16]. Fig. 11 shows the schematic illustration for bFGF

Y. Tabata et al. / Journal of Controlled Release 56 (1998) 135 – 148

143

Fig. 8. The subcutis of mice 6 days after treatment with DTPA-introduced amylopectin hydrogels incorporating bFGF with Cu 21 chelation or other agents: (A) PBS, (B) 100 mg of free bFGF, (C) DTPA-introduced hydrogel, (D) DTPA-introduced hydrogel incorporating 100 mg of bFGF, (E) DTPA-introduced hydrogel with Cu 21 chelation, and (F) DTPA-introduced hydrogel incorporating 100 mg of bFGF with Cu 21 chelation. The water content of hydrogels used is 93 wt%.

144

Y. Tabata et al. / Journal of Controlled Release 56 (1998) 135 – 148

Fig. 8. (continued)

Y. Tabata et al. / Journal of Controlled Release 56 (1998) 135 – 148

145

Fig. 9. The time course of neovascularization induced by amylopectin hydrogels or other agents. A dotted line indicates the hemoglobin level of untreated, control mice. (A) Amylopectin hydrogels incorporating bFGF with (open marks) and without Cu 21 chelation (closed marks): (s,d) DTPA-introduced and (n,m) DTPA-free hydrogels incorporating 100 mg of bFGF or PBS containing 100 mg of bFGF (h). *,†,‡: P,0.01 significant against the value of control mice, those treated with DTPA-introduced hydrogels incorporating bFGF without Cu 21 chelation, and those injected with PBS solution of bFGF. (B) bFGF-free amylopectin hydrogels with (open marks) and without Cu 21 chelation (closed marks): (s,d) DTPA-introduced and (n,m) DTPA-free hydrogels.

Fig. 10. Heparin HPLC chromatograms of the intact bFGF (A), bFGF in the presence of DTPA-introduced amylopectin with Cu 21 chelation (B), DTPA-free amylopectin with Cu 21 chelation (C), and Cu 21 ions (D).

146

Y. Tabata et al. / Journal of Controlled Release 56 (1998) 135 – 148

Fig. 11. Schematic illustration for the bFGF release from DTPA-introduced or DTPA-free amylopectin hydrogels with Cu 21 chelation.

release from amylopectin hydrogels with and without DTPA residues using Cu 21 chelation. Metal affinity chromatography is one of the very effective protein separation methods. This is based on the intrinsic nature of proteins to form coordinate bonds with metal through chelation [17]. Indeed, it has been demonstrated that various proteins can be separated and purified by this method with their biological activity remaining [18–20]. On the other hand, protein release from hydrogels has been intensively investigated, but the release period is mostly as short as 1 day, because of their diffusion-controlled characteristics [1,2,15,17]. Thus, the metal coordinate bond seems to be promising to incorporate protein molecules into hydrogels and reduce their diffusional release. However, to our knowledge, this metal affinity concept has never been applied for protein release and the present study will be the first

trial of protein release by taking advantage of metal coordination. In general, the coordinate bond once formed is not readily dissociated even by change in solution ionic strength [17]. That no bFGF release was observed upon increasing the solution ionic strength confirms that the interaction between bFGF molecules and DTPA residues of amylopectin hydrogel is not due to an electrostatic bond but to a metal coordinate bond (Fig. 4B). bFGF molecules coordinately immobilized to DTPA residues through Cu 21 chelation will not be released in PBS. As Cu 21 ions can be chelated also to hydroxyl groups of sugars [21], it is possible that bFGF is incorporated into the DTPA-free amylopectin hydrogel through Cu 21 chelation with the hydroxyl groups of amylopectin chains. However, since the Cu 21 chelation to hydroxyl groups is weaker than to DTPA residues, bFGF will be released from

Y. Tabata et al. / Journal of Controlled Release 56 (1998) 135 – 148

the DTPA-free amylopectin hydrogel even in PBS. As shown in Fig. 5, the HPLC peak profile was similar to that of the mixed bFGF and Cu 21 aqueous solution, indicating actual bFGF release in the denatured form from the hydrogel. It is also possible that bFGF molecules are released from the DTPA-free hydrogel with chelating Cu 21 ions because of weak coordination of Cu 21 to the hydroxy groups. For the DTPA-introduced hydrogel, no HPLC peak other than a small peak due to the intact bFGF was noticed, suggesting that bFGF coordinately interacted with DTPA-residues of hydrogel as strongly as it could not be released in an aqueous solution. The initial significant vascularization induced by DTPA-introduced hydrogels incorporating bFGF without Cu 21 chelation may be explained in terms of the Coulombic interaction between bFGF molecules and DTPA residues (Fig. 9). As positively charged bFGF can electrostatically interact with negatively charged DTPA residues [22]. However, the interaction will not be so strong as to retain bFGF molecules in the hydrogel for a long time period because of the low density of DTPA residues. The metal coordinate bond of bFGF to the DTPA-introduced hydrogel may be so strong as to immobilize bFGF in the hydrogel, so that bFGF will be released from the hydrogel only when the hydrogel is biodegraded. A similar time course of in vivo bFGF retention was observed for the Cu 21 -chelating hydrogels incorporating bFGF, irrespective of DTPA introduction (Fig. 6). This suggests that both the hydrogels with and without DTPA groups may induce prolonged vascularization, but the presence of DTPA residues was necessary in order to prolong the bFGF effect (Fig. 8), probably because of long-term retention of the bFGF activity. Cu 21 ions altered the heparin affinity of bFGF (Fig. 10), but the presence of DTPA-introduced amylopectin was effective in reducing this affinity change, in marked contrast to that of DTPA-free amylopectin. This suppressive effect of DTPA-introduced amylopectin on the Cu 21 -induced deactivation of bFGF was also observed in the in vitro proliferation assay of BHK cells. The detailed mechanism is unclear to us at present, but it is possible that bFGF was retained in the DTPA-introduced hydrogel for a long time period, being immobilized to the DTPA residues through metal coordination. With hydrogel

147

degradation, the bioactive bFGF coordinated with a DTPA-introduced amylopectin fragment will be released, resulting in prolonged vascularization. It is conceivable that the DTPA-free amylopectin hydrogel will release bFGF in a Cu 21 -chelating, inactive form. Chelating with other metal ions than Cu 21 and the toxicity of Cu 21 are currently underway.

