Download pdf - Protein Release From Gelatin Matrices REVIEW

LAdvanced Drug Delivery Reviews 31 (1998) 287–301

Protein release from gelatin matrices

*Yasuhiko Tabata, Yoshito IkadaResearch Center for Biomedical Engineering, Kyoto University, 53 Kawahara-cho Shogoin, Sakyo-ku, Kyoto 606, Japan

Abstract

Gelatin is a denatured, biodegradable protein obtained by acid and alkaline processing of collagen. This processing affectsthe electrical nature of collagen, yielding gelatin with different isoelectric points (IEPs). When mixed with positively ornegatively charged gelatin, an oppositely charged protein will ionically interact to form a polyion complex. This reviewarticle describes protein release from charged gelatin matrices on the basis of this polyion complexation. The biodegradablehydrogel matrices are prepared by chemical crosslinking of acidic or basic gelatin and are enzymatically degraded in thebody with time. The degradation is controllable by changing the extent of crosslinking, which, in turn, produces hydrogelswith different water contents. The time course of protein release is in good accordance with the rate of hydrogel degradation.It is very likely that the protein drug complexed with gelatin hydrogel is released as a result of its biodegradation. Thisgelatin hydrogel system releases the protein drug under maintenance of biological activity. This article will focus onexperimental data that sustained release of growth factor from the gelatin hydrogels is very effective in exerting thebiological functions of the growth factor. 1998 Elsevier Science B.V.

Keywords: Gelatin; Growth factor; Sustained release; Degradation; Polyion complexation; Neovascularization; Boneformation

Contents

1. Introduction ............................................................................................................................................................................ 2882. Preparation of gelatin hydrogels ............................................................................................................................................... 290

2.1. Block matrices ................................................................................................................................................................. 2902.2. Injectable matrices ........................................................................................................................................................... 290

3. Complexation of protein with gelatin ........................................................................................................................................ 2913.1. Polyion complexation in aqueous solution.......................................................................................................................... 2913.2. Interaction of protein with gelatin hydrogels ...................................................................................................................... 292

4. Degradation of gelatin hydrogels .............................................................................................................................................. 2935. bFGF release from gelatin hydrogels ........................................................................................................................................ 293

5.1. In vitro release ................................................................................................................................................................. 2935.2. In vivo release ................................................................................................................................................................. 294

6. Biological activity ................................................................................................................................................................... 2956.1. Neovasuclarization ........................................................................................................................................................... 2956.2. Bone formation ................................................................................................................................................................ 296

7. Conclusions ............................................................................................................................................................................ 297References .................................................................................................................................................................................. 298

*Corresponding author. Tel.: 075-751-4115; fax: 075-751-4144; e-mail: [email protected]

0169-409X/98/$19.00 1998 Elsevier Science B.V. All rights reserved.PII S0169-409X( 97 )00125-7

288 Y. Tabata, Y. Ikada / Advanced Drug Delivery Reviews 31 (1998) 287 –301

1. Introduction protein during the formulation process with a poly-mer matrix. When exposed to harsh environmental

Recent advances in biotechnology has made it changes, such as heating and exposure to sonicationpossible to produce various clinically useful peptides and organic solutions, protein is generally denatured,and proteins. While this technology has brought losing its biological activity [9,14,20]. Therefore, itabout the discovery and mass production of these is important to exploit a new formulation method ofbioactive macromolecules, several challenges need to protein carrier with polymers under mild conditionsbe addressed with regard to their sustained delivery to minimize protein denaturation. From this view-in a convenient, controlled manner, and targeting point, polymer hydrogel may be a preferable candi-formulations. In contrast to conventional synthetic date as a protein release matrix because of itspharmaceuticals, proteins are susceptible to biosafety and its high inertness towards protein drugsproteolysis, chemical change and denaturation during [72]. However, sustained release of protein over astorage and administration in the body [1,2]. Signifi- long time period will not be expected from hydro-cant efforts have been made to improve formulations gels, since the release rate of protein from hydrogelsfor better stabilization of proteins over a sufficiently is generally diffusion-controlled through aqueouslong storage time. Additional research has focused channels in the hydrogels. Thus, for achievingon the development of dosage forms that either effective protein release, it will be a key strategy toprolong the biological activity of protein in the body immobilize the protein drug to polymer carrieror assist in targeting the protein to a specific tissue. molecules constituting the hydrogel through someOne possible way to prolong activity is to incorpo- molecular interactions. For one trial, we have beenrate a protein drug into an appropriate matrix for attempting to take advantage of electrostatic interac-achieving sustained release of the drug at the site of tions between protein and polymer molecules for theaction over a long period of time. It is highly sustained release of protein from the polymer hydro-possible that protein is protected against proteolysis gel.and antibody neutralization, as far as it is, at least, It has been well recognized in polymer scienceincorporated in a release matrix for prolonged re- that a positively or negatively charged polyelec-tention of the protein activity in vivo. There have trolyte electrostatically interacts with an oppositelybeen a number of research reports on protein release charged partner to form a polyion complex [73,74].from polymer matrices: poly(L-lactic acid) (PLLA) It seems unlikely that all of the ionic interactionsand its copolymers with glycolic acid (PLGA) [3– between the two polyelectrolytes with many charged31], PLGA polymer blends [18,32,33], PLLA–poly- groups are dissociated at the same time. As a result,ethylene glycol (PEG) copolymers [34,35], poly- in contrast to low-molecular-weight electrolytes,(cyanoacrylates) [36,37], poly(anhydrides) [38–40], stable bonding will occur between the oppositelypoly(ortho esters) [41,42], polyphosphazene [43], charged polyelectrolytes, which will not be disso-poly(vinyl alcohol) [44], poly(vinyl pyrrolidone) ciated easily. In the research field of pharmaceutical[45], poly(acrylic acid) [46], poly(ethylene–co-vinyl science, this polyion complexation is not a newacetate) [47,48], cellulose derivatives [49–51], hy- technology but has been extensively explored foraluronic acid derivatives [52,53], alginate [54–58], drug coating and encapsulation. The application ofcollagen [59–61], gelatin [60–67], starch [68,69], this polyion complexation, which we will describedextran [70] and fibrin [71]. As is stated in other here, is ‘‘Drug complexation with polymer carriers’’.chapters of this special issue, the largest problem of This is a new trial that will allow us to pharma-protein release technology is the loss of biological ceutically modify a charged polymeric drug toactivity of the protein released from a protein–poly- increase its stability, targeting and sustained release,mer formulation. Thus, unless this problem is solved leading to enhanced therapeutic efficacy. Chargedby a breakthrough, it seems difficult to expect a drugs available for this trial include proteins andfurther research development in the area of protein oligo- and polynucleotides, while biodegradablerelease. It has been demonstrated that this activity polymers, such as proteins, polysaccharides andloss results from denaturation and deactivation of poly(amino acid)s, are applicable as the polymer

Y. Tabata, Y. Ikada / Advanced Drug Delivery Reviews 31 (1998) 287 –301 289

carriers. Another representative research field of is required for the carrier, the material to be used‘‘Drug complexation with polymer carriers’’ that has will be restricted to natural polymers with chargedbeen reported is gene therapy. It has been demon- groups, such as proteins and polysaccharides. There-strated that complexation with positively charged fore, as the carrier polymer, we have selectedpolymers enabled negatively charged DNA to have biodegradable gelatin, which is extensively used foran enhanced stability and transfection efficiency to industrial, pharmaceutical and medical purposes. Thecells [75–77]. However, it is unclear whether or not biosafety of gelatin has been proved through its longsuch a formulation also functions as a matrix for clinical usage as a plasma expander, in surgicalsustained release of polynucleotides. On the other biomaterials and as an ingredient in drugs [81].hand, few applications have been reported on poly- Another unique advantage of gelatin as a drug carrierion complexation for sustained release of macro- is the electrical nature of gelatin, which can bemolecular drugs from polymer matrices. Although changed by the collagen processing method [82]. Forlow-molecular-weight pharmaceuticals have been example, the alkaline process, through hydrolysis ofshown to release from polymer matrices on the basis amide groups of collagen, yields gelatin with a highof their ionic interaction [78–80], this is, however, density of carboxyl groups, which makes the gelatindifferent from polyion complexation. negatively charged. This reduces the isoelectric point

Fig. 1 shows a conceptual scheme of protein drug (IEP) of gelatin. In contrast, the electrostatic naturerelease from a biodegradable polymer carrier on the of collagen is hardly modified through the acidbasis of polyion complexation. A positively charged process because of a less invasive reaction to amideprotein drug is electrostatically complexed with groups of collagen. As a result, the IEP of the gelatinnegatively charged polymer chains, constituting a that is obtained will remain similar to that ofcarrier matrix. If an environmental change, such as collagen. In other words, a variety of gelatin samplesincreased ionic strength, occurs, the complexed drug with different IEP values are available (Fig. 2).will be released from the drug–carrier complex. If a protein to be released is acidic, basic gelatinEven if such an environmental change does not take with an IEP of 9.0 is preferable as the carrierplace, degradation of the polymer carrier itself will material, while acidic gelatin, with an IEP of 5.0,also lead to drug release. Because the latter is more will be applicable to the sustained release of a basiclikely to happen in vivo than the former, it is protein. Both gelatins are insolubilized in water topreferable that the drug carrier is prepared from prepare a hydrogel through chemical crosslinking,biodegradable polymers. The profile of drug release for instance, with water-soluble carbodiimides andin this drug–carrier system is regulated by the glutaraldehyde. It was reported that a model proteinchange of carrier biodegradation.

When we make use of polyion complexation forsustained release of a protein drug, it is absolutelynecessary to employ a highly bio-safe polyelectrolyteas the carrier matrix. In addition, if biodegradability

Fig. 1. Release of protein drug from biodegradable polymer carrier Fig. 2. Preparative process for acidic and basic gelatins fromon the basis of polyion complexation. collagen.


could be immobilized into albumin–heparin micro- water to deactivate and remove the unreacted WSC.spheres [83] or into a carrier of non-biodegradable The resulting hydrogels could be shaped into disks,synthetic polymer [84], through polyion complex- cubes or strips by punching out or cutting them withation, and that this protein was released from the a razor. Hydrogel tubes could also be prepared bycarriers upon environmental change. However, these chemically crosslinking the gelatin in a tube-shapedexperiments were conducted under in vitro con- mold. The hydrogels prepared were thoroughlyditions and the biological activity of the protein rinsed with double-distilled water, freeze-dried andreleased was not determined. Edelman et al. [54] sterilized using ethylene oxide gas. No big change inreported one trial of sustained release of basic hydrogel shape was observed before and after freeze-growth factor by using heparin incorporated into drying and sterilization. As a measure to evaluate thealginate beads. The sustained release of various extent of crosslinking of gelatin hydrogels, theirbioactive proteins from a collagen matrix has also water content was determined from the hydrogelbeen investigated [61,85,86] and it has been shown weight before and after swelling in a phosphate-that protein release was regulated by collagen swell- buffered saline solution (PBS, pH 7.4) for 24 h ating, but the contribution of ionic interactions be- 378C and expressed as the weight ratio of water intween the proteins and collagen was not studied. hydrogel to the whole wet hydrogel [65]. The waterProtein release from charged polysaccharides is content of gelatin hydrogels decreases with andiscussed in another chapter in this issue. Since increase in the concentration of crosslinking agentresearch on protein release based on polyion com- and gelatin and with the reaction time. The waterplexation has just started, this article mainly de- content can be changed over the range from 98 to 85scribes the preparation of biodegradable hydrogels wt%, irrespective of the gelatin’s IEP and the type offrom gelatin with two different IEP values and their crosslinking agent.efficacy as a sustained release carrier of a bioactiveprotein, together with our current findings on hydro- 2.2. Injectable matricesgel degradation and protein release.

Surgical invasion is required to implant blockmatrices in the body. However, this invasive opera-

2. Preparation of gelatin hydrogels tion can be avoided, as gelatin hydrogels can beeasily formulated into injectable shapes. For exam-

2.1. Block matrices ple, hydrogel microspheres can be prepared throughGA crosslinking of gelatin after an aqueous solution

The gelatin samples used were acidic gelatin with of gelatin is dispersed in an oil phase [67]. Briefly,an IEP of 5.0 and basic gelatin with an IEP of 9.0, aqueous gelatin solution is preheated and thenisolated from bovine bone by the alkaline process homogenized or sonicated at different input powersand from pig skin by the acid process, respectively. to yield a water-in-oil emulsion. The temperature ofBoth of them were chemically crosslinked to prepare the emulsion is lowered to approximately 208C,gelatin hydrogels with different biodegradation rates. followed by further continuous stirring for 30 min toBriefly, various amounts of glutaraldehyde (GA) [66] complete gelation of the gelatin solution in theor a water-soluble carbodiimide (WSC) [65] were dispersed phase. After gelatin dehydration by theadded to aqueous gelatin solutions and the cross- addition of acetone, the resulting microspheres arelinking reaction was allowed to proceed at 48C for collected by centrifugation, washed with acetone andvarious time periods. Following the crosslinking isopropyl alcohol, and dried under vacuum. Thereaction, GA-crosslinked hydrogels were immersed non-crosslinked, dried gelatin microspheres arein an aqueous solution of glycine at 378C for 1 h, to placed in an aqueous solution containing variousblock residual aldehyde groups of GA, and then were amounts of GA and are stirred to facilitate theirrinsed with water. On the other hand, WSC-cross- crosslinking. The resulting microspheres are treatedlinked hydrogels were immersed for 1 h in an with glycine, washed with distilled water and, final-aqueous HCl solution (pH 3.0) and washed with ly, freeze-dried. The size of the microspheres can be


regulated by altering the preparative conditions, e.g. IEP 5 4.3), soybean trypsin inhibitor (MW5 21,000;the gelatin concentration and the input power in the IEP 5 4.4), bovine pancreas trypsinogen (MW5

