Conjugates of stimuli-responsive polymers and proteins

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Prog. Polym. Sci. 32 (2007) 922–932 www.elsevier.com/locate/ppolysci

Conjugates of stimuli-responsive polymers and proteins Allan S. Hoffman, Patrick S. Stayton1 Bioengineering Department, University of Washington, Box 355061, Seattle, WA 98195, USA Received 17 April 2007; received in revised form 23 May 2007; accepted 24 May 2007 Available online 5 June 2007

Abstract This chapter focuses on the synthesis and applications of covalent conjugates of proteins and stimuli-responsive or ‘‘smart’’ polymers. Smart polymer–drug conjugates are included only when the drug is a protein. The conjugation of biotinylated smart polymers to streptavidin is included, as is the use of streptavidin to link a biotinylated protein and a biotinylated smart polymer. Proteins that are conjugated with poly(ethylene glycol) (PEG), or ‘‘PEGylated’’ proteins, are not included in this chapter since PEG is not utilized as a smart polymer in such conjugates. Smart polymer–protein conjugates immobilized on solid surfaces or within hydrogels are also not included, unless they represent the ‘‘phaseseparated state’’ of the reversible soluble–insoluble phases of the conjugate. r 2007 Elsevier Ltd. All rights reserved. Keywords: Smart polymers; Polymer–protein conjugates; Site-specific conjugation; Affinity precipitation

Contents 1. 2.

3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synthesis of ‘‘random’’ smart polymer–protein conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Smart polymers and structures of their protein conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Conjugation reactions between smart polymers and proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synthesis of site-specific smart polymer–protein conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of stimuli-responsive or ‘‘smart’’ polymers in protein conjugates . . . . . . . . . . . . . . . . . . . . . . . Some limitations of smart polymer–protein conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Synthetic polymers may be conjugated covalently to a variety of natural or synthetic biomolecules for Corresponding author. Fax: +1 206 616 3928. 1

E-mail address: [email protected] (A.S. Hoffman). With assistance of Mitsuhiro Ebara and Tomoyuki Oomura.

0079-6700/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.progpolymsci.2007.05.005

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many diverse uses. In 1975, Helmut Ringsdorf published his famous cartoon suggesting the use of a synthetic polymer backbone as a carrier for drug molecules [1–3]. In 1977, Abuchowski et al. [4] published the first paper on the conjugation of poly(ethylene glycol) (PEG) to protein drugs. PEG is known to shield the protein from recognition by the body’s immune system, which leads to

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2. Synthesis of ‘‘random’’ smart polymer–protein conjugates 2.1. Smart polymers and structures of their protein conjugates Smart polymers respond to relatively small changes in conditions with large and sharp changes

in the polymer solubility, i.e., they change from a fully hydrated, expanded, and soluble chain to a dehydrated, collapsed, and insoluble chain (Fig. 1). The conditions that can stimulate such changes include small changes in temperature, pH, concentration of specific ions, specific wavelengths of UV light, visible light, or in rare cases, the application of an electric field [5,10,68]. When the smart polymer molecules are stimulated to collapse, the conjugates may remain in solution if their concentration is dilute, or they may aggregate together and phase separate as a precipitate or as a gel if their concentration is high enough. The presence of free (unconjugated) smart polymer, which is often difficult to remove from the conjugation reaction solution, can enhance the phase separation of the conjugate [30,31]. The phase-separating conjugates may also bind to nearby surfaces, such as microfluidic channel walls or microparticles, especially when those surfaces are coated with the same smart polymer [69,70]. One of the most attractive advantages of smart polymers in general for use in protein conjugates is that they can be very diverse in composition, molecular structure, and molecular weight (MW). They may be designed to have either one or two reactive end groups, or an ‘‘average’’ number of pendant reactive groups. Conjugations via end groups, especially where there is only one reactive end group, usually yield conjugates that are the most clearly defined in structure and composition. Polymers with only one reactive end group may be synthesized using traditional chain transfer Small change in conditions 100

