Design of thermally responsive, recombinant polypeptide carriers for targeted drug delivery

Advanced Drug Delivery Reviews 54 (2002) 1093–1111 www.elsevier.com / locate / drugdeliv

Design of thermally responsive, recombinant polypeptide carriers for targeted drug delivery Ashutosh Chilkoti*, Matthew R. Dreher, Dan E. Meyer Department of Biomedical Engineering, Duke University, Durham, NC 27708 -0281, USA Received 25 May 2002; accepted 1 July 2002

Abstract In this article, we review recombinant DNA methods for the design and synthesis of amino acid-based biopolymers, and briefly summarize an approach, recursive directional ligation (RDL), that we have employed to synthesize oligomeric genes for such biopolymers. We then describe our ongoing research in the use of RDL to synthesize recombinant polypeptide carriers for the targeted delivery of radionuclides, chemotherapeutics and biomolecular therapeutics to tumors. The targeted delivery system uses a thermally responsive, elastin-like polypeptide (ELP) as the drug carrier to enhance the localization of ELP-drug conjugates within a solid tumor that is heated by regional hyperthermia. In the context of this drug delivery application, we discuss the design of ELPs and their recombinant synthesis, which enables the molecular weight and the thermal properties of the polypeptide to be precisely controlled. Finally, our results pertaining to the in vivo targeting of tumors with ELPs are briefly summarized.  2002 Elsevier Science B.V. All rights reserved. Keywords: Artificial polypeptide; Genetically encoded synthesis; Elastin-like polypeptide; Thermally responsive; Drug delivery; Thermal targeting; Cancer therapy; Block copolymers; Nanoparticles; Micelles

Contents 1. Introduction ............................................................................................................................................................................ 2. Methods for synthesis of biopolymers by DNA oligomerization.................................................................................................. 2.1. Genetic strategies for synthesis of protein-based polymers .................................................................................................. 2.2. Recombinant synthesis of ELPs by recursive directional ligation ......................................................................................... 3. Elastin-like homopolymers and pseudorandom copolymers ........................................................................................................ 3.1. Synthesis of elastin-like homopolymers and pseudorandom copolymers ............................................................................... 3.2. Thermal behavior of elastin-like homopolymers and pseudorandom copolymers ................................................................... 3.3. In vivo characterization of the elastin-like polypeptide delivery system ................................................................................ 4. Elastin-like block copolymers ..................................................................................................................................................

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Abbreviations: RDL, recursive directional ligation; ELP, elastin-like polypeptide; MW, molecular weight; EPR, enhance permeability and retention; Xaa, unconstrained amino acid residue; T t , transition temperature; T b , body temperature; T h , temperature in the heated tumor; RE, restriction endonuclease; BSA, bovine serum albumin; IB, iodobenzoate *Corresponding author. Tel.: 1 1-919-660-5373; fax: 1 1-919-660-5362. E-mail address: [email protected] (A. Chilkoti). 0169-409X / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 02 )00060-1

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4.1. Block copolymers ............................................................................................................................................................ 4.2. Synthesis of elastin-like block copolymers ......................................................................................................................... 4.3. Characterization of elastin-like block copolymers ............................................................................................................... 4.4. Future applications of elastin-like block copolymers ........................................................................................................... 5. Conclusions ............................................................................................................................................................................ Acknowledgements ...................................................................................................................................................................... References ..................................................................................................................................................................................

1. Introduction Protein-based polymers, which are composed of repeat units of natural or non-natural amino acids, have recently emerged as a promising new class of materials [1–4]. Protein-based polymers frequently have desirable mechanical, chemical and biological properties (e.g., general biocompatibility, biorecognition and biodegradation) that make their use appealing for in vivo applications as biomaterials and tissue engineering scaffolds. Protein-based polymers are degraded by the normal protein turnover pathways, and, as opposed to many synthetic polymers, the degradation products are nutrients that are excreted or utilized by physiological processes of the body [2]. The sequence and molecular weight (MW) of repetitive polypeptides are of particular importance because these two primary architectural variables determine the physicochemical properties of the macromolecule, and are also important for in vivo applications since they control pharmacokinetics, tissue and cellular transport phenomena, biological activity and the biodegradation of the polypeptide [5,6]. The precise and rapid synthesis of genes encoding a polypeptide of desired sequence and length is therefore a key requirement for producing genetically encoded, repetitive polypeptides for specific in vivo applications. We are interested in the use of genetically engineered polymers for delivery of cancer therapeutics because of the precise control of polymer architecture that is afforded by the recombinant synthesis of biopolymers, as well as their favorable biocompatibility properties. Because targeting is necessary for efficient tumor localization of systemically delivered therapeutics, substantial effort has been devoted in the past three decades to the design of carriers that can selectively localize the drug within the tumor. These drug carriers can be divided into two broad categories of affinity and passive target-

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ing. The affinity-targeting approach attempts to take advantage of over-expressed tumor associated antigens or receptors to selectively target the drug and an affinity-targeting carrier such as a peptide, antibody, or antibody fragment to the tumor [7]. In passive targeting, liposomes, nanoparticles or macromolecular carriers [8–10] exploit the enhanced permeability and retention (EPR) effect, which is a consequence of the increased vasculature permeability and decreased lymphatic function of tumors, to target the drug to the tumor [11,12]. Despite several decades of research and development, targeted delivery has not yet fulfilled its initial promise in the treatment of cancer. The targeted delivery of drugs to solid tumors is a complex problem because of the impediments to drug delivery that are posed by tumor heterogeneity [13]. Cancer cells typically occupy less than half of the total tumor volume. Approximately 1–10% is contributed by tumor vasculature, and the rest is occupied by a collagen-rich interstitium. The major impediments to drug delivery arise from heterogeneous distribution of blood vessels, combined with aberrant branching and tortuosity, which results in uneven and slowed blood flow. The leakiness of tumor vessels combined with the absence of a functional lymphatic system results in an elevated interstitial pressure, which retards the convective transport of high MW ( . 2000 Da) drugs [14]. The heterogeneity of antigen and receptor expression in tumors is an additional problem in affinity-targeted delivery of drugs to solid tumors. Hence, there is a clear need to explore alternative targeting methods to improve the delivery of drugs to tumors. In our research, we are studying a targeted delivery system using regional hyperthermia to improve accumulation of a drug–polymer conjugate within solid tumors. This thermal-targeting scheme uses a thermally responsive, recombinant elastin-like polypeptide (ELP) as the drug carrier. ELPs are biopoly-