emulsification reaction. Optical microscopic observa- 24,000; IEP 5 9.3), chicken egg lysozyme (MW5

tion reveals that GA-crosslinked gelatin hydrogel 11,400; IEP 5 11.0) and basic fibroblast growthmicrospheres are all spherical, with average diame- factor (bFGF; MW5 17,000; IEP 5 9.6). Fig. 3ters ranging from 3 to 200 mm, irrespective of the shows turbidimetric titration curves for basic bFGFIEP of the gelatin. The water content of gelatin at various concentrations of aqueous solution con-hydrogel microspheres is in the range from 98 to 85 taining acidic and basic gelatins at 258C. Whenwt%, depending on the concentration of gelatin and acidic gelatin was used for the titration, the mixedGA during the preparation of the microspheres. solution exhibited a turbidity maximum and the

Even if the gelatin hydrogel is of the block type, turbidity increased with complexation time. In con-the hydrogel with a high water content is soft when trast, basic gelatin does not cause an increase in theswollen in water and, hence, can be extruded from a turbidity of a mixed solution, irrespective of theneedle-attached syringe at a clinically acceptable bFGF concentration. A similar trend of increasingpressure. Such a fluid hydrogel will be applicable as solution turbidity with time was observed for basican injectable carrier, similar to microspheres. trypsinogen and lysozyme, but not for acidic lactal-

bumin and trypsin inhibitor. In contrast, solutionmixtures formed from basic gelatin and acidic pro-

3. Complexation of protein with gelatin teins become turbid, in contrast to those betweenbasic gelatin and basic proteins. This finding indi-

3.1. Polyion complexation in aqueous solution cates that a polyion complex of basic (or acidic)protein is formed with the acidic (or basic) gelatin

As a simple method to evaluate polyion com- but not with the basic (acidic) gelatin. High-per-plexation between gelatin and protein, turbidity of formance liquid chromatography (HPLC) with athe mixed solution is measured at different tempera- heparin affinity column (HPLC) can be used totures [87]. Proteins with different IEP values can be evaluate the change in heparin affinity of bFGFused for polyion complexation with charged macro- accompanied by gelatin complexation. The peak areamolecules: Bovine milk lactalbumin (MW5 14,400; of intact bFGF decreases with complexation time and

Fig. 3. Turbidimetric titrations of various concentrations of bFGF in 1/15 M phosphate-buffered solution (pH 7.0) containing 5.0 mg/ml ofgelatin at 258C. (A) Acidic gelatin with an IEP of 5.0, (s) 6, (d) 12, (n) 24, (m) 48 and (h) 72 h after mixing the gelatin and bFGF and(B) basic gelatin with an IEP of 9.0, (s) 6, (d) 12, (n) 24, (m) 48 and (h) 72 h after mixing the gelatin and bFGF.


a new peak appears at a shorter retention time, whenbFGF is mixed with acidic gelatin. In contrast,mixing bFGF with basic gelatin does not affect theHPLC peak of intact bFGF, indicating that theaffinity of bFGF for heparin has decreased due to theformation of a polyion complex with acidic gelatin,resulting in a shortened retention time. On the otherhand, basic gelatin does not interact ionically withbFGF, which is the reason for the lack of a new peakattributable to complex.

The turbidity of the mixed solution from basicbFGF and acidic gelatin increases with complexationtime, irrespective of the ionic strength of the solu-tion, however, the higher the ionic strength of the

Fig. 4. Influence of the temperature of the solution on bFGFmixed solution, the less significantly the turbidity sorption into hydrogels prepared from gelatin with IEP values ofincreases. A HPLC study has revealed that the peak 5.0 (open marks) and 9.0 (solid marks) in water at (s, d) 4, (n,

m) 25 and (h, j) 378C.area of the intact bFGF, 24 h after mixing withgelatin, decreases with a decrease in the ionicstrength, and reaches zero when double-distilledwater is used to mix. The peak area of the bFGF- molecule, with a molecular weight of 17,000, isgelatin complex increases with decreasing ionic small enough to freely diffuse into the interior ofstrength. Thus, complexation between bFGF and hydrogels with water contents of more that 87 wt%acidic gelatin is weakened by an increase in the ionic [88]. It should be noted that bFGF sorption to gelatinstrength of the mixed solution. It is obvious that the hydrogels depends on the temperature of the solu-electrostatic interaction between bFGF and gelatin tion. The amount of bFGF sorbed to acidic gelatinmainly contributes to the polyion complexation. hydrogels tends to increase with temperature. In

addition, bFGF sorption to basic gelatin hydrogels3.2. Interaction of protein with gelatin hydrogels was observed at 378C following an initial period

