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Surface structure of finely dispersed activated iron with immobilized interferon as derived from XPS
Journal of Electron Spectroscopy and Related Phenomena 76 (1995) 689-694
Surface structure of finely dispersed activated iron with immobilized interferon as derived from XPS O.M. Mikhailika, Yu.V. Pankratov a, I.S. Nikol'skya, Ed.A. Bakaia, A.I. Senkevichb, A.P. Shpak b aResearch SONAR Center for Biotechnical Systems, Ukrainian National Academy of Sciences, Peremogy Prospect 52/2, 252057 Kiev, Ukraine* bInstitute of Metal Physics, Ukrainian National Academy of Sciences, Vemadsky bulvar 36, 252142 Kiev, Ukraine The surface structure of freely dispersed iron (FDI) activated by 1,6-hexamethylene diisocyanate and that of resultant FDI with immobilized recombinant human a-interferon was examined by X-ray photoelectron spectroscopy (XPS). The organization of protein at the surface layer rather than the amount or thickness of protein films was shown to play a key part in the activity of immobilized interferon. The orientation of immobilized protein molecules in self magnetic field at the FDI surface was suggested to stabilize interferon activity. 1. INTRODUCTION Interferon is known to be an active immunopotentiator and antitumor factor. An attractive approach enabling one to obtain this cytokine at a desired concentration in a required part of the organism involves the formation of interferon immobilized to the F D I surface which acts as a magnetic substrate. A number of factors may govern the activity of immobilized protein. These include (a) the amount of protein adsorbed, (b) the thickness of the protein layer, (c) the degree of its binding to the surface, (d) the spatial arrangement of protein at the surface, (e) protein denaturation or alteration by adsorption or chemisorption and others [ 1]. X-ray photoelectron spectroscopy was proved to be the most valuable technique that is at present available for characterization of biomaterial surfaces. In this paper the XPS evidence has been used in an attempt to elucidate the reasons of drastic differences in *Acknowledgment- The support from the State Committee o n Sciences and Technologies o f the ~ is gratefully aeknowledged.
SSD1 0368-2048(95) 02501-4
antiviral activity of a series of immobilized interferon samples. 2. MATERIALS AND METHODS Spherical FDI samples with mean diameter ca 80 nm were stabilized by spontaneous adsorption of 1:1:0.1 2hydroxyethyl methacrylate, acrylamide and N,N'-metylen-b/s-acrylamide (Sample Ia), 25:1 2,3-epoxypropyl methacrylate and N,N'-methylen-bis-acrylamide (Sample IIa), dextran (Sample IIIa) from 5 wt~ solutions in an inert atmosphere. Sample IVa was obtained via plasma-condensation of v-amino-propyltriethoxysilane on the FDI surface. Activated FDI samples Ib-IVb have been prepared by treatment of samples Ia-IVa with 1,6-hexamethylene diisocyanate in a dry aprotonic solvent (methylene chloride) using tetrabutyltitanate as a catalyst [2]. The grafted activator amounts derived from the elemental analysis (I) are given in Table 1. Immobilized recombinant human c~interferon samples Ic-IVc were obtained by a 2 h storage of activated samples Ib-IVb
690
Table 1 XPS results, modifier film depth (d), nonmagnetic surface layer thickness (1), grafted 1,6-hexamethylene diisocyanate (I) and chemisorbed protein (P) amounts, antiviral activity, initial (Ao) and after 5 weeks keeping in suspension (A1) for FDI samples. Samples are the same as in MATERIALS AND METHODS
Sample
Relative surface elemental composition (ate)
Core level intensity ratios
Ao(A I)
Fe(2p) C
N
O
Si(S) Fc(3p) N/C C/S
Ia lb Ic
2.5 0.33 0.29
46.3 3.2 48.0 15.8 79.9 9.1 10.8 3.62 77.8 11.4 10.15 (0.37) 4.06
0.069 0.11 0.15 210
50 (50)
IIa lib IIc
8.3 1.37 0.28
34.8 2 . 2 4 54.6 21.5 76.8 3.6 18.2 11.9 79.05 2.71 1 6 . 7 (1.24) 1 2 . 0
0.064 0.046 0.034 64
0 (3)
IIIa IIIb IIIc
3.8 0.1 0.28
51.1 1.2 43.6 12.3 0.023 77.5 1.9 20.5 10.5 0.024 78.3 6.92 1 2 . 2 (2.37) 10.65 0.088 33
IVa IVb IVe
2.0 0.6 0.16
54.7 0 . 9 8 40.0 76.1 13.5 9.1 84.0 2.8 11.9
2.4 24.8 0.018 0.85 5.5 0.17 (1.1) 11.44 0.033 76