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mers with the pentapeptide repeat Val–Pro–Gly– Xaa–Gly, where the ‘guest residue’ Xaa can be any of the natural amino acids except Pro [15]. ELPs undergo an inverse temperature phase transition; they are soluble in aqueous solutions below their transition temperature (T t ), but hydrophobically collapse and aggregate at temperatures greater than T t [15,16]. We hypothesized that an ELP carrier with a T t that is intermediate between normal body temperature (T b ) and the temperature in a heated tumor (T h ) would enable thermally targeted drug delivery to a locally heated region. In this scenario, the ELP would be soluble systemically because its inverse transition temperature (T t ¯ 40 8C) is greater than physiologic body temperature (T b ¯ 37–38 8C), but would become insoluble and accumulate in locally heated regions where the temperature was increased to above the T t by externally targeted hyperthermia (T h ¯ 42–43 8C). This would combine thermal targeting through the ELP phase transition with the established advantages of both polymeric carriers, such as increased plasma half-life and high loading capacity [11], and those of hyperthermia as an anticancer treatment modality, which include increased sensitivity to therapeutics and greater tumor extravasation of macromolecules [12,17–23]. The design of ELP carriers is dictated by the different requirements for the method of incorporation (i.e., conjugation, chelation or encapsulation) of the drug into the carrier, and the different requirements for in vivo localization of the drug. There are three events in targeted delivery that are critical for effective therapy: first, targeting of the drug-carrier conjugate to the tumor microvasculature, second, extravasation from the tumor vasculature, and third, transport of the drug to the appropriate molecular site of action [14]. The relative importance of these three, sequential events depends greatly on the mode of action of the drug. When the drug is a radionuclide, targeting of the drug preferentially to the tumor microvasculature might be sufficient to gain a therapeutic advantage. In contrast, when the therapeutic agent has a site-specific mode of action such as most chemotherapeutics and biomolecular therapeutics (proteins, DNA, RNA, carbohydrates or their mimics), the polymer-drug conjugate must extravasate from the tumor microvasculature, and be transported to and internalized by tumor cells. Further-

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more, when these drugs are conjugated to a polymer carrier, intracellular release of the drug from the polymer carrier via the incorporation of proteolytically- and / or hydrolytically-labile linkages between the drug and the polymer may be necessary. ELP drug carriers can also be divided into three different classes based on their intended end-use as carriers: (1) ELP homopolymers and pseudorandom copolymers for the systemic delivery of chemically conjugated radionuclides and chemotherapeutics [24]; (2) block copolymers that thermally assemble into micelles or vesicles, designed for the encapsulation of hydrophobic drugs [25,26]; and (3) fusion proteins for the delivery of protein therapeutics. We describe the recombinant synthesis of the first two classes of ELP drug carriers, and our work to date in investigating the feasibility of thermally targeted drug delivery using these carriers. This article is focused on the design of the ELPs and their recombinant synthesis to enable important macromolecular properties such as the MW and aggregation state to be precisely controlled, as well as specifying the thermal properties of the polypeptide. The central results on in vivo targeting are briefly summarized in this article, and the reader is referred elsewhere for a more detailed presentation of these results [24].

2. Methods for synthesis of biopolymers by DNA oligomerization

2.1. Genetic strategies for synthesis of proteinbased polymers In synthesizing genes encoding repetitive proteinbased polymers, the techniques of molecular biology are typically employed to self-ligate monomer DNA fragments in a process of oligomerization. The monomer fragments must be oligomerized in a ‘head-to-tail’ orientation, and can be seamless in sequence or can contain intervening linkers between the desired repeats. Approaches to oligomerization can be broadly classified as either iterative, random or recursive, although these modes can be sequentially combined within the same implementation. Each of these methods is illustrated in Fig. 1. For iterative techniques, a DNA segment is oligomerized in a series of single, uniform steps; each step grows the

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Fig. 1. Schematic of three approaches to DNA oligomerization. Each line segment represents a DNA fragment encoding a monomer gene sequence.

oligomer by one unit length of the monomer gene. In random methods, an uncontrolled number of monomer DNA segments are oligomerized in a single step, creating a population of oligomerized clones of different lengths. This random approach of selfligation is referred to as ‘concatemerization’ because the DNA segment is concatenated in a reaction that is analogous to the propagation step in chemical polymerization. Finally, in recursive approaches, DNA segments are joined in sequential steps, with the length of the ligated segments growing geometrically in each step. Some of the earliest examples of the synthesis of genes encoding protein based polymers, reported by Protein Polymer Technologies of San Diego, CA [2] and by the Tirrell and coworkers at the University of Massachusetts [4], relied on concatemerization methods. Using a prototypical concatemerization approach, Cappello et al. produced a number of genes encoding silk-like and elastin-like copolymers, including one of approximately 160 repeats of a six amino acid repeat sequence, through self-ligation of a monomer sequence [2]. McGrath et al. also used a concatemerization technique to produce a gene encoding a 12-mer of a ((AlaGly) 3 ProGluGly) repeat, although they were unsuccessful in obtaining greater numbers of repeats by fractionation of the ligation products obtained from concatemerization [4]. Creel et al. similarly produced a gene encoding a 14-mer of an ((AlaGly) 4 ProGluGly) repeat. In a later paper, they successfully located larger oligomers encoding

up to 54 repeats of the (AlaGly) 3 ProGluGly nonapeptide [27]. McPherson et al. produced a gene encoding 19 pentapeptides of an ELP sequence by first creating a synthetic gene encoding 10 pentapeptides, which was then amplified by PCR and recombined by bluntended ligation [28]. In 1996, they reported a concatemerization-based technique to produce genes encoding 41, 121, 141 and 251 pentapeptides [29]. Chain-terminating sequences were used to enhance the population of DNA oligomers in the desired size range, and to minimize the circularization of the growing oligomers in the ligation reaction. Several other examples of ELP synthesis through genetic techniques have been reported. Kostal et al. produced ELP genes ranging from 10 to 78 pentapeptides by iteratively adding 10 pentapeptide segments which had been amplified from the monomer gene by PCR [30]. McMillan et al. used the ‘Seamless’ cloning technique [31] to produce two different ELP sequences [32]. They used a concatemerization method in which the monomer gene segment is produced by digestion with the type IIs restriction endonuclease (RE) Eam1104 I. The recognition sequence for the enzyme is located outside of the coding region, and thus the system can be used for any coding sequence without constraint. The oligomerized genes were cloned directly into an expression vector, which had been prepared using a PCR technique that yielded linearized vector that was prepared to receive the insert. This PCR technique obviates the need for a unique restriction site on the expression plasmid. Two clones of different sizes, one for each sequence, were isolated that were 3000 and 1350 bp in length, with the former encoding a | 90 kDa ELP. Kobatake et al. used concatemerization to synthesize a gene of a repeating hexapeptide sequence derived from elastin [33]. Directionality was ensured because the monomer was produced by digestion with nonpalindromic Ban I at sites located seamlessly at each end of the gene. After obtaining 12 repeats of the hexapeptide monomer sequence by random concatemerization, this larger gene was recursively, but not seamlessly, combined twice to yield the final product, a 48-mer. These synthetic genes for ELPs, created by a variety of oligomerization methods, have been successfully expressed in bacteria [28,34], fungi [35], chloroplasts [36] and

