Langmuir 2007, 23, 8531-8537
8531
Melting and Swelling Behaviors of UV-Irradiated Gelatin Gel Microcapsules Takao Yamamoto* Department of Physics, Faculty of Engineering, Gunma UniVersity, Kiryu, Gunma, 376-8515, Japan
Michiru Koike and Toshiaki Dobashi Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma UniVersity, Kiryu, Gunma 376-8515, Japan ReceiVed March 1, 2007. In Final Form: May 15, 2007 Gelatin gel microcapsules with a narrow size distribution have been prepared for the use of regenerative therapy by means of SPG (Shirasu porous glass) emulsification and UV-induced cross linking, and the melting and swelling behaviors of the gel membrane of the microcapsules were observed. The gel melting temperature was proportional to the 2/3 power of UV irradiation time t for t e1 h and deviated from the 2/3 power behavior for t g1 h. The average cross-sectional area of the microcapsules that remains insoluble normalized by that at 25 °C monotonically increased with temperature for t e1 h. It then decreased, passed a minimum, and increased at high temperature for t g1 h. Repeated quenching of the gel microcapsules between two temperatures (25 and 40 °C) induced a reversible size change, which was attributed to the helix-coil transition of collagen molecules locally. From a theoretical consideration of gel particles, the observed gel melting behavior was explained well, and the scaled volume of the microcapsules was expressed as a function of scaled temperature with four fitting parameters for t e1 h.
Introduction Gelatin is one of the most commonly used potential biopolymers for regenerative therapy because of its availability, nontoxicity, high adsorption to cells, and biodegradability.1 For such a use, it must be insoluble at physiological temperature (37 °C) in most cases. Because gelatin gels prepared from raw gelatin aqueous solution melt below 30 °C even at high concentrations of gelatin,2,3 raising the melting point above 37 °C is required for the application. UV-induced cross linking of gelatin molecules is one of the simplest methods of raising the melting point. Furthermore, this method is environmentally friendly and nontoxic in contrast to cross-linking methods using chemical reagents such as glutaraldehyde. Recent papers reported the suitability of UV-irradiated gelatin particles for pharmaceutical and medical use.4,5 We demonstrated that the microcapsules prepared by UV irradiation can be used as a smart cell culture scaffold because the cells are separated from the scaffold without centrifugation after trypsin treatment.6 For such use, we need to clarify the physicochemical properties and control the melting and swelling behavior of the gelatin-based particles prepared by UV-induced cross linking. In this article, we report an experimental and theoretical analysis of the swelling and melting behaviors of the gel microcapsules. Experimental Results Materials. Porcine gelatin (type APH-250, Nitta Gelatin Inc.) was dissolved in 40 °C Milli-Q water at 5 wt % to make gelatin * To whom all correspondence should be addressed. E-mail:
[email protected]. Fax: +81-277-30-1927. (1) Zekorn, D. Bibl. Haematol. 1969, 33, 131. (2) Joly-Duhamel, C.; Hellio, D.; Djabourov, M. Langmuir 2002, 18, 7208. (3) Joly-Duhamel, C.; Hellio, D.; Ajdari, A.; Djabourov, M. Langmuir 2002, 18, 7158. (4) Van Miller, J. P.; Hermansky, S. J.; Losco, P. E.; Ballantyne, B. Toxicology 2002, 175, 177. (5) Sugiyama, A.; Sugie, T.; Yanagawa, H. Jpn. Kokai Tokkyo Koho 1999, H11-47258. (6) Koike, M.; Kobayashi, K.; Tanaka, S.; Harano, A.; Yamamoto, T.; Dobashi, T. Trans. MRS J. 2006, 31, 823.
solutions. Aliquot of surface-active agent tetra glycerin fatty acid ester (SYglyster CR-310, Sakamoto Yakuhin Kogyo Co. Ltd) was added to isooctane (Wako Pure Chemical Industries Ltd) at 5 wt % to make a dispersion medium. A 5 wt % gelatin solution (1 mL) was added to 30 mL of the dispersion medium. Dispersions of gelatin droplets were prepared by the following two methods. (1) SPG emulsification. The suspension was made by SPG (Shirasu porous glass)7-10 with a pore size of 10.0 µm (sample A). (2) Simple stirring. The suspension was simply stirred at 40 °C at a stirring rate of 500 rpm to emulsify the solution (sample B). Sample B was used for investigating the size dependence of gel melting, and sample A was used for all other experiments. The emulsions were then incubated at 15 °C for 10 min to turn the droplets into gel particles (physical gel), which were collected as precipitates. The gelatin gel particles were washed in hexane (Wako Pure Chemical Industries Ltd) three times. Finally, we dispersed 1 g of the gelatin particles in 10 mL of hexane in a beaker with a diameter of 3.5 cm. The suspension was UV-irradiated at 1520 µW/cm2 at 254 nm (model UVG-11, Funakoshi Co Ltd) at 15 °C. The distance between the light source and the surface of the suspension was 5.5 cm. The UV irradiation time was varied from 0 to 10 h. The obtained gelatin particles in hexane were washed in 2-propanol three times and then in ethanol three times. Finally, they were dispersed in Milli-Q water. All organic solvents were reagent grade and were purchased from Wako Pure Chemicals Co. Ltd. Size Distribution. The gelatin particles were observed with an inverted microscope equipped with a water bath, and the digital data for the cross-sectional area were taken with a CCD camera. The particles were observed to be core-shell type (microcapsules), as shown in Figure 1. The size distribution was fairly sharp with a central peak at 26.7 ( 4 µm, as shown in Figure 2. Temperature Scanning Rate Dependence of the Gel Melting Point. The temperature of the water bath was raised from 25 to (7) Omi, S.; Katami, K.; Yamamoto, A.; Iso, M. J. Appl. Polym. Sci. 1994, 63, 1. (8) Omi, S.; Katami, K.; Taguchi, T.; Kaneko, K.; Iso, M. J. Appl. Polym. Sci. 1995, 57, 1013. (9) Omi, S.; Taguchi, T.; Nagai, M.; Ma, G.-H. J. Appl. Polym. Sci. 1997, 63, 931. (10) Ma, G. -H.; Nagai, M.; Omi, S. J. Appl. Polym. Sci. 1997, 66, 1325.
10.1021/la700606e CCC: $37.00 © 2007 American Chemical Society Published on Web 07/06/2007
8532 Langmuir, Vol. 23, No. 16, 2007
Figure 1. Optical micrograph of gelatin microcapsules prepared for a UV irradiation time t of 1.5 h for 5 wt % gelatin solutions emulsified with SPG emulsification.
Figure 2. Size distribution of gelatin microcapsules prepared with SPG emulsification for 5 wt % gelatin solutions in hexane. The average is 26.7 ( 4 µm.
Figure 3. Melting temperature of gelatin microcapsules measured at different temperature scanning rates V. The arrow shows a temperature scanning rate of 0.105 K/min, which was used in the other experiments. 75 °C at scanning rates of 0.105, 0.21, 0.42, and 0.83K/min, and the number of microcapsules remaining insoluble was measured. Here, the UV irradiation time was fixed at 0.5 h. The melting temperature of the microcapsules was determined as described in the next subsection. The temperature scanning rate V of the melting temperature Tm was S-shaped, as shown in Figure 3. Tm depends on V in the range of 0.2 K/min < V < 0.4 K/min but seems to be constant for V < 0.2 K/min. Therefore, the temperature scanning rate was fixed at V ) 0.105 K/min in all of the measurements below. UV Irradiation Time Dependence of the Gel Melting Point. The temperature of the water bath was raised from 25 to 75 °C at a rate of 0.105 K/min for the samples prepared with different UV irradiation times. Figure 4 shows the ratio of the number of microcapsules remaining insoluble at each temperature, N, and that at 25 °C, N0. As the UV irradiation time t increases, the decreasing rate of ratio N/N0 becomes smaller and almost unchanged at t > 1.34 h. The gel melting temperature Tm was determined from the intersection of the curves at N/N0 ) 0.5 for each UV irradiation time, as shown in Figure 4. As clearly seen in Figure 5, the 3/2 power of the melting temperature Tm is proportional to t for t e1 h and deviates
Yamamoto et al.
Figure 4. Number of microcapsules that remains insoluble as a function of temperature. The melting point was determined as the temperature when half of the particles melted in water. The symbols indicate UV irradiation times t of 0 h (b), 0.25 h (0), 0.5 h (2), 1 h (O), 1.17 h ((), 1.34 h (×), 1.5 h (4), and 10 h ()).