with an interferon solution in phosphate buffer at p H 7.0 and 4°C with subsequent thorough rinsing to remove unbound protein. Table 1 lists the amount of chemisorbed protein (P) estimated from the decrease o f protein in the contact solution aRer chemisorption with allowance for protein that is desorbed during sample rinsing. The antiviral activity (A) of suspensions of immobilized interferon was recurrently determined via suppression o f cytopathogenic effect [3] for 5 weeks (Table I). It is immobilized interferon rather than supel~atant that is responsible for the detected activity. The FDI sample containing h u m a n serum albumin adsorbed was chosen as the reference. X-ray photoelectron spectra were recorded with a Kratos Analytical Series-800 XPS electron spectrometer employing M g I ~ exciting radiation (1253.6 eV). An instrument vacuum o f at least 10-9 Torr was
d
I
(rel.un.) (nm) (nm)
P (mill)
7.0
-
-
0.15
-
9.7
8.0
-
0.6
2.9
0.35
15.5
5.0 5.5
0.4
5.7 3.2 (1.6) 19.5
8.0 8.5
0.75
1.1
6.O 16.2
6.O 6.7
0.47
O.75
3.2 (0)
7.2
I (mM/g)
-
maintained for all analyses. The instrument was calibrated for Au4fT/2 peak binding energy o f 84,0 eV. The binding energies for the XPS spectra were referenced to the hydrocarbon component of the Cls spectrum at 285.0 eV. The F D I P samples were attached to adhesive tape. By comparing the intensity ratios of two photoemission Fe2p and Fe3p peaks from iron atoms the thickness o f the overlayer (d) has been approximated (Table 1). The ESR spectra have been obtained on a EPA-10 mini spectrometer (St. Petersburg Instruments, Russia) at temperature 100 K and a superhigh frequency o f 9.30 GHz. Nonmagnetic layer thickness on the F D I surface (1) has been calculated from the ferromagnetic resonance line width using our model [4] for ferromagnetic resonance in aggregates of magnetic nanoparticles (Tablel). The XPS spectra are well-fitted by using Gaussian-Lorentzian peak shapes (50%
691 Table 2 XPS spectral features for finely dispersed iron, samples are the same as those in Table 1 and in MATERIALS A N D METHODS Fe3p s ~ c t r a Cls sncetra Ols (S2p) st~etra ~JL(SJ2II)~II~q,I~ Sample Ep, F W H M I, a Ep, FWHM I, cr Ep, FWHM I, cr Ep, F W H M I,
(ev)
(~;)
Ia
54.0, 55.0, 56.4, 57.5,
1.97 2.03, 1.99, 2.03,
1.9, 65.1, 19.0, 14.0,
Ib
53.6, 54.4, 55.7, 56.6, 58.1, 54.5, 55.8, 56.6, 58.5,
1.82 1.97 2.03 1.86 2.04 1.88 1.89 1.89 1.92
13, 5.6 46, 5.6 36, 5.6 0.2, 5.6 4.8, 5.6 39, 6.5 56, 6.5 1, 6.5 4, 6.5
51.9, 54.0, 55.6, 56.7, 57.4, 51.8, 52.9, 55.5, 57.2, 51.0,
1.98 2.05 1.96 1.99 1.96 2.00 2.01 2.02 2.02 1.98
15, 3.4 20, 3.4 42, 3.4 18, 3.4 5, 3.4 41, 2.8 11, 2.8 18, 2.8 6, 2.8 23, 2.8
IIc
51.8, 53.0, 55.7, 57.1, 50.6,
2.04 1.96 2.04 1.95 2.03
52, 2.4 7.2, 2.4 22, 2.4 6.8, 2.4 12, 2.4
IIIa
51.6, 53.4, 55.1, 56.4, 57.4,
1.99 2.04 2.07 1.97 1.97
27, 2.7 27, 2.7 29, 2.7 12, 2.7 5, 2.7
llIb
51.7, 53.2, 55.0, 57.2, 50.8,
2.01 1.97 2.01 1.97 1.96
46, 1.7 23, 1.7 11, 1.7 0.3, 1.7 21, 1.7
Ic
IIa
IIb
1.2 1.2 1.2 1.2
(~;)
(~;)
(eV)
284.4, 1.43, 285.0, 1.55 286.0, 1.57 288.1, 1.59 289.1, 1.56 284.7, 1.45 285.0, 1.52 287.5 1.54 285.7, 1.54 289.0, 1.54 284.4, 1.46, 285.0, 1.54, 286.0, 1.52, 287.5, 1.50, 288.0, 1.50, 289.2, 1.52, 283.6, 1.54 285.0, 1.54 287.5, 1.50 286.2, 1.54 288.7, 1.47 283.96, 1.49 284.96, 1.51 286.02, 1.50 287.47. 1.5 288.7, 1.5 290.44, 1.5 284.0, 1.52 285.0, 1.52 287.3, 1.48 286.0, 1.52 288.9, 1.48 290.4, 1.51 284.3, 1.51 285.1, 1.48 287.6 1.49 286.4 1.51 288.9 1.5 290.4 1.5 283.9 1.48 284.9 1.51 286.2 1.51 287.5 1.51 288.7 1.50 290.4 1.50
20, 2.6 46, 2.6 21, 2.6 11, 2.6 3,2.6 45, 2.6 9.0, 2.6 5.8, 2.6 31, 2.6 8.4, 2.6 19, 2.2 44, 2.2 24, 2.2 1.2, 2.2 3.3, 2.2 7.7, 2.2 19, 2.7 36, 2.7 13, 2.7 24, 2.7 8,2.7 16, 1.8 68, 1.8 13, 1.8 0.4, 1.8 1.8, 1.8 0.5, 1.8 18, 2.4 46, 2.4 2, 2.4 2.5, 2.4 5, 2.4 3.4, 2.4 21, 1.5 26, 1.5 13, 1.5 33, 1.5 5.5, 1.5 1.5, 1.5 16, 2.3 49, 2.3 23, 2.3 7.9, 2.3 3.7, 2.3 0.8, 2.3
529.9, 531.3, 531.9, 533.1,
1.87 1.89 1.91 1.84
63.3, 4.4 398.7, 1.83 19.4. 4.4 400.0, 1.98 17.3. 4.4 401.3, 1.96 5.0, 4.4
20.8, 1.7 57.9, 1.7 21.4, 1.7
529.3, 531.2, 531.9, 533.1,
1.79 1.8 1.8 1.87
29, 2.7 399.2, 1.95 45, 2.7 399.6, 1.86 14.7, 2.7 12, 2.7
16, 2.4 84, 2.4
530.0, 1.82 531.0, 1.95 531.8, 1.86 533.6, 1.85 (163.2, 2.0 164.9, 2.0) 529.1, 1.98 530.6, 2.03 531.7, 2.03 532.5, 1.97
20.8, 4.5 8.6, 4.5 54.1, 4.5 16.5, 4.5 (22, 17 78, 17) 7, 2.7 52, 2.7 22, 2.7 19, 2.7
399.5, 1.93 399.9, 1.94
31, 4.0 69, 4.0
399.3, 2.0 400.9, 2.04 402.4, 2.04
7, 5.0 56, 5.0 37, 5.0
530.49, 1.86 532.18, 1.8 533.28, 2.04
43, 3.5 39, 3.5 18, 3.5
399.38, 1.87 44, 3.4 402.47, 1.87, 0.25, 3.4 400.51, 1.86 55, 3.4
530.1, 1.85 530.6, 1.85 532.0, 1.91 533.4, 2.04 (165.5, 2.03 167.12, 2.03) 530.2, 1.88 531.2, 1.90 531.9, 1.89 533.3, 1.88
5.5, 2.8 34, 2.8 48, 2.8 13, 2.8 (88, 4.4 12, 4.4) 34, 3.0 2, 3.0 23, 3.0 41, 3.0
399.0, 2.04 402.5, 1.87 400.4, 1.96
21, 3.0 0.4, 3.0 79, 3.0
396.3, 2.02 400.0, 2.02 397.6, 1.98
33, 4.3 36, 4.3 31, 4.3
530.1, 530.7, 532.3, 533.3,