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plants [37] and have been used in different applications in biotechnology and medicine [24,30,38–44]. Genetic techniques have been used to produce many other protein-based polymers. For example, Feeney et al. produced wheat glutenin-based sequences encoding polypeptides up to 203 amino acids with an alternating hexapeptyl / nonapeptyl repeat [45]. Their iterative strategy employed a RE site within the coding sequence that was compatible with the overhangs of the oligonucleotide cassette, which itself contained the same RE site in the same location. When the cassette is ligated into the vector, the original site is destroyed leaving only the single site introduced in the middle of the newly inserted gene cassette. The process was repeated, incrementing the length of the gene by the same 90 bp in each step. There have been a number of examples of genetically directed synthesis of protein-based polymers using sequences from spider silk. Prince et al. located their synthetic monomer gene between two RE sites, Nhe I and Spe I, that cleave to form compatible, palindromic sticky ends [46]. For the first stages of oligomerization, they used a concatemerization approach, including both REs in the ligation reaction to favor head-to-tail orientation. By ligating an insert produced by double digestion into a vector prepared by digestion with only one of the enzymes, they were also able to recursively oligomerize previously generated gene segments. Winkler et al. subsequently used the same method to produce smaller silk-like proteins incorporating a redox trigger to control assembly [47]. For some applications, it can be useful to create protein-based block copolymers comprised of domains with different repeating sequence. For example, Petka et al. produced a block copolymer, containing two coiled–coil domains separated by flexible polyelectrolyte domain, that exhibited reversible gelation in response to environmental triggers [48]. The gene was produced by first constructing each separate domain in its entirety with synthetic oligonucleotides, and then joining them together by ligation using RE sites located in linker sequences adjacent to the helical and polyelectrolyte domains. Lee et al. created an AB block copolymer composed of two ELP domains that exhibited different T t values, and used the differences in thermal properties

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to drive the formation of micelle-like nanoparticles [49]. Each ELP domain was separately generated by concatemerization, and then separately ligated into a linker region on an engineered expression plasmid. In these examples, genes encoding protein-based polymers were joined together to form block copolymers using linkers that provided the necessary RE recognition sites. Although this approach is a convenient method to produce block copolymers, the introduction of extraneous intervening residues in the linker between the blocks can be disadvantageous. In summary, although a number of different strategies have been developed to assemble synthetic genes for protein-based polymers, most methods have focused on the simultaneous generation of a library of oligomeric genes by concatemerization of a monomer gene. Concatemerization has the advantage of creating, in a single ligation step, a library of genes of different length that encode oligomeric polypeptides with the same repeat sequence. Although concatemerization is rapid, it sacrifices precise control over the oligomerization process because it is a statistical process that yields a population of DNA oligomers with a distribution of lengths. Although the average degree of oligomerization can be partially controlled by varying the ligation conditions, concatemerization does not guarantee the synthesis of a specific gene of desired length. Therefore, concatemerization is a useful synthetic strategy when a range of MWs need to be rapidly generated but where synthesis of a gene encoding a specified number of peptide repeats is of secondary importance.

2.2. Recombinant synthesis of ELPs by recursive directional ligation In the synthesis of ELP genes, we have employed a method, which we term ‘recursive directional ligation’ that is generally applicable for the synthesis of repetitive polypeptides of specific and predetermined chain length. We developed RDL because we wished to study the effect of sequence, MW and the architecture (e.g., in ELP block copolymers) of the ELP on its thermal behavior for applications in drug delivery. The ability to precisely control these variables is crucial for thermal targeting because the T t of an ELP is independently related to both its

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Fig. 2. The molecular biology steps of RDL. (A) A synthetic monomer gene is inserted into a cloning vector. (B) The gene is designed to contain recognition sites for two different restriction endonucleases, RE1 and RE2. (C) An insert is prepared by digestion of the vector with both RE1 and RE2, and subsequently ligated into the vector that has been linearized by digestion with only RE1. (D) The product contains two head-to-tail repeats of the original gene, with the RE1 and RE2 sites maintained only at the ends of the gene. (E) Additional rounds of RDL proceed identically, using products from previous rounds as starting materials. Modified from [60].

sequence (i.e., the identity of the guest residues) and to its chain length [50,51]. A schematic of RDL is shown in Fig. 2. A synthetic oligonucleotide cassette encoding the monomer gene is first ligated into a cloning vector such as pUC19. The oligonucleotides are designed so that EcoR I and HinD III compatible cohesive ends are produced upon annealing, which enables the annealed product to be directly ligated into EcoR I and HinD III cleaved pUC19 (Fig. 2A). The monomer gene encodes a defined number of pentapeptide repeats, and the coding sequence is flanked on each end by two different restriction endonuclease recognition sites (Fig. 2B). These two sites, generically labeled RE1 and RE2 in Fig. 2, are used to oligomerize the gene by RDL as follows. An insert is produced by cleaving the plasmid that harbors the monomer gene with both RE1 and RE2, and a linearized vector is produced by separately digesting another aliquot of the same plasmid with only RE1 (Fig. 2C). The purified insert is ligated into the

linearized vector, resulting in dimerization of the gene (Fig. 2D). The monomer gene is designed such that gene oligomerization by RDL achieves three goals. First, the insert is ligated with its directionality preserved in a head-to-tail orientation upon ligation into the vector. Second, the ligation is seamless in that extraneous residues are not introduced at the ligation site. Third, the original recognition sites for RE1 and RE2 are maintained at each end of the dimerized gene, but neither recognition site is generated at the intervening ligation site. Therefore, the oligomer assembled in any round of RDL can be used in future rounds of RDL as the insert and / or the vector (Fig. 2E). Later rounds of RDL are identical to the first round, except that the product from a previous round serves as the source of the insert and vector. RDL requires the selection of a pair of restriction endonucleases that satisfy four requirements. First, they must have different recognition sequences so that the DNA can be selectively cleaved either by one or by both of the enzymes. Second, the two enzymes must produce complementary, singlestranded DNA overhangs upon cleavage. Third, at least one of the two sites (generically, RE1), after its introduction into the cloning vector in the first round of RDL, should be unique on the cloning vector so that digestion with the enzyme cleaves the plasmid only at a single site. Finally, the recognition sequences of both REs must be compatible with the coding sequence of the polypeptide such that, upon ligating together two gene segments, the repeat sequence of the polypeptide is not disrupted at the internal site of ligation. To fulfill these four requirement, PflM I and Bgl I were selected as RE1 and RE2, respectively, in our implementation of RDL for the oligomerization of ELP sequences. The different ELPs described in the following sections are named using the notation ELP[Xi Y j -n]. The bracketed capital letters are the single letter amino acid codes specifying the guest residues in the ELP sequence, and the corresponding subscripts designate the frequency of each guest residue in the monomer gene. Thus, the sum of the subscripts is equal to the number of Val–Pro–Gly–Xaa–Gly repeats in the monomer unit. n is the total length of the ELP gene in number of pentapeptides. For example, ELP[V5 A 2 G 3 -180] is an ELP of 180 penta-

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peptides in length that has a repeat unit composed of 10 pentapeptides with the guest residues Val, Ala and Gly in a 5:2:3 ratio, respectively. If n is unspecified, the notation refers to a MW library of a given sequence, rather than to an ELP construct of specific length.

3. Elastin-like homopolymers and pseudorandom copolymers

3.1. Synthesis of elastin-like homopolymers and pseudorandom copolymers In the design of ELPs for thermal targeting, the two critical parameters that we wished to specify were the MW of the polypeptide and the transition temperature of the carrier. Because the T t is related to both the amino acid composition (i.e., the identity and fraction of the fourth residue in the pentapeptide) as well as MW, libraries of ELPs were synthesized as a strategy to obtain a polymers of different MW, an important factor that partially determines the pharmacokinetic behavior of the carrier, but which also display the desired T t of | 40 8C. Within each library, genes encode the same repeat unit, but differ in the number of repeats, and hence MW. We designed three ELP gene libraries with the goal of selecting individual members that would provide ELPs with the target T t of | 40 8C but a different MW in the 10–100 kDa range. A secondary consideration in the design of these libraries was also to be able to create size-matched, thermally unresponsive controls for corresponding thermally responsive polymers. These controls are also ELPs, and therefore, have physicochemical properties that are similar to the thermally responsive carriers, but have a T t of . 50 8C so that they would undergo their transition in tumors heated to 42 8C. The DNA and corresponding amino acid sequences for all three libraries are shown in Fig. 3. The first library, ELP[V5 ], comprises of a set of Val–Pro–Gly–Val–Gly homopolymers ranging in length up to 120 pentapeptides ( | 50 kDa). The second library, ELP[V5 A 2 G 3 ], is a more complex copolymer that contains Val, Ala and Gly in a 5:2:3 ratio at the fourth residue of the ELP pentapeptide

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repeat. This library encodes polypeptides ranging up to 330 pentapeptides ( | 130 kDa). The third library, ELP[V1 A 8 G 7 ], is a set of copolymers with a Val:Ala:Gly guest residue ratio of 1:8:7, ranging up to 320 pentapeptides ( | 120 kDa). As shown in Fig. 3, for the two copolymers, we dispersed the different guest residues throughout each sequence in order to minimize repetition within the gene. We therefore refer to ELPs belonging to these two libraries as pseudorandom copolymers. Preferred E. coli codons were favored where possible [52], with exceptions made to reduce repetition of the nucleotide sequence. We chose different monomer gene lengths for each ELP sequence (see Fig. 3), depending on the desired incremental oligomerization step size during RDL. In order to illustrate the methodology by which these ELPs are produced, we summarize here the synthesis of ELP[V5 A 2 G 3 ], although similar results were obtained for the other two gene libraries. Fig. 4A shows the results of DNA agarose gel electrophoresis of the gene fragments for the entire ELP[V5 A 2 G 3 ] library, which was produced as follows. The monomer gene, which encodes 10 pentapeptides, was constructed by annealing chemically synthesized oligonucleotides that encode for the sense and antisense strands of the monomer. The annealed product was ligated into pUC19, yielding pUC19-ELP[V5 A 2 G 3 -10]. For the first round of RDL, the insert was prepared by digesting pUC19ELP[V5 A 2 G 3 -10] with PflM I and Bgl I, and purifying the insert after agarose gel electrophoresis. An ELP vector was prepared to receive the insert by linearizing another aliquot of the same pUC19-ELP[V5 A 2 G 3 -10] plasmid with only PflM I. The insert encoding the monomer gene was then ligated into the vector. Transformants yielded pUC19-ELP[V5 A 2 G 3 20], the gene dimer, and pUC19-ELP[V5 A 2 G 3 -30], a gene trimer. The trimer gene was the product of a trimolecular ligation, where two copies of the insert and one copy of the linearized vector were ligated together. These double inserts are functionally identical to two sequential RDL steps using the same insert, and therefore, the seam at the ligation joint and the head-to-tail orientation of both inserts are the same as for a RDL product from a single insert. We have observed that, for small inserts less than 500 bp, double inserts are commonly obtained for a small but significant fraction of transformants. This is

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Fig. 3. Gene and corresponding polypeptide sequences. (A) Monomer genes for the three ELP libraries are shown. The PflM I and Bgl I sites are shown with recognition sequences in bold, cleavage sites indicated by arrows, and cohesive ends underlined. (B) The gene sequences of three example expression vectors, showing the modifications that enable insertion of the ELP gene into the Sfi I site. The modifications encode for a unique Sfi I site, and also encode short leader and trailer peptides. Reprinted with permission from [60]. Copyright (2002) American Chemical Society.

useful because it reduces the number of RDL cycles required to build a larger library. In the second round of RDL, an insert encoding 30 pentapeptides was prepared from pUC19-ELP[V5 A 2 G 3 -30] by doubly digesting the vector with PflM I and Bgl I, and then ligating the ELP insert into PflM I linearized pUC19-ELP[V5 A 2 G 3 -30] vector, to yield the gene for 60 pentapeptide repeats. The 30 pentapeptide gene insert from the second round was then ligated into the pUC19-ELP[V5 A 2 G 3 -60] vector to form a gene encoding 90

pentapeptides. Next, the genes encoding 120, 150 and 180 pentapeptides were produced in parallel by ligating inserts encoding 30, 60 and 90 pentapeptides into the pUC19-ELP[V5 A 2 G 3 -90] vector, respectively. Finally, genes encoding 240 and 330 pentapeptides were created by ligating 60 and 150 pentapeptide gene inserts into the pUC19-ELP[V5 A 2 G 3 180] vector. The genes synthesized by RDL in the pUC19 cloning vector were transferred to the expression vector as follows. First, the pET25b( 1 ) T7 lac