when there was no release, although no bFGF wasThe interaction of protein with gelatin results in sorbed at lower temperatures. The sorption profile of

protein sorption to the gelatin hydrogel. Fig. 4 shows bFGF to hydrogels of poly(acrylic acid), which is athe time course of bFGF sorption to hydrogels synthetic polymer that has much more negativeprepared from gelatin with IEP values of 5.0 and 9.0 charges than gelatin, does not depend on the tem-at different temperatures. Basic bFGF is sorbed to perature of the solution. As it is well recognized thatthe hydrogel of acidic gelatin with an IEP of 5.0 at 4 electrostatic interaction does not depend theoreticallyand 378C with time, in contrast to basic gelatin on temperature, this finding indicates that bFGFhydrogel. When bFGF sorption is examined in sorption to poly(acrylic acid) hydrogels is primarilyaqueous solution containing different amounts of driven by electrostatic interaction. However, theNaCl, the amount of adsorption of bFGF decreases significant temperature dependence of bFGF sorptionwith an increase in the NaCl concentration, although to acidic gelatin hydrogels suggests that other fac-the addition of NaCl does not affect the time profile tors, such as conformational changes in the gelatinof bFGF desorption (bFGF release). No bFGF sorp- molecules, are also contributing to the interaction.tion is observed for basic gelatin hydrogels, irre- The rate of bFGF sorption to acidic gelatinspective of whether or not NaCl is added. When hydrogels is much lower than that to poly(acrylicacidic gelatin hydrogels with water contents ranging acid) hydrogels when compared at a similar waterfrom 98 to 85 wt% are used, the time profile of content. However, when the amino groups of gelatinbFGF sorption to every hydrogel is similar, irre- are chemically converted to carboxyl groups, bFGFspective of the water content. Probably, the bFGF sorption to the carboxylated gelatin tends to increase


with an increase in the extent of carboxylation. Thisindicates that the relatively slow sorption of bFGF isdue partially to the small amount of negative chargesin one gelatin molecule.

Scatchard analysis has demonstrated that the dis-sociation constant (K ) of bFGF for the acidicd

27gelatin hydrogel is 6.75 3 10 M. It is reported thatthe K of bFGF to a low affinity receptor, heparand

29sulfate, is 2.0 3 10 M [89]. It may be concludedthat the initial driving force of bFGF sorption to theacidic gelatin hydrogel is electrostatic interactionbetween the two molecules, although it is not asstrong as the bFGF–heparan sulfate interaction but issufficient to form a polyion complex. As is well Fig. 5. In vivo degradation profiles of bFGF-incorporating acidicrecognized, FGF has a strong affinity for acidic gelatin hydrogels with water contents of (s) 98.8 and (d) 96.9polysaccharides, such as heparin and heparan sulfate, wt% [65].

probably resulting in protection of FGF from denatu-ration and enzymatic degradation in vivo [90,91].This proposed hydrogel formulation mimics the hydrogels, the remaining radioactivity of the hydro-natural manner in which bFGF is stored in the gels was also measured, to evaluate the in vivo rateextracellular matrix. It is likely that gelatin com- of hydrogel degradation. This study has again dem-plexation enhances the in vivo stability of bFGF, onstrated that the radioactivity in hydrogels withsimilar to the bFGF–acidic polysaccharide complex- higher water contents decreases faster than in thoseation that occurs in biological systems. with lower water contents. The time profile of the

loss of radioactivity is in good agreement with theweight loss of hydrogels. Maintenance of the degra-

4. Degradation of gelatin hydrogels dation period of hydrogels from five days to fiveweeks is possible if the water content is changed,

Since gelatin hydrogels undergo enzymatic hy- and incorporation of bFGF into gelatin hydrogelsdrolysis in the body, it is too difficult to evaluate does not affect their profile of in vivo degradation.their degradation profile under in vitro conditions Gelatin hydrogels of the microsphere type are alsowithout any enzymes. Even if enzyme is present in degraded in vivo with time, irrespective of their size,the test solution, the in vitro result cannot simulate although the degradation rate is high compared withthe in vivo profile of hydrogel degradation because that of block gelatin hydrogels with a similar waterthe type and concentration of enzymes for collagen content, because of the high surface area [67].hydrolysis are not clear. Thus, gelatin hydrogelswere subcutaneously implanted into the backs ofmice and the weights of the hydrogels were mea- 5. bFGF release from gelatin hydrogelssured at different time intervals to evaluate the timeprofile of in vivo hydrogel degradation [92]. 5.1. In vitro release