0.8, 2.4 11, 2.4 34, 2.4 54, 2.4
399.1, 1.92 402.8, 1.88 399.9, 2.03
40, 2.7 0.5, 2.7 59.5, 2.7
1.86 1.86 1.86 1.97
(ev)
(~;)
(eV)
key: Ep, peak energy; FWHM, full width at half maximum height; I, relative intensity; c, mean deviation
692
Table 2 Continued
Sample
Fe3o soectra Ep, FWHM I, ~
Cls soectm Ep, FWHM
(eV)
(~¢)
(eV) 283.9, 285.0, 286.0, 287.3, 288.8,
IIIc
51.8, 53.2, 55.1, 57.1, 50.8,
2.02 1.96 2.02 1.98 2.01
57, 1.9 10, 1.9 9, 1.9 2.3, 1.9 22, 1.9
IVa
54.6, 55.9, 57.2, 58.8,
1.99 1.97 2.1 2.0
17, 3.2 45, 3.2 25, 3.2 13, 3.2
IVb
52.6, 54.4, 55.8, 56.9, 58.4,
2.03 2.04 1.95 2.03 1.89
51.8, 53.1, 55.6, 57.2, 50.6,
2.02 2.02 2.02 2.04 1.98
IVc
1.51 1.5 1.5 1.49 1.48
284.77 1.49 285.0 1.49 286.06 1.51 287.47 1.50 288.76 1.49 290.25 1.51 22, 4.4 284.3 1.50 32, 4.4 285.1 1.52 32, 4.4 287.9 1.51 16, 4.4 286.2 1.49 0.6, 4.4 288.9 1.49 290.6 1.51 49, 6.6 284.0 1.47 15, 6.6 285.1 1.52 9.7, 6.6 286.2 1.52 5.5, 6.6 287.6 1.48 20.8, 6.6 289.1 1.48 290.4 1.49
Ols (S2p) s_tmetm Ep, FWHM I, ~
I,a
(~)
7.8, 2.4 76, 2.4 13, 2.4 0.4, 2.4 2.7, 2.4 15, 3.9 35, 3.9 20, 3.9 7, 3.9 17, 3.9 5, 3.9 34, 1.5 36, 1.5 6.6, 1.5 15.5, 1.5 5.7, 1.5 1.7, 1.5 20, 2.6 49, 2.6 20, 2.6 4.2, 2.6 4.8, 2.6 2.4, 2.6
(eV)
(~)
530.0, 1.86 531.1, 1.86 532.2, 2.02 533.6, 2.03 (166.2, 2.03 167.4, 2.03) 530.69, 1.87 532.23, 1.89 532.79, 2.01
0.5, 2.7 22, 2.7 55, 2.7 23, 2.7 (66, 3.2 34, 3.2) 46, 3.9 31, 3.9 24, 3.9
529.7, 1.99 531.5, 1.93 531.8, 1.90 533.2, 2.00
13, 48, 26, 15,
530.3, 1.86 531.2, 2.03 531.8, 1.99 533.0, 2.04 (166.50, 1.98 167.6, 2.02)
9, 2.5 11, 2.5 53, 2.5 26, 2.5 (77, 4.1 23, 4.1)
Ep, FWHM
I,
399.1, 2.04 402.3, 1.86 400.2, 1.99
22, 3.8 8, 3.8 70, 3.8
399.11, 1.98 400.52, 1.98 401.84, 1.99 (100.7, 2.04 102.7, 1.97 103.8, 1.99) 399.2, 1.90 399.9, 1.94
20, 6.3 60, 6.3 20, 6.3 (40, 6.9 14, 6.9 46, 6.9) 28, 3.2 72, 3.2
(101.5, 1.87 102.7, 1.95 103.5, 1.92) 398.9,1.92 401.5, 1.93 400.1, 1.92
(54, 6.6 42, 6.6 3, 6.6) 5, 2.2 1, 2.2 94, 2.2
(eV)
2.5 2.5 2.5 2.5
(~)
key: Ep, peak energy; FWHM, full width at half maximum height; I, relative intensity; o, mean deviation Gaussian fraction), Shirley's base line correction function was subtracted, and the ESR spectra by a Lorentzian profile derivative. M i n i m u m deviation from the experimental spectrum was used as a "bestfit" criterion, widths o f the composite spectra were allowed to vary during the fitting procedure. The full curve is least squares fit o f the experimental points. The gradient m e t h o d was used for optimization o f the position and width o f lines. XPS binding energies are reported to i-0.1 eV (Table 2). 3. R E S U L T S A N D D I S C U S S I O N The data obtained indicate that immobilized interferon activity is dramatically
different. Sample lc exhibits a high antiviral activity which remained unchanged when it was stored in suspension for at least 5 weeks. At the same time no activity was observed or detected in trace amounts in suspensions containing samples IIc-IVc. These results were quite unexpected because the amount o f strongly bound protein (P) and immobilized layer thickness values determined by the XPS method for samples Ic-IVc were nearly the same. It should be noted that the adsorbed layer depth derived from XPS and ESR evidence were well correlated for a series of samples (Ia,b-IVa,b). High local pressure in contact points in aggregates can be responsible for a decrease in nonmagnetic layer depth in contact points o f particles in
693
A
B S
50
53
N
N
160
162
165
167
280 283 286 289
395 397 400 402
B I N D I N G ENERGY eV Figure 1.(A) Fe3p, S2P, Cls and Nls fragments of XPS spectra for finely dispersed iron samples with immobilized interferon: (a) Sample Ic, (b) Sample IIc, (c) Sample IIIc, (d) Sample IVc; (B) Nls fragments of XPS spectra for freely dispersed iron samples activated by 1,6-hexamethylene diisocyanate: (a) Sample lb, (b) Sample Ilb, (c) Sample IIIb, (d) Sample IVb. Samples are the same as in MATERIALS AND METHODS. samples with immobilized protein (Samples Ic-IVc). When samples are activated via an isocyanate procedure, tetrabutyltitanate catalyses the formation of an urethane bond which is stable toward hydrolysis. A simplified scheme for this activation reaction can be visualized as follows: I-OH + O=C=N(CH2)6NffiCffiO $ Ti(OR) 4 I-CO-NH(CH2) 6NffiCffiO A subsequent binding of protein occurs with participation of amino or thiol groups apt to interact with an isocyanate group. The Cls spectra for samples Ib-IVb activated via an isocyanate procedure (Figure 1B, Table 2)