Acknowledgements This work was supported by a grant of the ‘‘Research for the Future’’ Program from the Japan Society for the Promotion of Science (JSPSRFTF96100203).

References [1] W.R. Gombotz, D.K. Pettit, Biodegradable polymers for protein and peptide drug delivery, Bioconj. Chem. 6 (1995) 332–351. [2] A.K. Banga, Therapeutic Peptides and Proteins, Formulation, Processing, and Delivery Systems, Technomic, Basel, Switzerland, 1995. [3] H. Okada, Biodegradable microspheres for therapeutic peptide delivery, Adv. Drug Deliv. Rev. 28 (1997) 1–170. [4] S. Cohen, H. Bernstein, Microparticulate Systems for the Delivery of Proteins and Vaccines, Marcel Dekker, New York, 1996. [5] M.S. Hora, R.K. Rana, J.H. Nunberg, T.R. Tice, R.M. Gilley, M.E. Hudson, Release of human serum albumin from poly(lactide-co-glycolide) microspheres, Pharm. Res. 7 (1990) 1190–1194. [6] S. Cohen, T. Yoshioka, M. Lucarelli, L.H. Hwang, R. Langer, Controlled delivery systems for protein based on poly(lactic / glycolic acid) microspheres, Pharm. Res. 8 (1991) 713–720. [7] Y. Tabata, Y. Takebayashi, T. Ueda, Y. Ikada, A formulation method using D,L-lactic acid oligomer for protein release with reduced initial burst, J. Control. Release 23 (1993) 55–64. [8] K. Park, W.S. Shalaby, H. Park, Biodegradable Hydrogels for Drug Delivery, Technomic, Basel, Switzerland, 1993. [9] Y. Tabata, S. Hijikata, Y. Ikada, Enhanced vascularization and tissue granulation by basic fibroblast growth factor impregnated in gelatin hydrogels, J. Control. Release 31 (1994) 189–199. [10] Y. Tabata, Y. Ikada, Potentiated in vivo biological activity of basic fibroblast growth factor by incorporation into polymer hydrogel microsphere, Proc. Fourth Jpn. Int. SAMPE Symp., 4 (1995) 25–29. [11] K. Yamada, Y. Tabata, K. Yamamoto, S. Miyamoto, I.

148

[12]

[13]

[14]

[15] [16]

Y. Tabata et al. / Journal of Controlled Release 56 (1998) 135 – 148 Nagata, H. Kikuchi, Y. Ikada, Potential efficacy of basic fibroblast growth factor incorporated in biodegradable hydrogels for skull bone regeneration, J. Neurosurg. 86 (1997) 871–875. Y. Tabata, K. Yamada, S. Miyamoto, I. Nagata, H. Kikuchi, I. Aoyama, M. Tamura, Y. Ikada, Bone regeneration by basic fibroblast growth factor complexed with biodegradable hydrogels, Biomaterials (1998) in press. F.C. Greenwood, N.W. Hunter, T.C. Glover, The preparation of 125 I-labeled human growth hormone of high specific radioactivity, Biochem. J. 98 (1963) 114–123. T. Yamaoka, Y. Tabata, Y. Ikada, Body distribution profile of polysaccharides after intravenous administration, Drug Deliv. 1 (1993) 75–82. S. Dumitriu, Polysaccharides in Medical Applications, Marcel Dekker, New York, 1996. C. Nishi, N. Nakajima, Y. Ikada, In vitro evaluation of cytotoxicity of diepoxy compounds used for biomaterial modification, J. Biomed. Mater. Res. 29 (1995) 829–834.

[17] T.-T. Yip, T.W. Hutchens, Immobilized metal ion affinity chromatography, Mol. Biotechnol. 1 (1994) 151–164. [18] J. Porath, J. Carlsson, I. Olsson, G. Belfrage, Metal chelate affinity chromatography. A new approach to protein fractionation, Nature 258 (1975) 598–599. [19] E. Sulkowski, Purification of proteins by IMAC, Trends Biotechnol. 3 (1985) 1–7. [20] M. Kroiher, S. Raffioni, R.E. Steele, Single step purification of biologically active recombinant rat basic fibroblast growth factor by immobilized metal affinity chromatography, Biochim. Biophys. Acta 1250 (1995) 29–34. [21] J. Dolezal, K.S. Klausen, F.J. Langmyhr, Studies in the complex formation of metal ions with sugars. I. The complex formation of cobalt (II), cobalt (III), cupper (II) and nickel (II) with mannitol, Anal. Chim. Acta 63 (1973) 71–77. [22] Md. Munirzzaman, Y. Tabata, Y. Ikada, Complexation of basic fibroblast growth factor with gelatin, J. Biomater. Sci., Polym. Ed. (1998) in press.