The weight of the hydrogel was found to decreasewith implantation time and, finally, the mass dis- The preparation of hydrogels in the presence ofappeared from the implantation site, indicating that proteins will lead to a loss in their activity throughthe hydrogels were degraded in vivo. The degra- chemical crosslinking of gelatin. In contrast, thedation period for hydrogels depends on their water present method, in which an aqueous solution ofcontent (Fig. 5). The higher the water content of the bFGF is dropped onto freeze-dried gelatin hydrogels,hydrogels, the faster they degrade. Following the followed by leaving them under various conditions to

125subcutaneous implantation of I-labeled gelatin allow bFGF to sorb into the gelatin hydrogels, at


least will prevent protein from being chemically molecules are not ionically complexed with thedeactivated. This method is also effective in quan- acidic gelatin constituting the hydrogel, even aftertitatively incorporating bFGF into gelatin hydrogels overnight incubation at 48C. In addition, prolongedwith high reproducibility, irrespective of the water bFGF impregnation reduces the initial burst of bFGFcontent, because the volume of the bFGF solution is in the in vitro release test. It is probable that themuch less than that theoretically required to im- non-complexed bFGF is released from the hydrogelpregnate bFGF into the hydrogel. during the initial period of release, followed by no

The following fluorescent microscopic study has release of complexed bFGF, whereas no formationdemonstrated that fluorescent-labeled bFGF mole- between basic gelatin and basic bFGF leads tocules impregnated by this procedure are homoge- prompt release of all of the bFGF molecules from theneously distributed throughout the interior of the basic gelatin hydrogel, in contrast to the situationhydrogel [93]. with an acidic gelatin hydrogel.

bFGF was incorporated overnight into a gelatinhydrogel through impregnation at 48C. The bFGF- 5.2. In vivo releaseincorporating gelatin hydrogel was placed in PBS at378C and the bFGF concentration of the supernatant To assess the in vivo profile of bFGF release from

125was quantitated by HPLC at different time intervals, hydrogels, acid gelatin hydrogels containing I-to estimate the time profile of bFGF release. When labeled bFGF were implanted subcutaneously intoan acidic gelatin hydrogel was used to incorporate the backs of mice and the residual radioactivity was

125bFGF, up to about 40% of the initial loading was measured at different time intervals. The I-labeledreleased within one day, but, thereafter, no substan- bFGF-incorporating gelatin hydrogels showed de-tial release was observed. On the other hand, the creased residual radioactivity with implantation time,hydrogel prepared from basic gelatin exhibited al- while no radioactivity was observed in the blood,most complete release of the incorporated bFGF suggesting that bFGF is released in vivo from thewithin one day (Fig. 6). This demonstrates that bFGF-incorporating gelatin hydrogel. The decrementbFGF cannot be released from acidic gelatin hydro- pattern of radioactivity depends on the hydrogel’sgels under in vitro non-degradation conditions if degradability, in such a manner that the radioactivitybasic bFGF molecules are complexed with acidic is retained for longer when the water content of thegelatin. It is apparent from Fig. 4 that all bFGF hydrogel is lower. Also, the in vivo degradation

profile of hydrogels can be radiotraced throughsubcutaneous implantation of bFGF-incorporating

125hydrogels prepared from I-labeled acidic gelatin.The radioactivity of hydrogels decreases with im-plantation time, while the rate of decrease increasedwith an increase in the water content of the hydrogel.The decrement pattern of bFGF radioactivity in thehydrogel is in good agreement with that of gelatinradioactivity, irrespective of the hydrogel’s watercontent (Fig. 7). In addition, the half-life period forretention of bFGF in gelatin hydrogels of differentwater contents was found to be linearly related to theamount of hydrogel remaining. These findings indi-cate that bFGF is probably released from the gelatinhydrogel together with degraded gelatin fragments in

Fig. 6. In vitro release profiles of bFGF at 378C from bFGF- the body as a result of hydrogel degradation. As isincorporating hydrogels prepared from gelatin with IEP values of

apparent in Fig. 7, the amount of gelatin remaining5.0 (s) and 9.0 (d) in 1 /15 M phosphate-buffered solution (pHduring the initial degradation period is larger than the7.4). The water content of the gelatin hydrogels was 95.2 wt%,

irrespective of the type of gelatin used [65]. amount of bFGF remaining, irrespective of the water


proteins is whether or not the protein released in thebody actually retains its biological activity. Toevaluate protein activity, in vitro culture techniquesare normally employed because of their simplicityand convenience, compared with in vivo animalexperiments. However, any in vitro non-degradationsystem cannot be applied to evaluate the biologicalactivity of released bFGF, since protein release isinvolved with the in vivo degradation of hydrogelmatrices in our release system. Thus, to obtaininformation on the retention of bFGF activity, wedirectly assessed vascularization and bone formationafter implantation of bFGF-incorporating gelatin

Fig. 7. Relationship between the radioactivity of bFGF and the hydrogels in animals.125gelatin remaining after subcutaneous implantation of I-labeled

125bFGF-incorporating hydrogel prepared from I-labeled acidic 6.1. Neovasuclarization [65]gelatin. The water contents of the hydrogels were (s) 98.8 and(d) 96.9 wt%.

bFGF-incorporating gelatin hydrogels were sub-cutaneously implanted into the backs of mice and

content of the hydrogels. This can be explained in their effect on neovascularization was evaluated andterms of the initial release of free bFGF. As demon- compared with that of bFGF in solution. Histologicalstrated in Fig. 6, a certain amount of bFGF is examination demonstrated that vascularization wasreleased from the acidic gelatin hydrogel, probably remarkable around the implantation site of bFGF-because the impregnation conditions are not suffi- incorporating gelatin hydrogels, in contrast to sitescient to completely form a polyion complex between injected with an aqueous solution of bFGF. InjectionbFGF and gelatin. Therefore, it is possible that bFGF of bFGF in the form of a solution was not effectivemolecules that are not complexed with gelatin are in inducing vascularization at all and a bFGF-freereleased, even in vivo, from gelatin hydrogels during gelatin hydrogel alone did not induce any vasculari-the initial period after implantation. However, we zation effect. The amount of tissue hemoglobin,cannot completely rule out the possibility that bFGF which is a measure of bFGF-induced neovasculariza-is released from hydrogels through in vivo dissocia- tion, notably increased within one day of implanta-tion of bFGF–gelatin complexes, as is illustrated in tion of bFGF-incorporating gelatin hydrogels with aFig. 1. water content of 95.2 wt% and the increased level

was retained for one week, followed by a return tothe initial level of hemoglobin at day fourteen. On

6. Biological activity the other hand, injection of an aqueous solutioncontaining the same dose of bFGF, as a bFGF

The bFGF used here was originally characterized hydrogel, did not increase the amount of hemoglobinin vitro as a growth factor for fibroblasts and at the injection site over the time range studied; thecapillary endothelial cells and in vivo as a potent level of tissue hemoglobin remained at approximate-mitogen and chemoattractant for a wide range of ly the same level as that found on injection ofcells. In addition, bFGF is reported to have a variety bFGF-free PBS or in untreated mice (Fig. 8). Noof biological activities [90,91,94] and to be effective increase in the amount of hemoglobin was observedin enhancing wound healing through induction of even when the amount of bFGF in solution that wasneovascularization [95,96] and regeneration of bone injected was increased to 1 mg per mouse. This must[97–99], cartilage [100,101] and nerve [102,103], be due to a rapid elimination of bFGF from thewhen administrated in the form of a solution. The injection site. In contrast, incorporation of bFGF intomost important concern regarding the delivery of gelatin hydrogels enabled us to reduce the dose that


Fig. 8. Time-course of neovascularization induced by free bFGF and by a bFGF-incorporating acidic gelatin hydrogel. (A) Mice received asubcutaneous injection of a PBS solution containing 100 mg of bFGF (s) and bFGF-free PBS solution (d). (B) Mice received asubcutaneous implantation of hydrogel containing 100 mg of bFGF (s) and bFGF-free hydrogel (d). **indicates significance at P , 0.01against the value for each dosage at day zero [65].

was effective in inducing significant vascularization water contents, leading to a prolonged neovasculari-to 30 mg per mouse. This enhanced vascularization zation effect. Thus, these findings demonstrate thateffect was observed also on injection of gelatin the bFGF released from the gelatin hydrogel systemhydrogel microspheres that had bFGF incorporated may maintain biological activity, although the per-into them [67]. centage of the activity that remained could not be

The in vivo degradation profile of bFGF-incor- quantitatively evaluated from this in vivo study.porating gelatin hydrogels can be modified by chang-ing the water content. For example, a gelatin hydro- 6.2. Bone formation [66]gel with a water content of 95.2 wt% was degradedin the mouse subcutis with time and completely A rabbit model of a skull bone defect wasresorbed after fourteen days of implantation. At that employed to evaluate the in vivo efficacy of bFGF-time, neovascularization could no longer be detected incorporating gelatin hydrogels in bone formation.and the appearance of the tissue returned to that When implanted into a skull defect, the bFGF-incor-found in untreated mice. This indicates that the porating gelatin hydrogel accelerated bone regenera-retention period of the hydrogel-induced vascularization at the skull defect and almost closed the defecttion effect is in good agreement with the degree of after twelve weeks of implantation. In contrast,degradation of hydrogel. In addition, enhanced insignificant bone regeneration and remarkable in-neovascularization was observed for all types of growth of soft connective tissue were observed at theimplanted hydrogels, irrespective of the water con- bone defect when rabbits were treated with a bFGF-tent, but the time profile of vascularization depended free gelatin hydrogel and free bFGF or were lefton the water content of the hydrogels. The hydrogel- without treatment. The bFGF-free gelatin hydrogelinduced vascularization effect was prolonged when neither induced bone formation nor interfered withthe water content was decreased. This phenomenon bone regeneration at the skull defect. Table 1 sum-can be explained in terms of the sustained release of marizes the results of the measurement of bonebFGF. As described earlier, bFGF seems to be mineral density (BMD) at the skull defect of rabbitsreleased from the gelatin hydrogel as a result of eight and twelve weeks after different treatments.hydrogel degradation. It follows that the period of The BMD of intact rabbit skulls was 120610 mg/