contain components at 286 eV which can be assigned to the carbon atoms adjacent to nitrogen in the urethane or isocyanate groups, -OC(O)NH-CH 2- or -CH2-NffiCffiO, and the components at 289 eV to carbon involved in the methane groups, -OC(O)-NH- [5]. The N ls spectra for the same samples show components at ca 399.9 eV which can be assigned to the urethane bond nitrogen. A relative intensity of the components at 399.2 eV characteristic of nitrogen in the isocyanate groups [6,7] is rather high for all samples IbIVb (16-44~). Thus the activation was efficient for all samples. Appreciable discrepancies were observed only in a thin surface layer structure for
694
samples containing immobilized interferon. A great nitrogen/carbon and carbon/sulphur ratios were observed only for sample Ic displaying high interferon activity unlike samples IIc-IVc (Table 1). These data suggest that the spatial arrangement of immobilized interferon film is essentially different for active and inactive samples. Nitrogen present in the protein structure in active sample Ic is preferentially oriented toward outer surface and sulphur is oriented toward F D I iron core surface. Inactive samples IIc-IVc show a reverse tendency, namely, nitrogen atoms arc oriented toward the F D I surface. It is clearly seen from the differences observed in S2p and Fe3p photoelectron spectra given in Figure 1A. The S2p line for the sample Ic contain a component at 163.2 eV typical of sulphide [8]. In this case the spectrum does not show intense components with a low binding energy (51.8 eV) which can be associated only with iron nitride formed at the interface (Samples IIc-IVc) as a result of interaction with protein amino groups (Table 2) [9]. The mobility of functional groups on the polymer surface has been elegantly demonstrated. The solvent nature was shown to reversibly and markedly affect the surface concentration of amino groups involved in polyethylene surface modified in plasma [10]. At the same time it is also obvious from our data that the 'substrate' properties can to great extent influence the surface layer structure, organization, orientation and distribution of functional groups. A surprising stability of immobilized interferon (Sample IC) at least for 5 weeks upon storage in suspension based on physiological solution should be again emphasized. The orientation of protein frasments in self magnetic F D I field, which near the surface may attain 6T, can participate in protein structure stab'tUzation at the interface. The high-intensity component at a binding energy of ca 284 eV in the all samples studied correspondin8 to ~complexes of unsaturated adjacent molecules [II] is indicative of a high ordering of unsaturated molecules in adsorbed layer. A
prolonged (about year) activity of tripsin immobilized on fmely dispersed magnetite surface was observed and also attributed to a magnetic field effect [12]. CONCLUSIONS The spatial arrangement of protein in the adsorbed layer rather than protein amount on the surface is largely responsible for the activity or nonactivity of recombinant human interferon immobilized on the F D I surface. Unlike inactive samples, the active sample is characterized by preferential orientation of nitrogen-containing groups toward outer surface, whereas sulphur-containing groups toward the FDI substrate surface. The self magnetic F D I field can serve as a factor of stab'flization of immobilized protein structure and activity. REFERENCES 1. B.D. Ratner, T.A. Horbctt, D. Shuttleworth and H.R. Thomas, J. Colloid Interface Sci., 83 (1981) 630. 2. H.D. Lchmann. Method of binding of biologically active material to a carrier containing hydroxyl groups. EP No. 0052365 (1982). 3. W. E. Stuart II, Effect of interferon on Cells, Viruses and the Immune System (ed. A. Geraldcs), Academic, New York, 1975. 4. O. M. Mikhaflik, Yu.V. Pankratov and E.A. Bakai, J. Magn. Magn. Mater., 122 (1993) 379. 5. V.S. DA Costa, D. Bmer-Russel, E.W.Salzman and E.W. Merril, J. Colloid Interface Sci., 80 (1981) 445. 6. S. Shauwaert, R. I.azzaxony, J. Riga, et al., J.Chem Phys., 92 (1990) 2187. 7. E. T. Kang, K. G. Neon, S. H. Khor, et al., Polymer, 31 (1990) 202. 8. C.D. Wagner, J. Vac. Sci. Teclmol., 15 (1978) 518. 9. McIntyre N.S., Zetamk D.G., Anal. Chem. 49 (1977) 1521. 10. D. S. Everhait and C.N. Reilley, Surface Interface Anal., 3 (1981) 126. 11. O.M. Mikhailik, Yu.V. Pankmtov, V.I.Povstuga~ et al., Colloids Surfaces, A: 90 (1994) 111. 12. V.G. Bendikene, A.A. Razunas, B.A. Yuodka. In: Abstracts of Ill Conference on Magnetic Fluids in Biology and Medicine, Suhumy, 1989, P. 35.