Figure 5. Melting temperature of gelatin microcapsules prepared with different UV irradiation times. The dotted line represents Lindemann’s law of eq 16. for t g1 h, where Ta is the melting temperature of the unirradiated gel particles. Figure 6a shows the average cross-sectional area of the microcapsules that remains insoluble, S, normalized by that at 25 °C, S0. The average of the ratio 〈S/S0〉 monotonically increases with temperature for the samples prepared at t e1 h and, once it decreases, passes a minimum and increases at high temperatures for samples prepared at t g1 h. Temperature Dependence of the Cross-Sectional Area for Particles of Varying Initial Size. The size dependence of the gel melting behavior was studied using sample B, which has a wide size distribution. Figure 7 shows the temperature dependence of the crosssectional area for microcapsules with typical initial sizes from 30 to 500 µm. Temperature Dependence of the Cross-Sectional Area for Microcapsules in Hexane. The cross-sectional area of microcapsules prepared at a UV irradiation time of 1 h without washing in ethanol and water was unchanged in hexane. From this result, it was confirmed that the size change of the particles observed in Figure 7 is related to the inflow of the medium (i.e., swelling). Size Change for Quench. The size change in the time course of repeated quenching between two temperatures above and below the sol-gel transition temperature Ta of an unirradiated gelatin solution around 28 °C was performed for the sample prepared at a UV irradiation time of 3 h. The sample was quenched from 25 to 40 °C (first heating), held at 40 °C for 60 min, quenched to 25 °C, held for 60 min, quenched again to 40 °C, and held for 60 min, as shown in Figure 8. The average size of the microcapsules changed quickly, and the equilibrium size did not change by repeating the quench.
Theoretical Consideration Let us develop the theory for the melting and swelling behaviors of the microgel particles. A microgel particle having a coreshell structure consists of a core and a shell. The core part is filled with only a single gelatin molecule solution. Then, the core part is always a sol above Ta ) 28 °C and does not play an important role in melting and swelling behaviors above Ta. Therefore, we pay attention to the shell part having a gel structure.
Melting/Swelling BehaViors of Gel Microcapsules
Langmuir, Vol. 23, No. 16, 2007 8533
Figure 8. Average cross-sectional area of gelatin microcapsules in a time course of quenching temperature between 25 and 40 °C.
Figure 6. (a) Average cross-sectional area of gelatin microcapsules normalized by that at 25 °C as a function of temperature. The symbols indicate UV irradiation times t of 0 h (O), 0.25 h (b), 0.5 h (0), 0.75 h (9), 1 h (4), 1.17 h (2), 1.25 h (3), 1.34 h (1), 1.5 h ()), and 10 h ((). (b) Fitting of the average volume of gelatin microcapsules normalized by that at 25 °C as a function of temperature by theoretical eq 43. The symbols indicate UV irradiation times of 0 h (O), 0.25 h (0), 0.5 h (4), and 1 h ()).
molecules. The other is the cross linking between the polymerized gelatin molecules by hydrogen bonds (HB cross linking). HB cross linking binds the gelatin molecules weakly. The polymerized gelatin molecules form network structures, and thus gel structures, by HB cross linking. Effect of Ultraviolet Irradiation on the Number of CrossLinking Points. To simplify the UV cross-linking scheme, let us assume that a single gelatin molecule has two inactivated binding domains and that UV activates the binding domains to cross link the two molecules and finally yields molecules with high molecular weight. We denote the number of monomers in the initial state and the number of bonds after UV irradiation for t as N and B(t), respectively. Then, the number of unreacted binding domain pairs is N - B(t). The density of the unreacted domain F is expected to be proportional to the ratio of the number of unreacted binding domain pairs to the total number of binding domains, expressed as
c(t) )
N - B(t) B(t) )1N N
(1)
The probability p that we find an unreacted binding domain in the range where an activated domain can react is proportional to F because the monomer density is not very high. Therefore, we have
p ) p0F ) p1c(t)
(2)
where p0 and p1 are positive constants. Neglecting the probability of yielding ringlike molecules, we find that the increase in the number of bonds per unit time is proportional to the number of unreacted binding domains, and the proportionality coefficient is the product of the probability of the binding reaction per unit time and the probability of finding unreacted binding domains in the range where the binding reaction can occur
dB ) Rp(t)(N - B(t)) dt Figure 7. Cross-sectional area of gelatin microcapsules with various diameters normalized by that at 25 °C as a function of temperature. The symbols indicate initial cross-sectional areas at 25 °C of 500 µm (O), 300 µm (0), 220 µm (4), 120 µm (3), and 30 µm ()).