2bFGF release can be regulated by changing the rate cm . Clearly, both the bFGF-incorporating gelatinat which the hydrogel degrades. Hydrogels with hydrogels with water contents of 85 and 98 wt%lower water contents will be degraded and release enhanced the BMD of the skull defect, but the BMDbFGF in vivo more slowly than those with higher was significantly higher for rabbits that were treated


Table 1aBone mineral densities at the skull defect of rabbits eight and twelve weeks after different treatment regimens

2Treatment group Water BMD (mg/cm )content(wt%) 8 weeks 12 weeks

b c ,d ,ebFGF-containing 85 102.066.13 115.966.97f b ,ggelatin hydrogels 98 100.8611.3 101.5610.6

(100 mg bFGF/rabbit)Free bFGF NA 94.0611.4 82.6614.2

(100 mg bFGF/rabbit)Empty gelatin hydrogels 85 82.769.11 76.669.77

98 85.8616.1 80.567.75PBS NA 82.268.47 74.169.62a b c dSignificance levels were calculated for the following comparisons: P , 0.01; P , 0.001 versus PBS-treated group; P , 0.001 versus

e ffree bFGF-treated group; P , 0.01 versus 98 wt% water content bFGF-containing hydrogel-treated group; P , 0.05 versus PBS-treatedggroup and P , 0.05 versus free bFGF-treated group. Abbreviation: NA 5 not applicable.

with 85 wt.% hydrogel compared with the 98 wt% prolonged the retention period of cells by a fewhydrogel. On the other hand, free bFGF did not give weeks, but the number returned to the basal level inrise to any significant bone regeneration, although week eight. In contrast, the group of rabbits treatedthe BMD tended to be somewhat higher than that of with bFGF-incorporating gelatin hydrogels did notPBS-treated, control rabbits. The BMD at the defect exhibit such a rapid decrease in the cell number asof rabbits treated with bFGF-free, empty gelatin the group that received treatment with free bFGF,hydrogels was similar to that of control rabbits, and retained a significantly higher number of cellsindicating that implantation of hydrogels in the over the time range studied, irrespective of the typedefect did not disturb bone regeneration at the site. A of hydrogel used. However, the retention period ofsimilar trend was observed for bone formation after the enhanced cell level tended to increase with aeight weeks of implantation, but the efficacy of decrease in the water content of the hydrogel. ItbFGF-incorporating hydrogels in enhancing bone seems that bFGF was released in the biologicallyregeneration was not as clear as that of hydrogels active state from the gelatin hydrogel, activatingimplanted for twelve weeks. No significant differ- osteoblasts to induce bone regeneration at the skullences in the BMD were observed between these defect. This finding again suggests that the gelatinexperimental groups after four weeks of treatment. hydrogel system enables bFGF to be released with

The time course of the number of osteoblasts biological activity remaining.residing near the edge of the bone of the skull defectwas examined after different treatments. Interesting-ly, during the initial two weeks, the time course of 7. Conclusionscell number was similar among the differentlytreated rabbits. No differences were observed be- The need for sustained release of proteins willtween the rabbit groups treated with free bFGF and increasingly become larger in concert with theirbFGF-incorporating gelatin hydrogels in the distribu- production on an industrial scale. However, little hastion profile of the cells that were positively stained been reported on the technology that can achieve thewith alkaline phosphatase during the initial period. sustained release of proteins with their biologicalThe profile of the osteoblast number thereafter activity maintained. The main reason for this may bedepended on the treatment type. The cell number that recombinant bioactive proteins are at presentincreased for up to two weeks and then decreased for expensive and still difficult to obtain, even if theyboth non-treated rabbit groups and for those treated have been commercialized. Our technology for re-with bFGF-free, empty gelatin hydrogels, irrespec- leasing proteins is based on polyion complexation,tive of their water content. Free bFGF treatment which is commonly observed in the body between


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