80 % Response

prolonged circulation times in the body and enhanced efficacy of the drug [5]. Polymer–protein conjugates have been extensively investigated over the past 25–30 years [5]. Early publications on stimuli-responsive or ‘‘smart’’ polymer–protein conjugates appeared in the late 1970s and early 1980s. In those papers, proteins were conjugated to carboxylated polymers that phase-separated either at low pH or by the addition of calcium ions [6–9]. In the early 1980s, Hoffman and coworkers conjugated temperature-responsive polymers such as polyNIPAAm to proteins [10–13]. They synthesized polyNIPAAm–monoclonal antibody conjugates in order to develop a new thermally induced phase-separation immunoassay [11,13,14] (see applications below). Since then, many researchers have conjugated smart polymers to proteins for a great variety of applications in affinity separations [15–18], enzyme bioprocesses [19–39], drug delivery [40,41], diagnostics and biosensors [13,42] cell culture processes including tissue engineering [43,44], and DNA motors [45,46]. Smart polymer–biotin [47–49] and smart polymer– streptavidin conjugates have been extensively studied by Stayton et al. [50–57]. Streptavidin has also been used as a bridge for conjugating biotinylated smart polymers to biotinylated proteins and other biomolecules [54,55]. In one example, a biotinylated ssDNA chain was linked to streptavidin and the ssDNA was hybridized with a complementary ssDNA chain that was itself conjugated to a PNIPAAm chain. Following this, a biotinylated alkaline phosphatase was linked to remaining binding sites on the streptavidin, and the PNIPAAm–DNA–streptavidin–alkaline phosphatase conjugate was precipitated by the phase separation of the PNIPAAm [58] (see site-specific conjugates below for a similar example from references [54,55] using a pH-responsive polymer for targeted intracellular drug delivery). Maynard and coworkers have polymerized PNIPAAm directly from streptavidin by first linking a biotinylated ATRP initiator group to the streptavidin [59–61] (see site-specific conjugates below). A number of reviews of smart polymer–protein conjugates have appeared [5,62–67,97].

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Insoluble & collapsed

60 Soluble & hydrated

20 0 Stimulus (eg, T, hν , pH) Fig. 1. Schematic showing the special behavior of smart polymers.

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polymerization techniques with mercaptyl amines, carboxylic acids, or alcohols as chain transfer agents [13,15,17,26,28,30,31,71,72]. Once the smart polymer has been synthesized with one reactive end group (e.g., carboxyl, hydroxyl, or amine), rather than conjugate it directly by that end to a protein, one may conjugate a vinyl monomer to the reactive end group and form a ‘‘macro-monomer’’. This macro-monomer can be copolymerized with another smart polymer precursor monomer, to form a ‘‘doubly smart’’ graft copolymer. Then, reactive sites in either the backbone or the pendant graft polymer may be used to conjugate the graft polymer to the protein [73]. Novel linear random copolymers having both temperature- and pH-sensitivities or temperature- and photo-sensitivities have also been conjugated to proteins [33–35,38,74]. One may also conjugate vinyl monomer groups to protein lysine groups and then copolymerize the protein ‘‘macro-monomer’’ with free monomer to yield a polymer–protein conjugate [12,75,76]. More recently, RAFT and ATRP ‘‘living’’ free radical polymerization methods have been used to yield polymers with controlled MWs, narrow MW distributions, and one reactive end group [77,78]. Such controlled MW polymers have been conjugated to

Native protein

Native protein

Native protein

Conjugation of end-activated polymer

streptavidin [57]. One special feature of these controlled, ‘‘living’’ free radical polymerizations is that block copolymers may also be synthesized where one or both of the blocks are smart polymers. In the latter case, the two blocks can have different responses to different stimuli (see below) [79,80]. Another special feature of RAFT or ATRP polymerizations is that the polymers may also be directly grown from the protein surface, by first conjugating initiator groups onto cysteine thiol groups at specific sites on the protein surface [81]. This is similar in concept to the conjugation of the biotinylated initiator group to streptavidin mentioned earlier (see site-specific conjugations below). Figs. 2 and 3 show schematically the various ways that smart polymer–protein conjugates may be synthesized, either by direct polymer conjugation or by various polymerization techniques. Fig. 2 shows these reactions for native proteins and Fig. 3 shows them for genetically engineered mutant proteins designed for site-specific conjugations. Even simple end group conjugation of a polymer with only one reactive chain end can result in several polymer chains being linked to the same protein [10,62]. Multiple attachments are especially likely to occur if a polymer chain has multiple pendant

Polymer-protein conjugates

Conjugation of pendant-activated polymer

Conjugation of, or conversion to, polymerizationreactive group

Polymerization with monomer

Fig. 2. Schematic showing the various ways to synthesize random conjugates of smart polymers and native proteins. Only one reactive protein site is shown, but proteins will normally have several such sites, which are usually lysine amino groups. ‘‘Polymerization-reactive’’ groups include vinyl groups and free radical initiator or chain transfer groups.

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bangaru

Engineered protein

Engineered protein

Engineered protein

Conjugation of end-reactive polymer

Site-specific polymerprotein conjugates

Site-specific conjugation of pendant reactive polymer

Conjugation of, or conversion to, polymerizationreactive group

Site-specific polymerization with monomer

Fig. 3. Schematic showing the various ways to synthesize site-specific conjugates of smart polymers and engineered proteins. ