Genetic Engineering of Stimuli-Sensitive Silkelastin-like Protein Block

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Biomacromolecules 2003, 4, 602-607

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Genetic Engineering of Stimuli-Sensitive Silkelastin-like Protein Block Copolymers Ashish Nagarsekar,† John Crissman,‡ Mary Crissman,‡ Franco Ferrari,‡ Joseph Cappello,‡ and Hamidreza Ghandehari*,† Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, and Protein Polymer Technologies, 10655 Sorrento Valley Road, San Diego, California 92121 Received October 1, 2002; Revised Manuscript Received December 26, 2002

Differentially charged analogues of block copolymers containing repeating sequences from silk (GAGAGS) and elastin (GVGVP) were synthesized using genetic engineering techniques by replacing a valine residue with glutamic acid. The sensitivity to pH and temperature was examined at various polymer concentrations, ionic strengths, and polymer lengths. The polymers transitioned from soluble to precipitate state over narrow temperature ranges. The transition temperature Tt (the temperature at which half-maximal spectrophotometric absorption was observed) increased with increasing pH up to pH 7.0 and leveled off above this value for the Glu-containing polymer (17E)11. Tt was independent of pH for the Val-containing polymer (17V)11. It decreased with increasing ionic strength, polymer concentration, and polymer length for both polymers. These results suggest that by substituting charged amino acids for neutral amino acids at strategic locations in the polymer backbone and by control of the length of silkelastin-like block copolymers using genetic engineering techniques, it is possible to precisely control sensitivity to pH, temperature, and ionic strength. Introduction Polymers that are sensitive to specific stimuli such as pH, temperature, and the presence of biomolecules can be used, for example, as sensors, drug delivery devices, and actuators.1-3 The detailed molecular architecture of polymers influences their stimuli-sensitivity characteristics as well as physicochemical profile and biological fate.4,5 Recent advances in recombinant DNA technology have led to the evolution of a new methodology for the synthesis of polymers, namely, synthesis by genetic engineering techniques.6-10 The protein polymers synthesized by these techniques have a defined composition, sequence, stereochemistry, and molecular weight. Protein-based polymers can be designed to exhibit a variety of physicochemical properties such as stimuli sensitivity since amino acids offer a variety of structural options for use in the conceptual design of a polymer. A well-characterized example of control over physiological characteristics achieved by variations in copolymer composition using genetic engineering techniques is the family of silkelastin-like block copolymers (SELPs).9 SELPs are made up of repeats of GlyAla-Gly-Ala-Gly-Ser, the basic repeating sequence of silk fibroin,11 and Gly-Val-Gly-Val-Pro, a repeating sequence from mammalian elastin.12 Increasing the number of silklike blocks within a domain in a silkelastin-like block copolymer increases the rate of gelation and decreases the rate of resorption of the polymer.13,14 SELPs have potential in a * Author to whom correspondence may be addressed. Telephone: (410)706-8650. Fax: (410)-706-0346. E-mail: [email protected]. † University of Maryland School of Pharmacy. ‡ Protein Polymer Technologies.

variety of biomedical applications such as injectable urethral bulking agents for the treatment of female stress urinary incontinence,15 smart cell culture coatings containing cell attachment biorecognition sequences,9 and drug delivery systems.13,16-19 As such, introducing stimuli sensitivity to SELPs will broaden the range of applications of these biomaterials. In the past 2 or 3 decades, numerous studies have described chemically synthesized polymers with stimuli sensitivity for biomedical applications.1-3 Some of these polymers have been tested in clinical trials20 and clearly show advantages of stimuli sensitivity over conventional polymers. The successful design of stimuli-sensitive biomaterials requires control over the detailed structure of the polymers. Thus, the field could benefit from novel synthetic approaches and new biomaterials. The present work is an attempt to illustrate the potential utility of genetically engineered silkelastin-like copolymers in applications where pH and temperature sensitivities are desired. This study shows that by controlling the sequence and length of the polymers, it is possible to control their sensitivity to pH, temperature, and ionic strength. Stimuli sensitivity, coupled with control over degradation, and the ability to incorporate naturally occurring biorecognizable sequences using recombinant DNA techniques could be additional design features of new stimulisensitive SELPs for biomedical applications. In this article, we report the synthesis and characterization of SELPs with the structure [(Gly-Val-Gly-Val-Pro)4-GlyX-Gly-Val-Pro-(Gly-Val-Gly-Val-Pro)4-Gly-Ala-Gly-AlaGly-Ser]11, where X is either Glu or Val. As a result of the modification of one amino acid within the monomer, the

10.1021/bm0201082 CCC: $25.00 © 2003 American Chemical Society Published on Web 02/26/2003

Stimuli-Sensitive Silkelastin-like Protein Block Copolymers

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Scheme 1. Flow Chart for the Genetically Engineered Synthesis of Silkelastin-like Protein Block Copolymersa

TACCAGGCGTAGGCGTCCCTGGCGCTGGCGCGGGCTCTTCCGCTAAAGTCCTGCCGT-3′. The nucleotide sequences intended for polymerase chain reaction (PCR) primer hybridization are indicated in bold, the underlined sequence constitutes the recognition site for Ban II, and italicized nucleotides identify the locations at which two nucleotides were incorporated at the same positions. This produced oligonucleotides that would encode up to four different amino acids at this position: valine (GTA), isoleucine (ATA), glutamic acid (GAA), or lysine (AAA). The oligonucleotide was annealed and amplified by PCR. Linear acceptor plasmids and purified monomer gene segments were allowed to react in a 1:1 molar ratio, and recombinant plasmids were sequenced (Commonwealth Biotechnologies, Inc., Richmond, VA) to identify those containing codons for Glu or Val at the degenerate position. Each set of DNA segments was allowed to self-ligate at room temperature for 1 h in the presence of T4 DNA ligase, and this mixture was cloned into pPT358. Polymer gene size was estimated in each of the resulting transformants, and plasmids containing polymer gene of approximately 1600 kbp (1596 bp corresponds to 11 repeat polymer gene segment) were selected for protein polymer expression. Polymers were purified by temperature cycling (described below) and were dialyzed against distilled water using Float-A-Lyzer kits (molecular weight cutoff 15 kDa). Yields of 30 mg/L were obtained. Purification of Polymers by Temperature Cycling. Cell pellets were resuspended in lysis buffer (20 mL per gram wet weight of cells). Each 20 mL contained 2 mg of lysozyme (Sigma Chemical Company, St. Louis, MO), 3 mL of 0.5 M EDTA (Biosource International, Rockville, MD), and 50 µL of 1 M phenylmethylsulfonylfluoride (Sigma). Cells were lysed by sonication using a 100-W Fisher model 60 sonic dismembrator (Fisher Scientific, Suwanee, GA) on ice for three 30-s intervals. Cell debris was removed by centrifugation at 6000g for 15 min at 4 °C. The supernatant was centrifuged at 35000g for 30 min at 4 °C. Sodium chloride (Sigma) was added to the clarified supernatant with periodical checks for development of turbidity when immersed in a 35 °C water bath. When turbidity was observed, the lysate was incubated for 10 min on ice. The cooled lysate was then centrifuged at 10000g at 40 °C for 30 min, and the supernatant was discarded. The pellet was resuspended in distilled water and checked for turbidity with sodium chloride addition as before, and turbidity was centrifuged out. This “temperature cycle” was repeated until purified polymer was obtained. Structural Characterization of the Polymers. The polymers were analyzed by mass spectrometry, amino acid analysis, and gel electrophoresis. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS) was carried out by Commonwealth Biotechnologies, Inc. (Richmond, VA). MALDI-TOF-MS was carried out in the positive ion mode in a sinapinic acid matrix.22 Sodium dodecyl sulfate-poly(acrylamide) gel electrophoresis (SDSPAGE) was carried out using 4-20% PAGE gels run in trisglycine SDS buffer and negatively stained with copper stain. Amino acid analysis of the sample was carried out by Commonwealth Biotechnologies by chromatographic mea-

a In the oligonucleotide the Ban II recognition sites are underlined and polymerase chain reaction hybridization sites are shown in boldface.