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for Sfi I (Fig. 3B) We chose Sfi I as the insertion site because it has a partially degenerate recognition site, which can be specified to allow the single stranded overhang produced by cleavage of the expression vector with Sfi to be compatible with the ELP inserts excised from the pUC19 cloning vector by digestion with PflM I and Bgl I. Next, the pUC19 cloning vector containing the ELP gene was digested with PflM I and Bgl I, and ligated into a modified pET25b( 1 ) T7 lac expression vector that had been linearized with Sfi I. The expression vector was transformed into the E. coli strain BLR(DE3), and the cells were grown in shaker flasks at 37 8C, and induced using isopropyl b -Dthiogalactopyranoside. The expressed ELPs were present in the soluble fraction of the cell lysate, and were purified by inverse transition cycling [29]. Fig. 4B shows SDS–PAGE results for the ELP[V5 A 2 G 3 ] library. The ELPs consistently migrate | 20% larger than the protein standards, which is consistent with previous observations [29]. The mass of each ELP was independently confirmed by matrix-assisted laser desorption ionization mass spectrometry.

3.2. Thermal behavior of elastin-like homopolymers and pseudorandom copolymers

Fig. 4. ELP library produced by RDL. (A) Agarose gel (1.2%) electrophoresis of ELP[V5 A 2 G 3 ] genes visualized by ethidium bromide staining. The left lane contains a size standard, which is labeled in bp. Plasmids containing the ELP genes were digested with EcoR I and HinD III, producing a vector fragment (2635 bp) and an ELP gene fragment. The expected size of each ELP gene is labeled in bp on the right. The number of pentapeptide repeats encoded by each gene is labeled below each lane. (B) SDS–PAGE (4–20% gradient) visualized by copper staining of purified ELPs expressed from the genes in (A). The left lane contains a molecular weight standard, which is labeled in kDa. The expected molecular weight of each ELP is indicated on the right. The length in pentapeptides of each ELP is labeled below each lane. Reprinted with permission from [60]. Copyright (2002) American Chemical Society.

expression vector was modified by replacement of the region between Nde I and EcoR I by cassette mutagenesis to incorporate a unique recognition site

The thermal behavior of each member of the three ELP libraries was studied by measuring solution turbidity as a function of temperature. The T t is defined as the temperature at the onset of turbidity (5% maximum turbidity) in the upward thermal ramp. Fig. 5 shows a heating and cooling turbidity profile for the ELP[V5 A 2 G 3 -150] carrier. Below the T t , the polypeptide solution was clear, but upon further heating, the solution became turbid because of ELP aggregation. The rapid increase in turbidity upon reaching T t shows that the transition is very sharp with respect to temperature, occurring over a range of less than 2 8C. The inverse transition of each ELP was completely reversible, and no endpoint hysteresis was observed. The slight difference between the paths of the heating and cooling traces is attributed to slower kinetics of disaggregation as compared to aggregation. The data in Fig. 6 show that the T t is a function of two intrinsic parameters that are important for thermally targeted drug delivery, both of which can be

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Fig. 5. Typical, temperature-dependent turbidity profiles for ELP[V5 A 2 G 3 -150]. The turbidity profiles of the polypeptide (25 mM in PBS) were measured at a rate of 1 8C / min. Heating and cooling traces (marked by arrows) show that the transition is completely reversible.

We determined the T t at lower concentrations than Urry et al. because of our interest in concentrations that are relevant for drug delivery ( | 25 mM). Our synthesis method employs genetic techniques that yield nearly monodisperse ELPs while Urry utilized a chemical synthesis that results in a polydisperse population. Guest residue sequence is the second intrinsic parameter controlling the T t (Fig. 6). For a given MW, decreasing the hydrophobicity of the guest residues increases the T t , as has been previously reported by Urry et al. [50]. Based on these data, it is possible to design an ELP of specified sequence and MW to exhibit a desired T t for drug delivery applications The ELP concentration is also known to affect the T t [51]. Fig. 7 shows that the T t is a decreasing logarithmic function of ELP concentration. Changes in concentration may alter the extent of intermolecular hydrophobic interactions and the balance between waters of hydration and bulk waters. Achieving and maintaining the intended ELP plasma concentration is an important consideration for thermal targeting because if the in vivo concentration is too high, the T t will drop below T b and the carrier will undergo its phase transition systemically. This would eliminate selective targeting to the heated region and could potentially deliver an unintended dose of aggregated carrier to other organs. Conversely, if the in vivo concentration is too low, then the T t would be greater than T h , and the carrier will remain soluble

Fig. 6. ELP T t as a function of sequence and chain length. The T t was determined by temperature-dependent turbidity measurements for each member of three ELP libraries: ELP[V5 ], ELP[V5 A 2 G 3 ], and ELP[V1 A 8 G 7 ]. The ELP concentration was 25 mM in PBS. The dashed lines represent single-parameter fits to the equation: T t 5 C / Length, where the value of the constant, C, is determined independently for each library. Modified from [60].

precisely controlled at the gene level through RDL. First, the T t is inversely proportional to the ELP length, and therefore, this effect is most dramatic for lower MW ELPs. Although these results are qualitatively similar to previous results of Urry et al. [51], they observed a decreasing logarithmic relationship between T t and MW. The disparity in our results may arise from the different concentrations studied (Urry et al.: | 40 mg / ml; Chilkoti et al.: | 0.25–3.25 mg / ml) and / or from the methods of ELP synthesis.

Fig. 7. T t as a function of ELP[V5 A 2 G 3 -150] concentration, showing an decreasing logarithmic relationship (dashed line). The ELP dose was selected to result in a desired target T t of | 41 8C in vivo after dilution in plasma. Modified from [24].

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within tumor vasculature. Although this would eliminate thermal targeting to the heated tumor, this latter outcome is less problematic because there would also be no aggregation of the ELP carriers in non-targeted organs. During therapy, T t should therefore remain above T b , but more safely be allowed to increase to above T h as the carrier concentration decreases with time due to systemic clearance. Despite these constraints, the dependence of T t on ELP concentration does not severely limit the applicability of ELP thermal targeting because the effect is logarithmic, and thus, only a relatively small change in T t is caused by larger changes in ELP concentration. With ELP[V5 A 2 G 3 -150], for example, a T t can be achieved in the range that is clinically available for thermal targeting between 37 and 42 8C over a | 10-fold range in ELP concentration (Fig. 7). If higher or lower ELP doses are required for therapy, ELP sequences with different compositions can be readily synthesized such that they exhibit a T t between 37 and 42 8C in a different range of concentrations as compared to ELP[V5 A 2 G 3 -150]. We also note that if the total dose of drug needs to be limited below a specified threshold as defined by its systemic toxicity, free ELP carrier can be mixed with the ELP-drug conjugate to provide the necessary polypeptide concentration required to achieve the target T t . For our drug delivery studies, two polymers with similar MW but disparate thermal behavior, ELP[V5 A 2 G 3 -150] (59 kDa) and ELP[V1 A 8 G 7 -160] (61 kDa), were selected for further characterization. ELP[V5 A 2 G 3 -150] was chosen as the thermally responsive polymer because it exhibits a T t that is within the temperature range defined by T b ( | 37 8C) and T h ( | 42 8C) for concentrations between | 5 and 25 mM. In contrast, for a similar range of concentration, ELP[V1 A 8 G 7 -160], has a T t of | 70 8C that is significantly greater than the hyperthermia temperature of 42 8C, and therefore is a sizematched, thermally unresponsive control for ELP[V5 A 2 G 3 -150]. Because it is critical that the T t of the thermally sensitive carrier, ELP[V5 A 2 G 3 -150], remain between 37 and 42 8C after injecting the drug-ELP conjugate, prior to initiating in vivo studies of thermal targeting, we systematically studied the effect of conjugation of small molecule drug mimics and the effect of

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cosolutes on the thermal behavior of the ELP. These studies were motivated by the hypothesis that the inverse transition of ELPs is likely to be sensitive to drugs that are attached to the ELP, as well as by the presence of other solutes in aqueous solution (e.g., salts and proteins). Conjugation of reporters and drugs to the ELPs was achieved by designing the ELPs so that they have a short leader sequence that provides unique reactive groups to enable chemical conjugation via amine coupling chemistry. We commonly used a SKGPG leader peptide that is incorporated at the N-terminus of the ELP at the gene level, which provides an N-terminal amine and the ´ -amine in the Lys (K) residue for chemical conjugation. Typically, radionuclides or fluorophores are directly conjugated to these amine groups via a hydrolytically stable, amide linkage. In other experiments that involve conjugation to chemotherapeutics, we have attached the chemotherapeutic agent via a hydrolytically labile linker to enable intracellular release of the drug in lysosomes. Fig. 8 shows that reporter molecules such as iodobenzoate (IB), a radiolabeling agent used to conjugate 131 I to the ELPs, and rhodamine reduced the T t of the thermally sensitive carrier, ELP[V5 A 2 G 3 -150]. We hypothesize that this decrease in T t upon conjugation of reporter molecules is caused by hydrophobic interactions between the conjugated, nonpolar reporter molecules and the

Fig. 8. T t values of free ELP[V5 A 2 G 3 -150] or its conjugates to reporter molecules as a function of ELP concentration, either in PBS or in PBS supplemented with 0.9 mM BSA. Dotted lines represent logarithmic fits to the data.

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ELP. This effect may be analogous to the decrease in the T t observed with increasing hydrophobicity of the guest residue. Cosolutes, including salts and soluble plasma proteins, also have been shown to alter the T t of ELPs [16,40]. We found that murine plasma reduced the T t of ELP[V5 A 2 G 3 -150] by | 4 8C compared to pure water, and a similar effect is produced using 0.9 mM BSA as mock plasma Fig. 8. The decrease in T t after conjugation has important implications for the loading of ELPs as therapeutic carriers, particularly for drugs with a significant hydrophobic character. The decrease of T t by | 4 8C observed can be compensated through adjustment of the ELP plasma concentration during thermally targeted delivery. However, for drugs that are more hydrophobic or for higher drug-to-carrier loading ratios, the carrier can be designed to have a higher T t in anticipation of a larger downward shift caused by conjugation of the drug. Similar results were also obtained for the thermally insensitive control, ELP[V1 A 8 G 7 -160].

3.3. In vivo characterization of the elastin-like polypeptide delivery system Based on the results from in vitro characterization, we selected a target plasma concentration of 5 mM for the in vivo experiments. The accumulation of an ELP-rhodamine conjugate was studied in vivo as a function of temperature in a dorsal skin flap window chamber model. In this model, athymic mice are implanted with dorsal skin fold window chambers containing a small piece of tumor tissue, which is placed near the center of the window [18–20]. The preparations are typically used 7–8 days after implantation, when the tumors have grown to 2–3 mm in diameter. The mice are anesthetized and positioned laterally recumbent on a microscope stage, and the window chamber, which is connected to a temperature controlled water bath is maintained at either the physiologic subcutaneous temperature in mice, 34 8C, or at 42 8C. Under epi-illumination, fluorescence images for rhodamine are acquired by a CCD camera. Images are typically recorded continuously for 40 s after injection and for 10 s every 2 min for 60 min. In a typical experiment, 200 ml of 100 mM of the

ELP-Rhodamine conjugate in PBS ( | 1.2 mg / mouse) was injected into the cannulated tail vein. Fig. 9 shows representative videomicrographs acquired at 30 s and at 30 min post-injection for three groups of animals: ELP[V5 A 2 G 3 -150] at 42 8C, ELP[V1 A 8 G 7 -160] at 42 8C and ELP[V5 A 2 G 3 -150] at 34 8C. Within 1–2 min after the thermally responsive ELP1 carrier was injected into mice with window chambers heated to 42 8C, fluorescent particles were observed (Fig. 9, panel ii). No particles were observed for ELP[V1 A 8 G 7 -160] with hyperthermia (panel iii, iv) or for ELP[V5 A 2 G 3 -150] without hyperthermia (panel v, vi) at any time, strongly suggesting that these particles are ELP aggregates resulting from the inverse temperature transition. The aggregates often attached to the vessel wall, and grew in intensity and size over time. Once attached to the vessel wall, the aggregates were typically stable throughout the experiment. More rarely, as the particles grew larger, they were sheared from the vessel walls and carried away by the blood flow. Because the inverse transition is reversible, any particles washed from the heated tissue would be expected to rapidly disaggregate and resolubilize. The observation of ELP[V5 A 2 G 3 -150] aggregates only in the heated window chambers is the first in vivo demonstration, to our knowledge, that a thermal phase transition of a soluble polymer can be engineered to occur at a specified temperature within a complex physiological system. For all time points after 4 min, ELP[V5 A 2 G 3 -150] delivery to the heated tumor was greater than both the unheated ELP[V5 A 2 G 3 -150] (P , 0.05) and heated ELP[V1 A 8 G 7 -160] (P , 0.1) control groups. The results from the window chamber studies as well as complementary biodistribution studies using ELP radiolabeled with 131 I-labeled SIB showed that focused hyperthermia of a solid tumor results in a 2-fold increase in the accumulation of a systemically injected thermally sensitive ELP within the tumor. Furthermore, an additional 2–3-fold increase has been observed in the cellular uptake of ELP[V5 A 2 G 3 -150] with hyperthermia compared to unheated controls [53]. This increased uptake, when combined with increased tumor localization, should result in an even higher intracellular concentration of the therapeutic agent than thermally unresponsive polymers. In more recent studies, we have measured