We introduce two types of cross linking between the gelatin molecules. One is the cross linking induced by UV irradiation (UV cross linking). UV cross linking binds the “original” gelatin molecules strongly and induces the polymerization of gelatin
(3)
where the probability of making an activated domain per unit time is denoted by R. Combining eqs 2 and 3, we have
dc(t) ) -βc2(t) dt
(4)
where β ) Rp1. Because the initial condition of the above differential equation is c(0) ) 1, we have
8534 Langmuir, Vol. 23, No. 16, 2007
c(t) )
Yamamoto et al.
1 1 + βt
(5)
Using eqs 1 and 5, we obtain
〈|b| r 2〉 )
Nβt B(t) ) 1 + βt
N (P(t) - 1) P(t)
(7)
From eqs 6 and 7, the degree of polymerization P is obtained as
1 ) 1 + βt P(t) ) 1 - B(t)/N
(8)
Irradiation Time Dependence of the Melting Temperature. To estimate the melting temperature of the gel, we adopt the Lindemann picture for melting.11 The Lindemann picture is quite simple but powerful for predicting the melting point of solids. In the Lindemann picture, a solid is regarded as an assembly of particles bound with a particle-particle interaction potential and oscillating around an equilibrium point. At high temperatures, the thermal energy increases, and the thermal fluctuations enhance the particle oscillation. The solid can melt when the thermal average of the oscillation amplitude is comparable to the mean particle-particle distance; the thermal energy exceeds the particle-particle interaction potential energy. Thus, by comparing the oscillation amplitude with the particle-particle distance, we can estimate the melting temperature (Lindemann’s law11). The chainlike polymers (the polymerized gelatin molecules) yielded by UV irradiation are expected to fold and form a spherelike shape. We assume that the cross linking between the sphere-shaped polymers formed by hydrogen bonding (HB cross linking) induces gelation. The interaction energy between the sphere-shaped polymers is proportional to the number of crosslinking points Nc. Because only the hydrogen bonding sites on the surface contribute to the cross linking, the number is proportional to the surface area of the polymers: Nc ∝ P2/3. The potential energy when an arbitrary polymer shifts from the equilibrium position by b r is harmonically approximated as
1 r 2 U(b) r ) k|b| 2
(9)
The “elastic constant” (the strength of the potential) k in eq 9 depends on the hydrogen bonding interactions between polymers and is proportional to Nc and P2/3
k ) k0P
2/3
(10)
Lindemann’s law for the melting of gels suggests that gels melt (hydrogen bonds are broken) when the average positional b2|〉 is longer than the average distance between fluctuation x〈|r polymers lc, where the thermal average 〈‚‚‚〉 stands for
〈‚‚‚〉 )
(
r exp ∫ d3b(‚‚‚)
(
∫ d3br exp -
4 kB T 3 k
(12)
(6)
For simplicity, let us assume that all of the polymers yielded by UV irradiation have the same degree of polymerization P(t) at time t. Because the number of polymer chain is N/P(t), we have the number of total bonds B(t) as
B(t) )
where T is the absolute temperature and kB is the Boltzmann constant. From eq 9, we have
)
U(b) r kBT
)
U(b) r kBT
(11)
From Lindemann’s law, the melting temperature Tm is expressed as
〈|b| r 2〉 )
4 kBTm ) lc2 3 k
(13)
From eqs 10 and 13, the melting temperature of gel is expressed as
Tm ) TaP2/3
(14)
Note that Ta is the melting temperature when there is no UV irradiation. From eqs 8 and 14, the melting temperature of the polymer is rewritten as
Tm ∝ (1 + βt)2/3
(15)
The melting temperature ratio is obtained as
Tm ) (1 + βt)2/3 Ta
(16)
Temperature Dependence of the Volume of Polymers. The melting temperature discussed in the above corresponds to the temperature at which all of the hydrogen bonds are broken. The swelling behavior appears below the melting temperature. During the swelling process, the number of broken hydrogen bonds may increase with increasing temperature, and all of the hydrogen bonds are broken at the melting temperature. An increase in the number of the broken hydrogen bonds stands for the decrease in the number of the gelatin molecules constructing the gel network. Then, to analyze the swelling behaviors of the gelatin microcapsules, the molecules detached from the gel network should be taken into account. Let us assume that the gel part of the microcapsules consists of a gelatin-polymer network in a solution of non-cross-linking gelatin polymers and solvent. To use the Flory theory,12 all of the polymers are regarded as an assembly of the “segment”. Let us denote the average degree of polymerization of the non-crosslinking gelatin polymers as Pm (the segment number of noncross-linking gelatin polymers), the fraction of segments in solution in total polymer segments as γ, the volume fraction of the segments in gel as φ, and that of segments in solution as φm.