Surface structure of finely dispersed activated iron with immobilized interferon as derived from XPS O.M. Mikhailika, Yu.V. Pankratov a, I.S. Nikol'skya, Ed.A. Bakaia, A.I. Senkevichb, A.P. Shpak b aResearch SONAR Center for Biotechnical Systems, Ukrainian National Academy of Sciences, Peremogy Prospect 52/2, 252057 Kiev, Ukraine* bInstitute of Metal Physics, Ukrainian National Academy of Sciences, Vemadsky bulvar 36, 252142 Kiev, Ukraine The surface structure of freely dispersed iron (FDI) activated by 1,6-hexamethylene diisocyanate and that of resultant FDI with immobilized recombinant human a-interferon was examined by X-ray photoelectron spectroscopy (XPS). The organization of protein at the surface layer rather than the amount or thickness of protein films was shown to play a key part in the activity of immobilized interferon. The orientation of immobilized protein molecules in self magnetic field at the FDI surface was suggested to stabilize interferon activity. 1. INTRODUCTION Interferon is known to be an active immunopotentiator and antitumor factor. An attractive approach enabling one to obtain this cytokine at a desired concentration in a required part of the organism involves the formation of interferon immobilized to the F D I surface which acts as a magnetic substrate. A number of factors may govern the activity of immobilized protein. These include (a) the amount of protein adsorbed, (b) the thickness of the protein layer, (c) the degree of its binding to the surface, (d) the spatial arrangement of protein at the surface, (e) protein denaturation or alteration by adsorption or chemisorption and others [ 1]. X-ray photoelectron spectroscopy was proved to be the most valuable technique that is at present available for characterization of biomaterial surfaces. In this paper the XPS evidence has been used in an attempt to elucidate the reasons of drastic differences in *Acknowledgment- The support from the State Committee o n Sciences and Technologies o f the ~ is gratefully aeknowledged.
SSD1 0368-2048(95) 02501-4
antiviral activity of a series of immobilized interferon samples. 2. MATERIALS AND METHODS Spherical FDI samples with mean diameter ca 80 nm were stabilized by spontaneous adsorption of 1:1:0.1 2hydroxyethyl methacrylate, acrylamide and N,N'-metylen-b/s-acrylamide (Sample Ia), 25:1 2,3-epoxypropyl methacrylate and N,N'-methylen-bis-acrylamide (Sample IIa), dextran (Sample IIIa) from 5 wt~ solutions in an inert atmosphere. Sample IVa was obtained via plasma-condensation of v-amino-propyltriethoxysilane on the FDI surface. Activated FDI samples Ib-IVb have been prepared by treatment of samples Ia-IVa with 1,6-hexamethylene diisocyanate in a dry aprotonic solvent (methylene chloride) using tetrabutyltitanate as a catalyst [2]. The grafted activator amounts derived from the elemental analysis (I) are given in Table 1. Immobilized recombinant human c~interferon samples Ic-IVc were obtained by a 2 h storage of activated samples Ib-IVb
690
Table 1 XPS results, modifier film depth (d), nonmagnetic surface layer thickness (1), grafted 1,6-hexamethylene diisocyanate (I) and chemisorbed protein (P) amounts, antiviral activity, initial (Ao) and after 5 weeks keeping in suspension (A1) for FDI samples. Samples are the same as in MATERIALS AND METHODS
Sample
Relative surface elemental composition (ate)
Core level intensity ratios
Ao(A I)
Fe(2p) C
N
O
Si(S) Fc(3p) N/C C/S
Ia lb Ic
2.5 0.33 0.29
46.3 3.2 48.0 15.8 79.9 9.1 10.8 3.62 77.8 11.4 10.15 (0.37) 4.06
0.069 0.11 0.15 210
50 (50)
IIa lib IIc
8.3 1.37 0.28
34.8 2 . 2 4 54.6 21.5 76.8 3.6 18.2 11.9 79.05 2.71 1 6 . 7 (1.24) 1 2 . 0
0.064 0.046 0.034 64
0 (3)
IIIa IIIb IIIc
3.8 0.1 0.28
51.1 1.2 43.6 12.3 0.023 77.5 1.9 20.5 10.5 0.024 78.3 6.92 1 2 . 2 (2.37) 10.65 0.088 33
IVa IVb IVe
2.0 0.6 0.16
54.7 0 . 9 8 40.0 76.1 13.5 9.1 84.0 2.8 11.9
2.4 24.8 0.018 0.85 5.5 0.17 (1.1) 11.44 0.033 76
with an interferon solution in phosphate buffer at p H 7.0 and 4°C with subsequent thorough rinsing to remove unbound protein. Table 1 lists the amount of chemisorbed protein (P) estimated from the decrease o f protein in the contact solution aRer chemisorption with allowance for protein that is desorbed during sample rinsing. The antiviral activity (A) of suspensions of immobilized interferon was recurrently determined via suppression o f cytopathogenic effect [3] for 5 weeks (Table I). It is immobilized interferon rather than supel~atant that is responsible for the detected activity. The FDI sample containing h u m a n serum albumin adsorbed was chosen as the reference. X-ray photoelectron spectra were recorded with a Kratos Analytical Series-800 XPS electron spectrometer employing M g I ~ exciting radiation (1253.6 eV). An instrument vacuum o f at least 10-9 Torr was
d
I
(rel.un.) (nm) (nm)
P (mill)
7.0
-
-
0.15
-
9.7
8.0
-
0.6
2.9
0.35
15.5
5.0 5.5
0.4
5.7 3.2 (1.6) 19.5
8.0 8.5
0.75
1.1
6.O 16.2
6.O 6.7
0.47
O.75
3.2 (0)
7.2
I (mM/g)
-
maintained for all analyses. The instrument was calibrated for Au4fT/2 peak binding energy o f 84,0 eV. The binding energies for the XPS spectra were referenced to the hydrocarbon component of the Cls spectrum at 285.0 eV. The F D I P samples were attached to adhesive tape. By comparing the intensity ratios of two photoemission Fe2p and Fe3p peaks from iron atoms the thickness o f the overlayer (d) has been approximated (Table 1). The ESR spectra have been obtained on a EPA-10 mini spectrometer (St. Petersburg Instruments, Russia) at temperature 100 K and a superhigh frequency o f 9.30 GHz. Nonmagnetic layer thickness on the F D I surface (1) has been calculated from the ferromagnetic resonance line width using our model [4] for ferromagnetic resonance in aggregates of magnetic nanoparticles (Tablel). The XPS spectra are well-fitted by using Gaussian-Lorentzian peak shapes (50%
691 Table 2 XPS spectral features for finely dispersed iron, samples are the same as those in Table 1 and in MATERIALS A N D METHODS Fe3p s ~ c t r a Cls sncetra Ols (S2p) st~etra ~JL(SJ2II)~II~q,I~ Sample Ep, F W H M I, a Ep, FWHM I, cr Ep, FWHM I, cr Ep, F W H M I,
(ev)
(~;)
Ia
54.0, 55.0, 56.4, 57.5,
1.97 2.03, 1.99, 2.03,
1.9, 65.1, 19.0, 14.0,
Ib
53.6, 54.4, 55.7, 56.6, 58.1, 54.5, 55.8, 56.6, 58.5,
1.82 1.97 2.03 1.86 2.04 1.88 1.89 1.89 1.92
13, 5.6 46, 5.6 36, 5.6 0.2, 5.6 4.8, 5.6 39, 6.5 56, 6.5 1, 6.5 4, 6.5
51.9, 54.0, 55.6, 56.7, 57.4, 51.8, 52.9, 55.5, 57.2, 51.0,
1.98 2.05 1.96 1.99 1.96 2.00 2.01 2.02 2.02 1.98
15, 3.4 20, 3.4 42, 3.4 18, 3.4 5, 3.4 41, 2.8 11, 2.8 18, 2.8 6, 2.8 23, 2.8
IIc
51.8, 53.0, 55.7, 57.1, 50.6,
2.04 1.96 2.04 1.95 2.03
52, 2.4 7.2, 2.4 22, 2.4 6.8, 2.4 12, 2.4
IIIa
51.6, 53.4, 55.1, 56.4, 57.4,
1.99 2.04 2.07 1.97 1.97
27, 2.7 27, 2.7 29, 2.7 12, 2.7 5, 2.7
llIb
51.7, 53.2, 55.0, 57.2, 50.8,
2.01 1.97 2.01 1.97 1.96
46, 1.7 23, 1.7 11, 1.7 0.3, 1.7 21, 1.7
Ic
IIa
IIb
1.2 1.2 1.2 1.2
(~;)
(~;)
(eV)
284.4, 1.43, 285.0, 1.55 286.0, 1.57 288.1, 1.59 289.1, 1.56 284.7, 1.45 285.0, 1.52 287.5 1.54 285.7, 1.54 289.0, 1.54 284.4, 1.46, 285.0, 1.54, 286.0, 1.52, 287.5, 1.50, 288.0, 1.50, 289.2, 1.52, 283.6, 1.54 285.0, 1.54 287.5, 1.50 286.2, 1.54 288.7, 1.47 283.96, 1.49 284.96, 1.51 286.02, 1.50 287.47. 1.5 288.7, 1.5 290.44, 1.5 284.0, 1.52 285.0, 1.52 287.3, 1.48 286.0, 1.52 288.9, 1.48 290.4, 1.51 284.3, 1.51 285.1, 1.48 287.6 1.49 286.4 1.51 288.9 1.5 290.4 1.5 283.9 1.48 284.9 1.51 286.2 1.51 287.5 1.51 288.7 1.50 290.4 1.50
20, 2.6 46, 2.6 21, 2.6 11, 2.6 3,2.6 45, 2.6 9.0, 2.6 5.8, 2.6 31, 2.6 8.4, 2.6 19, 2.2 44, 2.2 24, 2.2 1.2, 2.2 3.3, 2.2 7.7, 2.2 19, 2.7 36, 2.7 13, 2.7 24, 2.7 8,2.7 16, 1.8 68, 1.8 13, 1.8 0.4, 1.8 1.8, 1.8 0.5, 1.8 18, 2.4 46, 2.4 2, 2.4 2.5, 2.4 5, 2.4 3.4, 2.4 21, 1.5 26, 1.5 13, 1.5 33, 1.5 5.5, 1.5 1.5, 1.5 16, 2.3 49, 2.3 23, 2.3 7.9, 2.3 3.7, 2.3 0.8, 2.3
529.9, 531.3, 531.9, 533.1,
1.87 1.89 1.91 1.84
63.3, 4.4 398.7, 1.83 19.4. 4.4 400.0, 1.98 17.3. 4.4 401.3, 1.96 5.0, 4.4
20.8, 1.7 57.9, 1.7 21.4, 1.7
529.3, 531.2, 531.9, 533.1,
1.79 1.8 1.8 1.87
29, 2.7 399.2, 1.95 45, 2.7 399.6, 1.86 14.7, 2.7 12, 2.7
16, 2.4 84, 2.4
530.0, 1.82 531.0, 1.95 531.8, 1.86 533.6, 1.85 (163.2, 2.0 164.9, 2.0) 529.1, 1.98 530.6, 2.03 531.7, 2.03 532.5, 1.97
20.8, 4.5 8.6, 4.5 54.1, 4.5 16.5, 4.5 (22, 17 78, 17) 7, 2.7 52, 2.7 22, 2.7 19, 2.7
399.5, 1.93 399.9, 1.94
31, 4.0 69, 4.0
399.3, 2.0 400.9, 2.04 402.4, 2.04
7, 5.0 56, 5.0 37, 5.0
530.49, 1.86 532.18, 1.8 533.28, 2.04
43, 3.5 39, 3.5 18, 3.5
399.38, 1.87 44, 3.4 402.47, 1.87, 0.25, 3.4 400.51, 1.86 55, 3.4
530.1, 1.85 530.6, 1.85 532.0, 1.91 533.4, 2.04 (165.5, 2.03 167.12, 2.03) 530.2, 1.88 531.2, 1.90 531.9, 1.89 533.3, 1.88
5.5, 2.8 34, 2.8 48, 2.8 13, 2.8 (88, 4.4 12, 4.4) 34, 3.0 2, 3.0 23, 3.0 41, 3.0
399.0, 2.04 402.5, 1.87 400.4, 1.96
21, 3.0 0.4, 3.0 79, 3.0
396.3, 2.02 400.0, 2.02 397.6, 1.98
33, 4.3 36, 4.3 31, 4.3
530.1, 530.7, 532.3, 533.3,
0.8, 2.4 11, 2.4 34, 2.4 54, 2.4
399.1, 1.92 402.8, 1.88 399.9, 2.03
40, 2.7 0.5, 2.7 59.5, 2.7
1.86 1.86 1.86 1.97
(ev)
(~;)