polymer achieves positive, or neutral charge status, allowing the evaluation of the influence of sequence on stimuli sensitivity. The characterization of the polymers with respect to sensitivity to pH, temperature, ionic strength, and concentration is described. In addition to the comparisons between the analogues, the Glu containing 11mer is also compared to the Glu containing 16mer reported previously,21 to elucidate the influence of polymer length on stimuli sensitivity. Experimental Section Materials. The acceptor and expression plasmids, pPT358 and pPT317, respectively, were constructed by Protein Polymer Technologies, Inc. (San Diego, CA). Float-A-Lyzers were obtained from Spectrum Laboratories (Rancho Dominguez, CA). Precast tris-glycine gels (4-20%) were purchased from iGels (Salt Lake City, UT). Tris-glycine SDS buffer and copper stain were obtained from Bio-Rad (Hercules, CA). All centrifugation steps were carried out on an Allegra centrifuge (Beckman Instruments, Palo Alto, CA). Synthesis of Polymers. The polymers were synthesized by the methodology described earlier.21 The synthetic process is outlined in Scheme 1. Briefly, the procedure involved first the synthesis of an oligonucleotide with the following sequence: 5′-ATGGCAGCGAAAGGGGACCGGGCTCTGGAGTAGGTGTGCCGGGTGTAGGAGTTCCAGGTGTAGGTGTCCCGGGTGTAGGTGTTCCTGGA (A/G)(A/T)A GGTGTTCCGGGCGTAGGCGTTCCGGGAGTTGGAG-

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Figure 1. (A) SDS-PAGE analysis of polymers: lane 1, molecular weight marker; lane 2, polymer (17E)11; lane 3, polymer (17V)11; (B) MALDITOF spectrum of polymer (17E)11; (C) MALDI-TOF spectrum of polymer (17V)11.

surement of derivatized peaks after hydrolysis of the sample in 6 N HCl at 110 °C for 20 h. Characterization of Stimuli Sensitivity. The polymers were dissolved in buffers (Micro Essential Laboratories, Brooklyn, NY) of pH values ranging from 3.0 to 12.0, ionic strength (I) values 0.2 to 1.0, adjusted with sodium chloride, at a concentration of 0.5 mg/mL. The absorbances of the resulting solutions were evaluated at 300 nm over a temperature range of 20-100 °C and a heating rate of 3 °C/ min, using a spectrophotometer (Ultrospec 4000, Amersham Pharmacia Biotech, Piscataway, NJ) fitted with a programmable Peltier cell (Amersham Pharmacia Biotech). The absorbance profiles of the polymers were also studied at various concentrations in pH 7.0 buffer (I ) 0.2) over the same temperature range at a concentration of 3.0 mg/mL. The reversibility of the transition was studied by measuring the absorbance of polymer solution (0.5 mg/mL, pH 3.0, ionic strength 0.2-1.0) over 20-100 °C. Absorbance readings were taken after equilibrating the polymer solution at the desired temperature for 5 min. Results and Discussion Structural Characterization of the Polymers. SDSPAGE data (Figure 1, panel A) indicate that the polymers have molecular weights around 47 kDa. MALDI-TOF data show the presence of a peak at 45132 Da and 47359 Da for polymers (17E)11 and (17V)11 respectively (Figure 1, panels B and C). The theoretical sequences of the polymers are (17E)11 (the glutamic acid-containing polymer with 11 repeat units) MDPVVLQRRDWENPGVTQLVRLAAHPPFASDPMGAGAGSGAGAGS[(GVGVP)4GEGVP(GVGVP)3GAGAGS]11GAGAMDPGRYQDLRSHHHHHH (theoretical MW: 47614 Da) (17V)11 (the valine containing polymer with 11 repeat units) MDPVVLQRRDWENPGVTQLVRLAAHPPFASDPMGAGAGSGAGAGS[(GVGVP)8GAGAGS]11GAGAMDPGRYQDLRSHHHHHH (theoretical MW: 47284 Da) On the basis of peak width at half-height calculations, the error of the TOF instrument was calculated to be ap-

Table 1. Amino Acid Composition Analysis of the Polymers (17E)11

(17V)11

amino acid

R/M Tha

R/M Oba,b

R/M Tha

R/M Oba,b

G V A S P E+Q H R D+N T Y M W F I L K

219 168 31 15 94 15 7 5 7 1 1 3 1 1 0 3 0

244.1 181.4 30.2 12.3 81.5 16.8 0.0 2.3 5.6 1.4 1.2 0.3 c 1.3 1.6 2.7 1.9

219 179 31 15 94 4 7 5 7 1 1 3 1 1 0 3 0

216.5 178.0 29.4 12.7 103.8 4.8 6.5 4.3 7.2 1.1 0.9 3.3 c 0.9 0.0 3.7 0.0

a R/M Th, theoretical number of residues per mole; R/M Ob, residue per mole observed. b Based on observed molecular weight. c Not determined.

