Introduction
As described in Chapter 2, I obtained α-elastin cross-linked nanoparticles.
However, I could not obtain any information about the difference in efficiency to obtain stable cross-linked nanoparticle size with a narrow size distribution-specific heating rate.
It is difficult to analyze the aggregation and cross-linking procedure for α-elastin through different heating processes, because the protein has various types of amino acids (hydrophobic or hydrophilic amino acids and positively or negatively charged amino acids). To obtain the details of the aggregation and cross-linking procedure, I used 3 types of the elastic model polypeptide, namely, polypeptide I: [(GVGVP)251] (containing GVGVP whose repeat caused aggregation), polypeptide II: [(GVGVP GVGFP GEGFP GVGVP GVGFP GFGFP)17(GVGVP)] (containing Glu), and polypeptide III: [(GVGVP GVGFP GKGFP GVGVP GVGVP GVGVP)22(GVGVP)]
(containing Lys). These polypeptides were synthesized with Escherichia coli recombinant DNA technology described by Urry et al. Polypeptide II and III were synthesized as a controlled release delivery system for amphiphilic drugs and therapeutics. These polypeptide designs use charged side chains with increase in
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hydrophobicity by replacing Val with a much more hydrophobic residue, namely, Phe.
As a result, there is a systematic shift in pKa values of the polypeptides. This is based on the polar-polar repulsive free energy of hydration [1]. I attempted to prepare aggregated polypeptide I, II, and III in an aqueous solution above their CP and to form nanoparticles of the polypeptides by various heating methods.
Materials and Methods
Materials
Elastic model polypeptide I: (GVGVP)251, II: (GVGVP GVGFP GEGFP GVGVP GVGFP GFGFP)17(GVGVP), and III: (GVGVP GVGFP GKGFP GVGVP GVGVP GVGVP)22(GVGVP) were supplied from Prof. Urry (Minnesota University) and BRL (Bioelastic Research Ltd.) through Bioelastic Japan Co. [1]. Sodium phosphate, dibasic, anhydrous (NaHPO4); sodium phosphate, monobasic (NaH2PO4 ・ 2H2O); and hydrochloric acid were obtained from Wako Pure Chemicals Industries Ltd.
Evaluation of CP of polypeptide solution
Transmittances were measured with a HITACHI U-3210 UV-Vis spectrophotometer under various temperatures maintained using a water bath (EYELA
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SB-350) with a magnetic stirrer/hot plate (CORNING). Turbidity was assessed by the change in absorbance at 300 nm for a 1 mg/ml solution [2]. At a given temperature, the disposable plastic cuvette containing the sample was allowed to stand until a constant turbidity value was obtained. This constant value is considered to be the actual turbidity of the sample at the given temperature.
Preparation of polypeptide I, II, and III aggregates in an aqueous solution heated above CP
The aggregates of Polypeptide I, II, and III were prepared by heating the aqueous solutions from 4ºC to 60ºC at various heating rates (1–6ºC/min) as described in Chapter 2. Moreover, “Heat shock,” procedure was employed as an additional heating process, in which the polymer solution at a high concentration was dropped into hot water (above CP) with constant stirring in a water bath (EYELA SB-350) by using a magnetic stirrer (EYELA RCN-3D) at 600 rpm.
Measurement of size distribution of aggregated polypeptide I, II, and III
Particles size was measured by dynamic light scattering (DLS) (Particle Sizing Systems, Nicomp 370) at a 90° scattering angle. The polymer solution described above was diluted to 1 mg/ml with deionized water. The solution was placed into a disposable
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plastic cuvette (Kartell ART.01961-00) and incubated at 42°C or 50°C in the cuvette holder of Nicomp 370.
Gamma irradiation of aggregated polypeptide I, II, and III
The heated polymer samples were placed in thermos bottles (TIGER MWE-C350) and irradiated with 60Co gamma-rays at doses of 8.5–30 kGy by using 60Co gamma irradiation pool at Osaka Prefecture University (dose rate: 10.5 kGy/h). The temperature of the sample was maintained at 42°C or 50°C during irradiation.
Transmission electron microscopy
Samples for transmission electron microscopy (TEM) were dissolved in deionized water to a final concentration of 10 mg/ml. The sample solution was added to carbon-coated 400-mesh copper grids (VECO) on ice, excess sample was absorbed with a filter paper, and the grid was dried at room temperature before observation by TEM.
The prepared samples were viewed using a HITACHI EF2000 (HITACHI Ltd.) operating at 200 keV.
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Atomic force microscopy
Samples for Atomic force microscopy (AFM) were dissolved in deionized water to a final concentration of 1 mg/ml. The samples were deposited on mica on ice and dried at room temperature. AFM analysis was performed in air by using a digital instrument multimode (SII Nano Technology Inc., SPI 3800N, SPI 4000 NanoNavi) using SI-DF20 cantilevers working in the tapping mode with a resonant frequency of less than 200 kHz.
Results and Discussion
Preparation of polypeptide I nanoparticles
Effect of the heating process on aggregation of polypeptide I
I examined the effect of the heating process on particle formation of polypeptide I in aqueous solution. The polypeptides in the solution aggregated at a temperature greater than the CP (30°C) irrespective of the heating rate from 1.3 to 3.8°C/min. I attempted to obtain aggregated polypeptide I at a concentration of 5 mg/ml and temperature of 42°C. This concentration and temperature were the optimum for forming aggregated polypeptide I particles. The average size of the aggregated particles was within the range of 380–440 nm for all the heating processes before irradiation according to the DLS measurement (Table 3-1).
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Table 3-1. Effect of the heating process on polypeptide I particle size. Mean size of the polypeptide I measured by DLS analysis at 42°C.
Heating process Irradiation Size (nm)
Slow heating Before 440(±100)a
After No stable particles
Fast heating Before 380(±70)a
After No stable particles
astandard deviation
I also investigated the effect of gamma irradiation on the aggregated polypeptides prepared by each heating process at a temperature greater than the CP. After irradiation,
“Slow heating” (1.3°C/min) yielded a gel of the aggregated polypeptides, while “Fast heating” (3.8oC/min) yielded sediments of the aggregated polypeptides (aggregated
polypeptide could not form nanoparticles), which did not dissolve again at temperatures lower than the CP.
Therefore, I employed the “Heat shock (40oC/min)” heating process and attempted to obtain cross-linked nanoparticles. In “Heat shock,” 25 mg/ml of the polypeptide I solution was dropped into the water at 42oC. This concentration and temperature were the optimum to form polypeptide I aggregates. The size of aggregated polypeptide I was ca. 370 nm before irradiation. After irradiation, “Heat shock” yielded a clouded
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suspension of nanoparticles (ca. 150 nm). The results of the DLS measurements are shown in Fig. 1. The nanoparticles did not dissolve at temperatures lower than the CP.
Fig. 3-1 Mean size of the polypeptide I particles measured by DLS analysis. The sample is prepared using the “Heat shock” process at 42°C in deionized water. The shaded square and blank square in the figure are before irradiation and after irradiation, respectively.
These results strongly suggest that the nanoparticles were cross-linked by irradiation with gamma-rays. Fig. 3-2(A) shows the TEM image of cross-linked nanoparticles. As seen in the figure, almost all the cross-linked nanoparticles were spherical. I measured the size of the 120 particles that appeared in the TEM image by using the scale bar. The obtained size distribution histogram (average size and standard deviation: 150 ± 60 nm)
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(Fig. 3-2(B)) was consistent with the DLS result (average size and standard deviation:
150 ± 30 nm). The AFM image was also consistent with the DLS result (Fig. 3-3). The yield of the cross-linked nanoparticles increased with an increase in radiation doses, as shown in Table 3-2. The size of the cross-linked nanoparticles decreased slightly with an increase in radiation doses, as shown in Table 3-3.