(eV)
key: Ep, peak energy; FWHM, full width at half maximum height; I, relative intensity; c, mean deviation
692
Table 2 Continued
Sample
Fe3o soectra Ep, FWHM I, ~
Cls soectm Ep, FWHM
(eV)
(~¢)
(eV) 283.9, 285.0, 286.0, 287.3, 288.8,
IIIc
51.8, 53.2, 55.1, 57.1, 50.8,
2.02 1.96 2.02 1.98 2.01
57, 1.9 10, 1.9 9, 1.9 2.3, 1.9 22, 1.9
IVa
54.6, 55.9, 57.2, 58.8,
1.99 1.97 2.1 2.0
17, 3.2 45, 3.2 25, 3.2 13, 3.2
IVb
52.6, 54.4, 55.8, 56.9, 58.4,
2.03 2.04 1.95 2.03 1.89
51.8, 53.1, 55.6, 57.2, 50.6,
2.02 2.02 2.02 2.04 1.98
IVc
1.51 1.5 1.5 1.49 1.48
284.77 1.49 285.0 1.49 286.06 1.51 287.47 1.50 288.76 1.49 290.25 1.51 22, 4.4 284.3 1.50 32, 4.4 285.1 1.52 32, 4.4 287.9 1.51 16, 4.4 286.2 1.49 0.6, 4.4 288.9 1.49 290.6 1.51 49, 6.6 284.0 1.47 15, 6.6 285.1 1.52 9.7, 6.6 286.2 1.52 5.5, 6.6 287.6 1.48 20.8, 6.6 289.1 1.48 290.4 1.49
Ols (S2p) s_tmetm Ep, FWHM I, ~
I,a
(~)
7.8, 2.4 76, 2.4 13, 2.4 0.4, 2.4 2.7, 2.4 15, 3.9 35, 3.9 20, 3.9 7, 3.9 17, 3.9 5, 3.9 34, 1.5 36, 1.5 6.6, 1.5 15.5, 1.5 5.7, 1.5 1.7, 1.5 20, 2.6 49, 2.6 20, 2.6 4.2, 2.6 4.8, 2.6 2.4, 2.6
(eV)
(~)
530.0, 1.86 531.1, 1.86 532.2, 2.02 533.6, 2.03 (166.2, 2.03 167.4, 2.03) 530.69, 1.87 532.23, 1.89 532.79, 2.01
0.5, 2.7 22, 2.7 55, 2.7 23, 2.7 (66, 3.2 34, 3.2) 46, 3.9 31, 3.9 24, 3.9
529.7, 1.99 531.5, 1.93 531.8, 1.90 533.2, 2.00
13, 48, 26, 15,
530.3, 1.86 531.2, 2.03 531.8, 1.99 533.0, 2.04 (166.50, 1.98 167.6, 2.02)
9, 2.5 11, 2.5 53, 2.5 26, 2.5 (77, 4.1 23, 4.1)
Ep, FWHM
I,
399.1, 2.04 402.3, 1.86 400.2, 1.99
22, 3.8 8, 3.8 70, 3.8
399.11, 1.98 400.52, 1.98 401.84, 1.99 (100.7, 2.04 102.7, 1.97 103.8, 1.99) 399.2, 1.90 399.9, 1.94
20, 6.3 60, 6.3 20, 6.3 (40, 6.9 14, 6.9 46, 6.9) 28, 3.2 72, 3.2
(101.5, 1.87 102.7, 1.95 103.5, 1.92) 398.9,1.92 401.5, 1.93 400.1, 1.92
(54, 6.6 42, 6.6 3, 6.6) 5, 2.2 1, 2.2 94, 2.2
(eV)
2.5 2.5 2.5 2.5
(~)
key: Ep, peak energy; FWHM, full width at half maximum height; I, relative intensity; o, mean deviation Gaussian fraction), Shirley's base line correction function was subtracted, and the ESR spectra by a Lorentzian profile derivative. M i n i m u m deviation from the experimental spectrum was used as a "bestfit" criterion, widths o f the composite spectra were allowed to vary during the fitting procedure. The full curve is least squares fit o f the experimental points. The gradient m e t h o d was used for optimization o f the position and width o f lines. XPS binding energies are reported to i-0.1 eV (Table 2). 3. R E S U L T S A N D D I S C U S S I O N The data obtained indicate that immobilized interferon activity is dramatically
different. Sample lc exhibits a high antiviral activity which remained unchanged when it was stored in suspension for at least 5 weeks. At the same time no activity was observed or detected in trace amounts in suspensions containing samples IIc-IVc. These results were quite unexpected because the amount o f strongly bound protein (P) and immobilized layer thickness values determined by the XPS method for samples Ic-IVc were nearly the same. It should be noted that the adsorbed layer depth derived from XPS and ESR evidence were well correlated for a series of samples (Ia,b-IVa,b). High local pressure in contact points in aggregates can be responsible for a decrease in nonmagnetic layer depth in contact points o f particles in
693
A
B S
50
53
N
N
160
162
165
167
280 283 286 289
395 397 400 402
B I N D I N G ENERGY eV Figure 1.(A) Fe3p, S2P, Cls and Nls fragments of XPS spectra for finely dispersed iron samples with immobilized interferon: (a) Sample Ic, (b) Sample IIc, (c) Sample IIIc, (d) Sample IVc; (B) Nls fragments of XPS spectra for freely dispersed iron samples activated by 1,6-hexamethylene diisocyanate: (a) Sample lb, (b) Sample Ilb, (c) Sample IIIb, (d) Sample IVb. Samples are the same as in MATERIALS AND METHODS. samples with immobilized protein (Samples Ic-IVc). When samples are activated via an isocyanate procedure, tetrabutyltitanate catalyses the formation of an urethane bond which is stable toward hydrolysis. A simplified scheme for this activation reaction can be visualized as follows: I-OH + O=C=N(CH2)6NffiCffiO $ Ti(OR) 4 I-CO-NH(CH2) 6NffiCffiO A subsequent binding of protein occurs with participation of amino or thiol groups apt to interact with an isocyanate group. The Cls spectra for samples Ib-IVb activated via an isocyanate procedure (Figure 1B, Table 2)
contain components at 286 eV which can be assigned to the carbon atoms adjacent to nitrogen in the urethane or isocyanate groups, -OC(O)NH-CH 2- or -CH2-NffiCffiO, and the components at 289 eV to carbon involved in the methane groups, -OC(O)-NH- [5]. The N ls spectra for the same samples show components at ca 399.9 eV which can be assigned to the urethane bond nitrogen. A relative intensity of the components at 399.2 eV characteristic of nitrogen in the isocyanate groups [6,7] is rather high for all samples IbIVb (16-44~). Thus the activation was efficient for all samples. Appreciable discrepancies were observed only in a thin surface layer structure for
694
samples containing immobilized interferon. A great nitrogen/carbon and carbon/sulphur ratios were observed only for sample Ic displaying high interferon activity unlike samples IIc-IVc (Table 1). These data suggest that the spatial arrangement of immobilized interferon film is essentially different for active and inactive samples. Nitrogen present in the protein structure in active sample Ic is preferentially oriented toward outer surface and sulphur is oriented toward F D I iron core surface. Inactive samples IIc-IVc show a reverse tendency, namely, nitrogen atoms arc oriented toward the F D I surface. It is clearly seen from the differences observed in S2p and Fe3p photoelectron spectra given in Figure 1A. The S2p line for the sample Ic contain a component at 163.2 eV typical of sulphide [8]. In this case the spectrum does not show intense components with a low binding energy (51.8 eV) which can be associated only with iron nitride formed at the interface (Samples IIc-IVc) as a result of interaction with protein amino groups (Table 2) [9]. The mobility of functional groups on the polymer surface has been elegantly demonstrated. The solvent nature was shown to reversibly and markedly affect the surface concentration of amino groups involved in polyethylene surface modified in plasma [10]. At the same time it is also obvious from our data that the 'substrate' properties can to great extent influence the surface layer structure, organization, orientation and distribution of functional groups. A surprising stability of immobilized interferon (Sample IC) at least for 5 weeks upon storage in suspension based on physiological solution should be again emphasized. The orientation of protein frasments in self magnetic F D I field, which near the surface may attain 6T, can participate in protein structure stab'tUzation at the interface. The high-intensity component at a binding energy of ca 284 eV in the all samples studied correspondin8 to ~complexes of unsaturated adjacent molecules [II] is indicative of a high ordering of unsaturated molecules in adsorbed layer. A
prolonged (about year) activity of tripsin immobilized on fmely dispersed magnetite surface was observed and also attributed to a magnetic field effect [12]. CONCLUSIONS The spatial arrangement of protein in the adsorbed layer rather than protein amount on the surface is largely responsible for the activity or nonactivity of recombinant human interferon immobilized on the F D I surface. Unlike inactive samples, the active sample is characterized by preferential orientation of nitrogen-containing groups toward outer surface, whereas sulphur-containing groups toward the FDI substrate surface. The self magnetic F D I field can serve as a factor of stab'flization of immobilized protein structure and activity. REFERENCES 1. B.D. Ratner, T.A. Horbctt, D. Shuttleworth and H.R. Thomas, J. Colloid Interface Sci., 83 (1981) 630. 2. H.D. Lchmann. Method of binding of biologically active material to a carrier containing hydroxyl groups. EP No. 0052365 (1982). 3. W. E. Stuart II, Effect of interferon on Cells, Viruses and the Immune System (ed. A. Geraldcs), Academic, New York, 1975. 4. O. M. Mikhaflik, Yu.V. Pankratov and E.A. Bakai, J. Magn. Magn. Mater., 122 (1993) 379. 5. V.S. DA Costa, D. Bmer-Russel, E.W.Salzman and E.W. Merril, J. Colloid Interface Sci., 80 (1981) 445. 6. S. Shauwaert, R. I.azzaxony, J. Riga, et al., J.Chem Phys., 92 (1990) 2187. 7. E. T. Kang, K. G. Neon, S. H. Khor, et al., Polymer, 31 (1990) 202. 8. C.D. Wagner, J. Vac. Sci. Teclmol., 15 (1978) 518. 9. McIntyre N.S., Zetamk D.G., Anal. Chem. 49 (1977) 1521. 10. D. S. Everhait and C.N. Reilley, Surface Interface Anal., 3 (1981) 126. 11. O.M. Mikhailik, Yu.V. Pankmtov, V.I.Povstuga~ et al., Colloids Surfaces, A: 90 (1994) 111. 12. V.G. Bendikene, A.A. Razunas, B.A. Yuodka. In: Abstracts of Ill Conference on Magnetic Fluids in Biology and Medicine, Suhumy, 1989, P. 35.