proximately 700 Da for each determination. The actual difference between the theoretical and observed molecular weights was 2482 Da for polymer (17E)11 (5.21%) and 75 Da for (17V)11 (0.16%). The discrepancy in the molecular weights for (17V)11 is well within the error limits, but that for (17E)11 is not. The results of the amino acid analysis are listed in Table 1. In general, the theoretical and observed amino acid compositions are in agreement. Since the amino acid analysis of (17E)11 did not show any His, it can be assumed that part of the tail sequence of polymer (17E)11 had degraded, giving a lower molecular weight product. Arg and Met residues, also, show a substantially lesser percent content (Table 1). This suggests the absence of the carboxyterminal sequence LRSHHHHHH and the amino-terminal sequence MDPVVLQRRD possibly due to degradation by aspartic proteases, which would account for a loss of 2407 Da. The mass spectrum for polymer (17E)11 does show a

Stimuli-Sensitive Silkelastin-like Protein Block Copolymers

Figure 2. Plots of absorbance (λ ) 300 nm) of 0.5 mg/mL solutions of (17E)11, (17V)11, and (17E)1621 at pH 6.0 and ionic strength 0.2. Filled symbols represent heating data and open symbols represent cooling data. Each data point is a mean ( standard deviation of triplicate readings.

smaller peak at 47545 Da (Figure 1, panel B), which may be the full length protein, since it differs by only 69 Da (0.14%) from the theoretical molecular weight for polymer (17E)11. The other minor peaks in the spectra are most likely degradation products or truncated polymer. It should be noted that a discrepancy of 442 Da (less than 1% of the theoretical molecular weight) was observed for polymer (17E)16.21 The results from SDS-PAGE analysis (Figure 1, panel A) suggest that polymer (17E)11 has a higher apparent molecular weight than polymer (17V)11 whereas the MALDI-TOF results suggest the opposite. Anomalous migration in SDSPAGE may be due to a number of physical properties of the polymer independent of molecular weight, such as the predominance of negative charge, its conformational state even under denaturing conditions, and its ability to bind SDS. One or more of these factors could explain the difference in migration and, thus, the apparent molecular weight differences between (17E)11 and (17V)11 as inferred from SDSPAGE. Characterization of Stimuli Sensitivity. The polymers were evaluated for stimuli sensitivity by monitoring changes in spectrophotometric absorption of polymer solutions at 300 nm23 in response to various pH, temperature, and ionic strengths. In general, aqueous solutions of the polymer showed a transition from solution state to a turbid state with suspended precipitate upon increasing temperature. A representative plot of the absorbance of the polymer solutions at pH 6.0 and ionic strength 0.2 is shown in Figure 2. The absorbance of (17E)16, a previously synthesized21 higher molecular weight analogue of (17E)11, is also shown. The inverse transition temperature values (Tt, the temperature at which half-maximal absorption is observed23) for (17V)11 and (17E)11 were 34 and 83 °C, respectively. The Tt value for (17E)16 was 59 °C under the same conditions of pH, ionic strength, and concentration. Inverse temperature-dependent solubility transitions depend on the reversible hydration of the hydrophobic portions of the polymer chain. In the soluble state, the hydrophobic moieties within the polymer are surrounded by ordered water (water of hydrophobic hydration). As the temperature is raised, this water becomes less ordered bulk water, and the polymer folds by hydrophobic self-assembly.23 Tt values reflect the interaction of polymers with solvent, the solventsolvent interaction, and inter- and intrapolymer interaction. The water of hydrophobic hydration which keeps the

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hydrophobic residues of the polymer in solution converts to bulk water at higher temperature, thus desolvating the polymer.23 Ionized and polar groups are solvated by water of ionic hydration and water of polar hydration, respectively, which require higher energy to destructure.24 Consequently, the more hydrophilic the polymer, the higher the temperature needed to precipitate it, and the higher is the Tt. In Figure 2, Tt is seen to decrease in the order (17E)11 > (17E)16 > (17V)11. Polymer (17V)11 does not have an ionizable group in the repeating unit; therefore it shows the lowest Tt (34 °C). Polymer (17E)16 is a higher molecular weight analogue of (17E)11, with 16 repeats of the silkelastin-like monomer instead of 11. The lower Tt value (59 °C) shown by (17E)16 as compared to (17E)11 suggests that there are more intrapolymer interactions for (17E)16 because of the increase in the polymer chain length. This result is in agreement with similar studies with elastin-like polymers not containing ionic groups where Tt was found to decrease with increase in molecular weight of the polymer.25 The transition is fully reversible and the heating and cooling curves are essentially superimposable (Figure 2). Presumably this reversibility occurs because the waters of hydrophobic hydration reorganize themselves on the polymer upon cooling, and the rehydrated polymer goes back into solution. Differential scanning calorimetry studies of homopolymers of elastin [poly(VPGVG)] have shown that for this class of polymers the transition is reversible.26 Consistent with these reports, reversibility was noted for silkelastinlike block copolymers as well, in the present study. Complete reversibility of stimuli-sensitive biomaterials is important for applications such as on/off solute release from gels,27 reversible cell culture coatings,28 as well as diagnostics and affinity separation tools.29 The influence of polymer concentration (0.5 mg/mL and 3.0 mg/mL) on Tt is tabulated in Table 2, which includes corresponding data from polymer (17E)16.21 The lesser Tt value at the higher polymer concentration could arise from the increased interpolymer contact at the higher concentration. Table 2 also shows that the difference in the Tt values for the two concentrations, 0.5 and 3.0 mg/mL, at any given ionic strength differs among the three polymers. For example, at ionic strength 0.2, for (17V)11, this difference is about 3 °C, whereas it is more than 30 °C for (17E)11 and 10 °C for (17E)16. The trend among the Tt differences [(17E)11 > (17E)16 > (17V)11] mirrors the trend in the solubility of the polymers, as reflected by the Tt values (Figure 2). The lowering of Tt is more pronounced for more soluble (ionized) polymers, such as polymer (17E)11, than for less-watersoluble (un-ionized) polymers such as (17V)11. Figure 3 illustrates the influence of ionic strength on 0.5 mg/mL aqueous solutions of polymers (17E)11 and (17V)11 over pH 3.0-12.0. The behavior of (17E)11 correlates with the degree of ionization of the glutamic acid side chains from pH 3.0 to pH 7.0 (Figure 3). Beyond pH 7.0, the glutamic acid side chains are essentially fully ionized. Correspondingly, the polymer precipitates at markedly higher temperatures when the pH is increased from 3.0 to 7.0. Beyond pH 6.0, when the state of ionization of glutamic acid does not change appreciably, the temperature range in which the

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Table 2. Concentration Dependence of the Transition Temperature of the Polymers at pH 7.0 over Ionic Strengths 0.2-1.0a (17V)11

(17E)16b

(17E)11

I

0.5 mg/mL

3.0 mg/mL

0.5 mg/mL

3.0 mg/mL

0.5 mg/mL

3.0 mg/mL

0.2 0.4 0.6 0.8 1.0

34.2 ( 0.1 31.7 ( 0.2 29.6 ( 0.3 27.0 ( 0.2 24.6 ( 0.2

31.3 ( 0.1 28.6 ( 0.3 26.3 ( 0.2 23.2 ( 0.1 d

c 74.3 ( 1.5 61.2 ( 1.0 56.4 ( 0.5 50.3 ( 0.2

69.1 ( 1.2 55.7 ( 1.0 49.8 ( 0.8 44.8 ( 0.8 41.3 ( 0.7

64.1 ( 0.1 54.0 ( 1.8 48.2 ( 0.8 44.2 ( 0.8 41.7 ( 0.2

54.6 ( 0.6 47.4 ( 0.2 43.1 ( 0.1 39.7 ( 0.3 36.4 ( 0.2

a T values are mean ( standard deviation of triplicate readings. b For ease of comparison, values for (17E) 20 are incorporated in the table. c T t 16 t technically difficult to calculate since it approaches 100 °C. d Tt lower than 20 °C, temperature below range of instrument.

Figure 3. Influence of ionic strength on Tt of 0.5 mg/mL solutions of polymers (17E)11 and (17V)11: white, pH 3.0; gray, pH 4.0; black, pH 5.0; horizontal lines, pH 6.0; checkered pattern, pH 7.0; crosshatch lines, pH 8.0; vertical lines, pH 9.0; hatch lines rising left, pH 10.0; brick pattern, pH 11.0; hatch lines rising right, pH 12.0. (a) Tt values are technically difficult to measure since they approach 100 °C. (b) Tt values below 20 °C, temperature range not accessible by instrument. All Tt values are mean ( standard deviation of triplicate readings.

polymer precipitates remains essentially constant. Figure 3, panel A shows Tt values increase from pH 3.0 to 6.0 for (17E)11 and level off above pH 7.0. The leveling off effect is less pronounced at lower ionic strengths. At ionic strength 0.2, for example, there is a sharp increase in Tt values from pH 3.0 to 6.0, above which the polymer stays soluble in the entire aqueous range (marked “a” in Figure 3, panel A). At higher ionic strength, it is likely that the counterions shield the negatively charged E residue, and therefore the polymer becomes more hydrophobic and precipitates at lower Tt, leading to less pronounced effect at lower ionic strength. Polymer (17V)11, the polymer without the ionizable group in the repeat sequences, did not show appreciable sensitivity

to pH (Figure 3, panel B). This is because there is no ionization within the repeat sequences of the polymer to increase hydrophilicity and thereby modulate Tt. As pH increases from 3.0 to 5.0, polymer (17V)11 shows a slight increase in Tt values (Figure 3, panel B). This behavior can be due to the ionizable groups present in the pre- and postpolymer sequences of the polymer as well as the free terminal amino and carboxyl groups. For both (17E)11 and (17V)11, a distinct decrease in Tt with increasing ionic strength is observed. An increase in ionic strength results in a decrease in the tendency of water molecules to hydrate the polymer resulting in the precipitation of the polymer at lower temperatures. Polymer (17E)11

Stimuli-Sensitive Silkelastin-like Protein Block Copolymers

Figure 4. Influence of molecular weight on the solution transition behavior of 0.5 mg/mL solutions of polymers (17E)11 (open bars) and (17E)1621 (filled bars) at pH 7.0, and ionic strength ranging from 0.2 to 1.0. All Tt values are mean ( standard deviation of triplicate reading.

showed a higher degree of sensitivity to ionic strength than (17V)11. For example, the decrease in Tt upon increasing the ionic strength from 0.2 to 0.4 for (17E)11 at pH 6.0 is about 20 °C (Figure 3, panel A), whereas that for (17V)11 is about 2 °C (Figure 3, panel B). An increase in the ionic strength shields the charges on the polymer and decreases its solubility. The Tt values of the polymers decreased with increasing ionic strength, due mainly to the charged residues on both polymers. It should be noted that in addition to the effects due to charged residues, the nature of the salt can influence hydrophobic interactions.30 In the present study, the buffers used varied in salt composition, which may also have contributed to the effects observed. Results of this research indicate that it is possible to control the sensitivity of SELPs by substituting charged amino acids for neutral amino acids at precise locations along the polymer backbone and by control over polymer length using genetic engineering techniques. For (17V)11, transition in the physiological range can be achieved over a broad range of pH values (3.0-12.0) and ionic strengths (0.2-1.0). For (17E)11 and (17E)16, this range is narrower, since the polymer tends to remain in solution in the physiologic temperature range at pH values above 4.0 unless high ionic strengths are used. The results obtained in this work are in general agreement with the results of studies on elastin-like polymers.22,25 However, since this work was carried out at lesser polymer concentration (0.5 mg/mL) than the studies mentioned, direct comparisons cannot be made, for example, to delineate the influence of silk units on Tt. Conclusions In conclusion, silkelastin-like block copolymers with glutamic acid or valine substitutions at strategic positions were synthesized using genetic engineering techniques and characterized. The resulting biomaterials were differentially sensitive to pH, temperature, ionic strength, and concentration. The polymers showed a reversible transition from solution to precipitate at various pH, temperature, ionic strength, and concentration values. For the glutamic acid containing polymer, Tt (the temperature of half-maximal spectrophotometric absorption) increased with an increase in the pH range of 3.0-7.0, and the increase leveled off