Fig. 3-2 TEM image of polypeptide I particles cross-linked by gamma irradiation (A) and the size distribution histogram obtained from the TEM image (B). The scale bar in the micrograph represents a length of 600 nm. The size distribution histogram was obtained by measuring the diameter of 120 particles in the TEM image by using the scale bar.
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Fig. 3-3 AFM image of the polypeptide I particles cross-linked by gamma irradiation.
Table 3-2. Radiation dose response of the yield of the polypeptide I particles. Deposit of the particles obtained by centrifugation of the particle solution at 19,000 rpm at 4°C for 1 hour (HITACHI himac CR21, rotor no. 46) and dissolving in deionized water. Yield is determined by measuring the dry weight of the deposit.
Radiation doses (kGy) Yield (%)
8.5 40
17 50
34 60
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Table 3-3. Effect of the dose rate of gamma-rays. Mean sizes of polypeptide Iparticles were determined by DLS analysis.
Radiation doses (kGy) Size (nm) 42oC
Size (nm) 15oC
8.5 380(±50)a 200(±30)a
15 200(±20)a 120(±10)a
30 180(±30)a 170(±50)a
astandard deviation
During the “Heat shock” process, because the polypeptide concentration was 25
mg/ml, the initial polypeptide concentration might be higher than that during any other heating process during particle formation of the polypeptides when the polypeptides were dropped into hot deionized water at a temperature greater than the CP, although the final concentration of the polypeptide after particle formation was 5 mg/ml. In contrast, the polypeptide concentration was maintained at 5 mg/ml all the time during
“Slow heating” and “Fast heating.” Therefore, I compared particle formation by “Slow heating” and “Fast heating” in aqueous solutions with different concentrations of polypeptides (5 and 25 mg/ml). Both concentrations of the polypeptide solutions
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yielded clouded suspensions after “Slow heating” and “Fast heating,” and no significant
difference was observed before irradiation. After irradiation, nanoparticles were not formed in the aggregated polypeptide solutions in both 25 mg/ml solution and 5 mg/ml solution, as described above. These results suggest that the “Heat shock” process was the most important process with respect to the formation of nanoparticles. I also attempted to increase the sample volume of the droplet of the polypeptide solution during the “Heat shock” process by using needles of different gauge sizes. The cooled
polypeptide I solution (25 mg/ml cooled at 4°C) was dropped into hot deionized water at 42°C with a tuberculin syringe using 18-G (droplet volume, 20 μl) and 27-G (droplet volume, 5 μl) needles, and the final concentration after aggregation of polypeptide I was 5 mg/ml. A stable particle was obtained when the 27-G needle was used but not obtained when the 18-G needle was used. The average size of the particles was 150 nm when the 27-G needle was used. Thus, the 27-G needle was more effective than the 18-G needle in forming stable cross-linked nanoparticles distributed within a narrow size range.
These results suggest that in cases of “Slow heating” and “Fast heating,” the hydrophobic side-chain interaction of the polypeptides might be poor and might not be effective to form the cross-linked structure. Indeed, “Slow heating” and “Fast heating”
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yielded gels and sediments, respectively. It was thought that the inter-molecular contact between hydrophobic side chains was important to form cross-linked structures.
Moreover, I reconfirmed that repeat of hydrophobic domains such as (GVGVP) was important to form α-elastin aggregation and cross-linked structure.
Preparation of polypeptide II and III nanoparticles
Effect of the heating process on the aggregation of polypeptide II and III
To study the effect of charge within α-elastin, I used 2 types of elastic model polypeptide II and III. I attempted to dissolve polypeptide II and III in deionized water.
However, solubility of both polypeptide II and III were low in the deionized water at 4oC and any CP could not be observed at any temperature in deionized water. Therefore, polypeptide II and III were prepared in 10 mM phosphate buffer (PB). In the solution of polypeptide II in PB (7 mg/ml), polypeptide II underwent aggregation at a temperature corresponding to 30oC with an increase in temperature. However, polypeptide III did not show CP in deionized water and PB at any temperature.
I examined the effects of the three heating processes on the particle formation of polypeptide II in PB, in which the time of the heating was varied from 4oC to 50oC. I attempted to obtain aggregated polypeptide II at a concentration of 7 mg/ml and temperature of 50°C. This concentration and temperature were the optimum to form
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aggregated polypeptide II. As shown in Table 3-4, polypeptide II was aggregated in PB (7 mg/ml) with heating at 50oC (above CP) by any of the heating processes, but the main peak size distribution of the aggregates varied among “Slow heating (1.5oC/min)”
(70 ± 15 nm), “Fast heating (4.6oC/min)” (60 ± 10 nm), and “Heat shock (50oC/min)”
Table 3-4. Effect of the heating process on polypeptide II particle sizes. Mean size of polypeptide II particles measured by DLS analysis at 50°C.
Heating process Irradiation Size (nm)
Slow heating Before 20(±3)a 12% ,70(±15)a 88%
After 60(±10)a
Fast heating Before 10(±2)a 4% ,60(±10)a 96%
After 55(±10)a
Heat shock Before 80(±20)a
After 40(±10)a 56% ,90(±20)a 44%
astandard deviation
(80 ± 20 nm), as shown in Fig. 3-5. All the aggregates were dissolved again by reducing the temperature below CP.
On the other hand, after 30-kGy irradiation, the particles could not be dissolved by reducing the temperature to 15oC (below CP), and transmittance of the solution was almost zero at all the temperatures examined. These results indicate that the aggregates
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stabilized and lost their thermosensitivity by gamma-ray irradiation cross-linking. I collected the aggregates by centrifugation at 4oC after the irradiation and analyzed their size distributions by DLS (Table 3-4). As shown in Fig. 3-6, the mean of the size distributions was shifted to lower than 100 nm for the preparation conditions “Slow heating” and “Fast heating”; both these conditions yielded narrow size distribution
containing a single peak around 60 nm, while the other conditions yielded broader and unstable size distributions containing several peaks. However, the yield rate of particles for “Slow heating” was more than that of “Fast heating” after gamma irradiation (Table 3-5). Figure 3-7 shows the cross-linked nanoparticle image obtained from AFM. On the basis of these observations, I assume that nanoparticles were formed from the aggregated polypeptide II particles by gamma-ray irradiation cross-linking and that
“Slow heating” was the optimum process to obtain crosslinked nanoparticles with a narrow size distribution.
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Fig. 3-5 Mean size of the aggregated polypeptide II particles by the three heating processes measured by DLS before gamma irradiation ((A): Slow heating (B): Fast heating (C): Heat shock).
Fig. 3-6 Mean size of the aggregated polypeptide II particles by the three heating processes measured by DLS after 30 kGy gamma irradiation ((A): Slow heating (B):
Fast heating (C): Heat shock).
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Table 3-5. Yield rate of polypeptide II nanoparticles after gamma irradiation by using different heating methods. Supernatant obtained by centrifugation of the suspension at 19,000 rpm at 4°C for 1 hour (HITACHI himac CR21, rotor no.46) was dried. The yield was determined by measuring its dry weight.
Heating process Yield (%)
Slow heating 60
Fast heating 30
Fig. 3-7 AFM image of polypeptide II nanoparticles cross-linked by gamma irradiation (Preparation by “Slow heating”).
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Table 3-6. Radiation dose response of the yield of polypeptide II nanoparticles. Deposit of the particles was obtained by centrifugation of the particle solution at 19,000 rpm at 4°C for 1 hour (HITACHI himac CR21, rotor no.46) and dissolved in deionized water.
The yield was determined by measuring the dry weight of the deposit.
Radiation doses (kGy) Yield (%)
8 60
32 70
Table 3-7. Effect of the dose rate of gamma-rays. Mean sizes of polypeptide II particles were determined by DLS analysis.
Radiation doses (kGy) Size (nm) 50oC
Size (nm) 15oC
8.5 50(±10)a 40(±10)a
15 40(±10)a 35(±10)a
30 60(±10)a 70(±20)a
astandard deviation
I also investigated the effect of gamma irradiation dose on the formation of stable nanoparticles by using the “Slow heating” condition. At 8 and 32 kGy irradiation doses, the yield of the cross-linked nanoparticles collected at 4oC after irradiation was approximately 60% and 70%, respectively (Table 3-6). For each dose, the size
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distributions of the particles were within the order of less than 100 nm at 15oC (Table 3-7).
From these results, it is suggested that the heating process might be crucial to form stable nanoparticles of aggregated polypeptide II by gamma irradiation. Generally, polymer chains should be close to each other to obtain efficient cross-linking. “Slow heating” might contribute to efficient aggregation to help cross-linking. In cases of
“Heat shock” and “Fast heating,” the hydrophobic side-chain interaction of the
polypeptides might be poor and might not be effective to form the cross-linked structure.
The inclusion of charged amino acids within the polypeptides would take a longer time for interaction of hydrophobic side chains because of repulsion of charges.
I also attempted to prepare aggregated polypeptide III by each heating process at a temperature greater than the CP. However, polypeptide III did not show CP in deionized water and PB at any temperature. Therefore, aggregated polypeptide III could not be prepared like aggregated polypeptide II. Thus, I could not obtain polypeptide III nanoparticles.
From these results, it is suggested that the “Slow heating” process is the optimum process to aggregate polypeptides containing charged amino acids, because it causes repulsion between charged amino acids. Moreover, the present study showed that
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cross-linked nanoparticles could not be obtained without aggregation of the polypeptides. To efficiently obtain cross-linked nanoparticles of α-elastin, “Slow heating" is required because α-elastin contains charged amino acids.
In the next chapter, to obtain information about aggregation of the hydrophobic domain (GVGVP) within α-elastin, I focus on the heating process for optimizing nanoparticle formation. I changed the heating rate and monitored the aggregation process of polypeptide I by circular dichroism (CD) spectrometry in order to yield the nanoparticles more efficiently. Moreover, I used molecular dynamics (MD) simulation to analyze the mechanism of aggregate formation. I also used simple amino acids such as Val, Pro, and Gly for analyzing cross-linked points.
Conclusions
Stable nanoparticles of polypeptide I were successfully obtained by gamma-ray cross-linking on increasing the temperature to a value greater than the CP with the
“Heat shock” process and by using a needle with a small droplet volume before
irradiation and maintaining the temperature during gamma irradiation. The size of the nanoparticles was ca. 150 nm.
Stable nanoparticles of polypeptide II were successfully obtained by gamma
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irradiation through choosing the appropriate heating rate before irradiation and maintaining the temperature during irradiation. Although stable cross-linked nanoparticles of polypeptide II were formed by gamma irradiation with the “Slow heating” process, the size of the nanoparticles was approximately less than 100 nm.
References
[1] Urry, D. W., Biorefinery (34), In: Kamm, B., Gruber, P. R., Kamm, M. (eds) Biorefineries- industrial process and products, Status quo and future directions, 2 volumes. Wiley VCH, Weinheim, 2006, pp. 217-253.
[2] Urry, D. W., Nichol., A., Mcpherson, D. T, Harris, C. M., Parker, T. M., Xu, J., Gowda, D. C., Shewry, P. R., Properties, preparations, and applications of bioelastic materials. In: Wiseman, D. M.., Trantolo, D. J., Altobelli, D. E., Yaszemski, M. J., Gresser, J. D., Schwartz, E. R., (eds), Encyclopedic handbook of biomaterials and bioengineering-Part A-Materials, Volume 2. Marcel Dekker, New York, 1995, pp.1619-1673.
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