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Fig. 9. Window chamber fluorescence videomicrographs of tumor tissue at 30 s and 30 min post-injection of the ELPs. The thermally responsive carrier, ELP[V5 A 2 G 3 -150], was injected and the window was maintained at 42 8C (panels i and ii). For the high temperature control group, the thermally unresponsive carrier, ELP[V1 A 8 G 7 -160] was injected and the window was maintained at 42 8C (panels iii and iv). For the low temperature control group, the thermally responsive carrier, ELP[V5 A 2 G 3 -150] was injected but the window was not heated (panels v and vi). ELP aggregates (panel ii) were observed only for ELP1 in tumors heated to 42 8C, and demonstrate that the inverse temperature phase transition occurred in vivo. Increased extravasation was also observed for ELP[V5 A 2 G 3 -150] with hyperthermia compared to the controls. Modified from [24].

the cytotoxicity of an ELP conjugated to a chemotherapeutic agent, doxorubicin, and found that the ELP-doxorubicin conjugate displayed near equivalent cytotoxicity towards a human squamous carcinoma cell line (FaDu) in a tissue culture assay as compared to free doxorubicin (manuscript in preparation). Although these results are promising, much remains to be done to move this delivery method toward clinical application. Outstanding issues that need to be addressed include characterization of the resolubilization kinetics of the ELP in the tumor after cessation of hyperthermia, characterization of the toxicity and immunogenicity of the polymer, as well as optimization of the MW, concentration and mode of administration (e.g., bolus versus infusion) to maximize the localization of the drug within tumors.

Future work will also explore the feasibility of combining thermal targeting with affinity targeting by incorporation of short, affinity targeting peptides, which are specific for cell surface receptors that are overexpressed in tumors, into the ELP sequence at the N and C-termini [54–56]. In this dual thermalaffinity targeting scheme, the thermally sensitive ELP serves as a first stage, systemic targeting method to rapidly increase concentration of the polymer-drug conjugate in the tumor, followed by affinity targeting of the resolubilized ELP-drug conjugate after cessation of hyperthermia. The facile incorporation of multiple repeats of a targeting sequence into recombinant ELP by standard molecular biology approaches is attractive because it further enables multivalency effects to be exploited for delivery [57].

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4. Elastin-like block copolymers

4.1. Block copolymers There has been recent interest in the use of block copolymers as drug delivery vehicles [58]. This is because block copolymers can be designed to exhibit a number of different supramolecular architectures such as micelles and vesicles which have many desirable properties for drug delivery such as a low critical micelle concentration, good stability and control of aggregate size. The reader is also referred to the article by Conticello and colleagues appearing in the same issue of Advanced Drug Delivery Reviews that discusses the synthesis and properties of polypeptide-based block copolymers in greater detail. Motivated by these reports, we decided to investigate the prospect of creating thermoreversible micelles composed of ELP blocks with different transition temperatures. Here, we describe one example of the synthesis and characterization of an ELP-based block copolymer, and suggest methods by which these block copolymers might find application in drug delivery. This example also highlights the simple and facile synthesis of block copolymers by RDL, which enables the sequence and length of the individual blocks, as well as the overall block architecture of the copolymer to be readily controlled.

4.2. Synthesis of elastin-like block copolymers We synthesized and expressed a gene encoding an AB diblock copolymer, in which ELP[V1 A 8 G 7 -64] is followed seamlessly by ELP[V5 -60] (Fig. 10). This was achieved in one cycle of RDL using the PflM I /Bgl I-digested ELP[V5 -60] gene as the insert and pUC19-ELP[V1 A 8 G 7 -64] as the PflM I-linearized vector. Both of these genes had been previously generated during synthesis of the ELP libraries. We chose these two genes because we hypothesized that a copolymer of these two blocks would form a nanoparticle in solution as a function of temperature driven by the disparity in the T t of each block (ELP[V5 -60] T t 5 35 8C; ELP[V1 A 8 G 7 -64] T t . 90 8C). We hypothesized that if the two blocks exhibited independent transition behavior when

Fig. 10. Schematic representation of the ELP block copolymers. Upon increasing the solution temperature, the ELP[V-60] block, with a T t much lower than the ELP[V1 A 8 G 7 -64] block, undergoes its transition to a more hydrophobic state, driving the formation of ELP nanoparticles.

joined as a single molecule, this would lead to the formation of a micelle-like structure because the ELP[V5 -60] block would hydrophobically collapse and aggregate at temperatures above its T t while the ELP[V1 A 8 G 7 -64] block would remain hydrophilic and solvated [26,49]. The two sequences were also selected because of the availability of a pseudorandom analogue in ELP[V5 A 2 G 3 -120] as a control sequence. The ELP[V1 A 8 G 7 -64]-ELP[V5 -60] block copolymer is 124 pentapeptides in length and its guest residues are composed of 51.6% Val, 25.8% Ala, and 22.6% Gly, with the Val residues located in a leading block followed by an Ala and Gly rich block. Similarly, the pseudorandom ELP[V5 A 2 G 3 -120] polymer is 120 pentapeptides in length and its guest residues are composed of 50% Val, 20% Ala and 30% Gly. In contrast to the block copolymer, however, the guest residues are all dispersed evenly throughout the polymer. Thus, comparison of the block and pseudorandom sequence was expected to provide insight into the effect of the distribution of guest residues within an ELP sequence on its thermal behavior.

4.3. Characterization of elastin-like block copolymers We independently monitored solutions of the block copolymer and the pseudorandom control copolymer by turbidimetry and by dynamic light

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scattering (DLS) as a function of temperature. The pseudorandom copolymer, ELP[V5 A 2 G 3 -120], exhibits a T t of 44 8C as seen by turbidity measurements (Fig. 11A), and the DLS results confirm that a single transition occurs resulting in a near step-wise increase in hydrodynamic radius (R h ) from ELP monomers (4.861.1 nm; mean R h 6polydispersity) to micron-sized aggregates (1.260.26 mm). The change in R h of the polymer from monomer to micron-size aggregates as a function of temperature observed by DLS superimposes upon the turbidity results, indicating that the turbidity observed when the temperature is increased above the T t is caused by the formation of ELP aggregates. We term this

Fig. 11. Solution turbidity and particle size as a function of temperature for the (A) pseudorandom and (B) block ELP copolymers. The hydrodynamic radii of each ELP were measured by dynamic light scattering as a function of temperature (mean6polydispersity of the particle size distribution). These results suggest that the block copolymer forms a micelle-like structure at temperatures intermediate between the initial collapse of the more hydrophobic ELP segment at 40.0 8C and the collapse and subsequent aggregation of the less hydrophobic segment at 50.8 8C. Modified from [60].

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transition the ‘bulk transition’ because it results in a sudden and dramatic change in turbidity due to the formation of large, micron-sized aggregates. In contrast, the thermal behavior of the block copolymer is significantly more complex (Fig. 11B). At lower temperatures, two inflection points in the turbidity profile of the block copolymer are observed that are absent in the turbidity profile of the pseudorandom control ELP[V5 A 2 G 3 -120] copolymer. The first inflection point is manifested as a small but constant increase in turbidity that occurs at 40.0 8C. At 47.5 8C, a second inflection point is observed in the turbidity versus temperature plot. As the temperature is increased further, there is a significantly larger change in turbidity at 50.8 8C that is similar to the ‘bulk transition’ observed for the pseudorandom copolymer. Upon cooling the solution, the turbidity profile overlays the heating curve nearly perfectly, showing that all three inflections at 40.0, 47.5 and 50.8 8C are due to fully reversible, thermodynamic transitions. The appearance of two additional transitions at lower temperature that precede the bulk transition are not observed in the thermal profiles of the pseudorandom control copolymer or other homopolymer ELPs that we have studied. The block copolymer was studied by DLS as a function of temperature to further elucidate these thermally dependent effects. DLS shows that particles of four distinct sizes are observed for the block copolymer as the solution is heated (Fig. 11B). As the temperature is increased from 35 8C, individual ELP molecules (4.461.6 nm) are the sole species in solution. At 39 8C, a new larger particle with a R h of 20.465.8 nm is observed that persists up to 47 8C. At 47 8C, a discontinuous jump in the R h of the particles to 54.5620.3 nm is observed. These particles are stable up to 50 8C, above which larger aggregates form with a R h of 1.460.35 mm. The temperatures at which the new particles are first detected by DLS align precisely with the inflection points observed in turbidity versus temperature plots, which were independently measured in a UV-visible spectrophotometer. The DLS results provide a structural rationale for the observed changes in turbidity as a function of temperature. We hypothesize that at 40.0 8C, the previously solvated ELP[V5 -60] block of the copolymer undergoes its inverse temperature transition,

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and desolvates resulting in hydrophobic collapse of this segment. Individual ELP[V5 -60] blocks then aggregate with other ELP[V5 -60] blocks to form a hydrophobic core surrounded by a hydrophilic shell composed of solvated ELP[V1 A 8 G 7 -64] blocks. The appearance of these micelles causes a slight increase in turbidity between 40 and 47.5 8C because monomers convert to micelles in this temperature range. These micelles, which have a constant diameter of | 40 nm over this temperature range, undergo a structural rearrangement at 47.5 8C, leading to the formation of larger nanoparticles that are | 110 nm in diameter. The increase in size results in increased light scattering, resulting in an increased slope of the solution turbidity as a function of temperature observed by UV-visible spectrophotometry. The rearrangement from | 40 nm micelles to | 110 nm diameter nanoparticles may be driven by a change in the micelle shape or the formation of vesicles, although at this time the exact mechanism of this transition is not known at this time. Finally, at 50.8 8C, the outer ELP[V1 A 8 G 7 -64] block undergoes its inverse temperature transition, which drives the aggregation of the 100 nm nanoparticles to form micron-sized aggregates.

4.4. Future applications of elastin-like block copolymers The formation of thermally reversible nanoparticles suggests several potential applications of ELP block copolymers in drug delivery. For example, the formation of nanoparticles of 40–100 nm size is intrinsically attractive for delivery of therapeutics to solid tumors, because previous work with liposomes has shown that extravasation from tumor vessels is sensitive to particle size, and that the pore-size cutoff of the tumor endothelium is a few hundred nm [59]. Furthermore, the thermally triggered transition from ELP monomers to nanoparticles might also be used to load hydrophobic drugs by entrapment within the hydrophobic core of the nanoparticles prior to in vivo injection by designing the T t of the monomer to micelle transition to occur below body temperature. Triggered disassembly after localization in the tumor to release the encapsulated drug is a major challenge that remains to be addressed in the use of these nanoparticles for targeted delivery of chemo-

therapeutics. This can be achieved, in principle, by focused hypothermia to cool the tumor below the T t of micelle formation to drive disaggregation into ELP monomers. Alternatively, a different trigger might be incorporated into the ELP, such that the disassembly would be achieved by the use of other stimuli such as light or pH [15,16]. The modular nature of RDL also provides a convenient and powerful method to vary the physicochemical properties of block copolymers for drug delivery [10,58]. For example, RDL should be useful in precisely engineering the properties of micelleforming ELP block copolymers to optimize drug loading capacity, thermally triggered micelle formation, and micelle size. Studies are currently underway to systematically vary these parameters through the selection of ELP blocks of different lengths and sequences. The loading of drugs may depend upon noncovalent interactions between the hydrophobic core segment of the block copolymer and the drug. Alternatively, sites for chemical conjugation of the drug could also be engineered into either the low or high T t block. Altering the particle size would enable control of the pharmacokinetics of systemically injected, drug-loaded micelles. RDL should also allow the presentation of affinity targeting peptides or proteins at the termini of the solvent-exposed hydrophilic segments to enable receptor-mediated targeting of the micelle to physiological targets of interest. Mixing of block copolymers containing targeting peptide with polymers lacking the targeting sequence would further enable the number of targeting moieties to be precisely specified in the micelles, and would thereby enable polyvalency effects to be precisely exploited in targeted drug delivery [57].

5. Conclusions In conclusion, we believe that thermosensitive polymeric carriers are promising for the targeted delivery of therapeutics to solid tumors. We recognize, however, that the development of this approach is still in its infancy, and that the road ahead presents a number of exciting opportunities but also pitfalls that will need to be circumvented to realize the potential of this new targeting modality for cancer therapy.

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Acknowledgements This research was partially supported by research grants from the Whitaker Foundation and the Duke Comprehensive Cancer Center to A.C. We also thank the Whitaker Foundation for support of D.E.M. through a graduate student fellowship. The research described in this article is an ongoing collaboration that involves many other individuals besides the authors. We thank Michael Zalutsky (Radiology), Mark Dewhirst (Radiation Oncology), and Michael Colvin and Susan Ludemann (Duke Comprehensive Cancer Center) for collaborating on various aspects of the studies described here. We also thank Wenge Liu and Garheng Kong for help in performing the animal studies, and Drazen Raucher for performing cell uptake studies of the ELPs.

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