φm ) γφ
(17)
According to the Flory theory,12 the free energy of mixing per lattice site is assumed to be
f(φ) ) (1 - φ) ln(1 - φ) +
γφ ln γφ + χφ(1 - φ) (18) Pm
where χ ) B/kBT and B is a constant. We also assume that the non-cross-linking polymers do not get out from the gel part. The elastic energy of the gel part of a gel microcapsule is given by13 (11) Lindemann, F. A. Z. Phys. 1910, 11, 609. (12) Flory, P. J. Principles of Polymer Chemistry; Cornell University Press: Ithaca, NY, 1953.
Melting/Swelling BehaViors of Gel Microcapsules
Langmuir, Vol. 23, No. 16, 2007 8535
()
3 φi Ael ) nc 2 φ
2/3
(19)
where nc is the number of active chains and φi and φ are the volume fractions of gel segments before and after swelling, respectively. Because non-cross-linking polymers do not contribute to the elasticity, we have
nc = nc
0
(
)
(
Pac ∆Vi Pac 1- γ ) νc0 1 - γ Pm Vc Pm
)
(
3 Ael ) νc0 2
) ()
2/3
(
)( )
3 ∆Vi γ φi 2/3 ∆V f(φ) + νc0 1 - Pac ) Vc 2 Vc Pm φ ∆Vi φi γ φi 3 f(φ) + νc0 1 - Pac Vc φ 2 Pm φ
[
∆Vφ ) ∆Viφi
(
νc(T) ≡ νc0 1 - Pac
∂A )0 ∂φ
(
1-
(
[
(24)
)]
-
1 b0 T Pac νc0 Tm
(32)
(
)
(33)
1 b0 Pac νc0
(34)
b0 1 1≈1 Pac νc0
Then, introducing the parameter
(
)( )
γ φ νc0 1 - Pac Pm φ i
1/3
) 0 (25)
Let us consider the gel melting process when heating the solution. As the temperature increases, the gel partially melts, and non-cross-linking polymers are finally yielded; the number of active chains nc in the gel is a decreasing function of temperature. Let the temperature dependence of nc be expressed by the “renormalized number” of active chains per lattice site (the coefficient of the term proportional to (φ/φi)1/3 in eq 25):
(
(31)
The first term on the right-hand side in eq 32 is a temperatureindependent term, and the second is a temperature-dependent term. Because we could expect Pac . 1, the temperatureindependent term is reasonably approximated as
1-
νc ≡ νc0 1 - Pac
(30)
)
b0 γ 1 ) 11Pm Pac νc0
Then, we have
)
)
The coefficient of the φ linear term in eq 25 is rewritten as
(23)
γ φ + χφ2 + Pm
) (
γ T ≈ b0 1 Pm Tm
b0 b0 T 1 γ ≈ 1+ Pm Pac νc0 νc0 Tm
(22)
Because at equilibrium A should have its minimum value as a function of φ, the value of φ at equilibrium is derived from
(
(29)
It is noted that ν′c (T0) < 0 because the number of active chains is a decreasing function of temperature. At T ) Tm0, the renormalized number of active chains vanishes; νc(T) ) 0. This shows that at this temperature the elasticity of the gel disappears. Then, let the temperature Tm0 be regarded as the gel melting temperature Tm and be denoted simply by Tm from now on. Hence,
2/3
where ∆V is the gel volume after swelling. From mass conservation, we have
ln(1 - φ) + 1 -
ν′c(T0) 1 )Tm0 νc(T0) - ν′c(T0)T0
and
Note that the validity of the identification of Tm0 with Tm depends on the validity of the linear approximation (eq 27). Equation 30 gives
)( ) ]
(
(28)
(21)
The total free energy of the shell part is given by
A)
b0 ) νc(T0) - ν′c(T0)T0
(20)
where Pac is the average degree of polymerization of active chains (the number of the segments of active chains), nc0 is the number of active chains when no non-cross-linking polymers exist, νc0 is the number of active chains per lattice site before swelling when no non-cross-linking polymers exist, Vc is the volume per lattice site, and ∆Vi is the gel volume (thus the shell part volume of a gel microcapsule) before swelling. Then we have
γ ∆Vi φi 1 - Pac Pm Vc φ
where
)
γ Pm
(26)
Around a temperature T0, which is not very far from the melting temperature of the gel, νc is linearly expanded as
(
νc(T) ≈ νc(T0) + ν′c(T0)(T - T0) ) b0 1 -
T Tm0
)
(27)
(13) Doi, M. Introduction to Polymer Physics; Oxford University Press: New York, 1996; translated by See, H. from Japanese.
λ≡
we have an approximate expression
1-
T γ ≈1-λ Pm Tm
(35)
From eqs 30 and 35, eq 25 is rewritten as
(
)
T ln(1 - φ) + 1 - λ φ + χφ2 + Tm
(
b0 1 -
)( )
T φ Tm φ i
1/3
) 0 (36)
To make a simple comparison between the result from theory and that from the experiment, we introduce a scaled expression of eq 36. Because the volume V of the gel microcapsule is expected to be proportional to the volume ∆V of the shell part of the microcapsule, the swelling behavior of the shell part is equivalent
8536 Langmuir, Vol. 23, No. 16, 2007
Yamamoto et al.
to that of the gel microcapsule itself. Denoting the volumes of the gel microcapsule at T and T0 as V and V0, respectively, we can relate the volume fraction φ at T to the volume fraction φ0 at T0 as
φ)
V0 1 φ ≡ V 0 aVˆ
(37)
where Vˆ ) V/V0 is the scaled gel particle volume and a ) 1/φ0. We further introduce the scaled temperature Tˆ ) T/T0, the scaled gel melting temperature Tˆ m ) Tm/T0, the scaled parameter Bˆ ) B/kBT0a, and a new parameter b ) ab0φi-1/3a-1/3. Then, eq 36 is reduced to
[ (
Bˆ Tˆ -2 + aVˆ ln 1 -
1 λ + Vˆ + bVˆ 5/3 Tˆ -1 - Vˆ aVˆ Tˆ m 1 b Vˆ 5/3 ) 0 (38) Tˆ m
]
)
Using the expressions
(
1 + Vˆ + bVˆ 5/3 aVˆ
)
(39)
λ 1 Vˆ - b Vˆ 5/3 Tˆ m Tˆ m
(40)
C1 ) aVˆ 2 ln 1 and
C2 ) -
we finally have an equation for the scaled temperature
Bˆ Tˆ -2 + C1Tˆ -1 + C2 ) 0 Solving the above equation with respect to Tˆ
Tˆ
-1
(41) -1,
we obtain
-C1 ( xC12 - 4 Bˆ C2 ) 2Bˆ
(42)
Because C2 < 0, Bˆ > 0, and C1 < 0, we have the relationship between the scaled temperature and the scaled gel microcapsule volume (thus, the swelling behavior of the gel microcapsule) as
Tˆ
-1
-C1 + xC12 - 4Bˆ C2 ) 2Bˆ
(43)
Because Vˆ ) 1 at Tˆ ) 1, we have
(
Bˆ ) -a ln 1 -
1 λ b -1-b+ + a Tˆ m Tˆ m
)
(44)
Hence, we find that the fitting parameters in the expression (eq 43) to be derived from the experimental data are a, b, λ, and Tˆ m.
Discussion Ultraviolet rays have been known to induce radicals at specific residues such as tyrosine and phenylalanine in collagen (gelatin) molecules, resulting in the scission and cross linking of molecules.14-18 At low UV doses, it is difficult to form a network because of a deficiency in the number of radicals. However, at (14) Fujimori, E. Biopolymers 1965, 3, 115. (15) Cooper, D. R.; Davidson, R. J. Biochem. J. 1965, 97, 139. (16) Cooper, D. R.; Davidson, R. J. Biochem. J. 1966, 98, 655. (17) Weadock, K. S.; Miller, E. J.; Bellincampi, L. D.; Zawadsky, J. P.; Dunn, M. G. J. Biomed. Mater. Res. 1995, 29, 1373. (18) Lee, J.-E.; Park, J.-C.; Hwang, Y.-S.; Kim, J. K.; Kim, J.-G.; Suh, H. Yonsei Med. J. 2001, 42, 172.
a high UV dose the mobility of gelatin molecules decreases. In the latter case, further cross linking is difficult to obtain, but scission becomes predominant because the diffusion of polymer radicals is required for cross linking. Therefore, cross linking is predominant only in a limited range of UV dosage. The melting point temperature increases with increasing UV irradiation time t in the experimental range, suggesting that cross linking by UV irradiation is predominant for t < 10 h at 1520 µW/cm2 of 254 nm. The optical micrograph as shown in Figure 1 clearly indicates that the gelatin emulsion became core-shell-type particles (microcapsules), not close-packed spheres, after UV irradiation. This result is attributed to a low penetration of UV rays in gelatin solution. The microcapsules have a fairly narrow size distribution, as shown in Figure 2. It should be noted that the melting behavior depends significantly on the size of the microcapsule, as discussed below on the basis of Figures 7 and 8. As shown in Figure 4, microcapsules melt in a small range of temperature (i.e., the number of microcapsules that remains insoluble N decreases to zero sharply with increasing temperature when the UV irradiation time t is small, whereas N gradually decreases with temperature when t is large). This suggests that large t induces inhomogeneity in the microcapsule membranes. Therefore, it is meaningless to compare the experimental results with theories at large t. The 3/2 power of gel melting temperature Tm determined from the intersection of the curves when N/N0 ) 0.5 is proportional to UV irradiation time for t < 1 h, as shown in Figure 5. This is consistent with the theoretical result of eq 16. The molecular weight dependence of the melting temperature of our model is much different from the logarithmic dependence derived by Nishinari and Tanaka.19 The difference comes from the models for the cross-linking reaction in gelation. In the case of UV irradiation of a gelatin emulsion in UV nonabsorbed organic solvent, the cross linking occurs on the surface of gelatin molecules or their aggregates, and the number of cross-linking points is assumed to be proportional to the surface area of the polymers: Nc ∝ P2/3 in eq 10. It is, however, difficult to judge which equation is better to explain the experimental results because only a small range of data for t < 1 h are available in the current experiment. The observed reduced cross-sectional area S with increasing temperature for t < 1 h in Figure 6a was fitted to the scaled eq 43 fairly well, as shown in Figure 6b. We chose 25 °C as the normalization temperature T0. Within the four parameters a, b, λ, and Tˆ m in eq 43, the three parameters a, b, and λ were determined by the least-squares method because the values of Tˆ m, the melting temperatures normalized by T0, were used. The set of the four parameters (a, b, λ, Tˆ m) that we obtained were (9.5, 0.08, 0.0007, 1.0147) for t ) 0 h, (4.4, 0.15, 0.004, 1.031) for t ) 0.25 h, (5.9, 0.66, 0, 1.0587) for t ) 0.5 h, and (4.2, 0.06, 0, 1.0755) for t ) 1 h, respectively. Because λ ∝ 1/Pac, the small values of λ seems to be reasonable. However, the curves with a minimum for t > 1 h in Figure 6 cannot be derived from the functional form of the theoretical eq 43. Such swelling behavior also appeared for the initial size dependence as shown in Figure 7, where small initial size corresponds to large t. These results indicate that the reason the minimum appears is related to the UV cross-linking density because both small initial size and large t result in a large UV cross-linking density, which could yield a significant elastic force of the active chains in the gel membrane. As shown in Figure 8, the size change between 25 and 40 °C is reversible. According to circular dichroism measurements, the conformation of gelatin (collagen) molecules changes from triple helix to random coil locally by changing the temperature from 25 to (19) Tanaka, F.; Nishinari, K. Macromolecules 1996, 29, 3625.
Melting/Swelling BehaViors of Gel Microcapsules
40 °C (not shown). Thus, the initial decrease in size with increasing temperature for the samples prepared at large t or small initial size can be consistently explained by a decrease in the persistence length from the triple helix to random chain of active chains.
Conclusions The melting temperature and swelling behavior of gelatin gel microcapsules prepared by UV-induced cross linking were consistently explained by a theoretical model for small UV
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irradiation times of t < 1h or a large initial size. Elastic properties characteristic of a gel were significant at large t or a small initial size. Acknowledgment. This work was partially supported by a Grant-in-Aid for Science Research from The Ministry of Education, Culture, Sports, Science and Technology of Japan (grant no. 16540366). LA700606E