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above this pH. Overall, the range of Tt values varied from around 25 °C to greater than 100 °C. The Tt values for the valine-containing polymer remained within a 10 °C range over the entire pH (3.0-12.0) and ionic strength (0.2-1.0) ranges studied. In the pH range studied, an increase in ionic strength resulted in a decrease in Tt. These results indicate that it is possible to adjust the temperature sensitivity of these genetically engineered biomaterials by the incorporation of amino acids with ionizable side chains and variation in parameters such as pH, concentration, ionic strength, and polymer length. These polymers show promise for applications where stimuli sensitivity of the polymer is desired. References and Notes (1) Kost, J.; Langer, R. AdV. Drug DeliVery ReV. 2001, 46, 125-148. (2) Yuk, S. H.; Bae, Y. H. Crit. ReV. Ther. Drug Carrier Syst. 1999, 16, 385-423. (3) Gaalev, I. Y.; Mattiasson, B. Trends Biotechnol. 1999, 17, 335340. (4) Nagarsekar, A.; Ghandehari, H. J. Drug Targeting 1999, 7, 11-32. (5) Ghandehari, H.; Cappello, J. Pharm. Res. 1998, 15, 813-815. (6) McGrath, K. P.; Tirrell, D. A.; Kawai, M.; Mason, T. L.; Fournier, M. J. Biotechnol. Prog. 1990, 6, 186-192. (7) Cappello, J.; Crissman, J. W.; Dorman, M.; Mikolajczak, M.; Textor, G.; Marquet, M.; Ferrari; F. Biotechnol. Prog. 1990, 6, 198202. (8) Fournier, M. J.; Creel, H. S.; Krejchi, M. T.; Mason, T. L.; Tirrell, D. A.; McGrath K. P.; Atkins, E. D. T. J. Bioact. Compat. Polym. 1991, 6, 326-338. (9) Cappello J.; Ferrari, F. In Plastics from Microbes; Mobley, D. P., Ed.; Hanser: New York, 1994; pp 35-92. (10) Cappello, J., Ghandehari, H., Eds. AdV. Drug. Del. ReV. 2002; 54. (11) Lucas, F.; Shaw J. T. B.; Smith, S. G. AdV. Protein Chem. 1958, 13, 107-242. (12) Sandberg, L. B.; Soskel, N. T.; Leslie, J. B. N. Engl. J. Med. 1981, 304, 566-579. (13) Cappello J.; Crissman, J. W.; Crissman, M.; Ferrari, F. A.; Textor, G.; Wallis, O.; Whitledge, J. R.; Zhou, X.; Burman, D.; Aukerman, L.; Stedronsky, E. J. Controlled Release 1998, 53, 105-117. (14) Cappello, J. In Controlled Drug DeliVery: Challenges and Strategies; Park, K., Ed.; American Chemical Society: Washington, DC, 1997; pp 439-453. (15) http://www.ppti.com (accessed Dec 2002). (16) Dinerman, A. A.; Cappello, J.; Ghandehari, H.; Hoag, S. Biomaterials 2002, 23, 4203-4210. (17) Dinerman, A. A.; Cappello, J.; Ghandehari, H.; Hoag, S. J. Controlled Release 2002, 82, 277-287. (18) Megeed, Z.; Cappello, J.; Ghandehari, H. Pharm. Res. 2002, 19, 954959. (19) Megeed, Z.; Cappello, J.; Ghandehari, H. AdV. Drug. DeliVery ReV. 2002, 54, 1075-1091. (20) Schindler, A. E.; Oberhoff, C.; Rohr, U. D.; Zentner, G. M.; McRea, J. C. Tenth international symposium in recent adVances in drug deliVery systems; Salt Lake City, UT, 2001; pp 82-83. (21) Nagarsekar, A.; Crissman, J.; Crissman, M.; Ferrari, F.; Cappello, J.; Ghandehari, H. J. Biomed. Mater. Res. 2002, 62, 195-203. (22) Beavis, R. T.; Chait, B. T. Rapid Commun. Mass Spectrom. 1989, 3, 432-435. (23) Urry, D. W. J. Phys. Chem. B 1997, 101, 11007-11028. (24) Franks, F. Protein Hydration. In Protein Biotechnology; Franks, F., Ed.; Humana: Totowa, NJ, 1993; pp 437-465. (25) Urry, D. W.; Trapane, T. L.; Prasad, K. U. Biopolymers 1985, 24, 2345-2356. (26) Luan, C.-H.; Harris, R. D.; Prasad, K. U.; Urry, D. W. Biopolymers 1990, 29, 1699-1706. (27) Bae, Y. H.; Okano, T.; Kim, S. W. Pharm. Res. 1991, 8, 531-537. (28) Okano, T.; Yamada, N.; Sakai, H.; Sakurai, Y. J. Biomed. Mater. Res. 1993, 27, 1243-1251. (29) Hoffman, A. S. Clin. Chem. 2000, 46, 1478-1486. (30) Blokzijl, W.; Engberts, J. B. F. N. Angew. Chem., Intl. Ed. Engl. 1993, 32, 1545-1579.

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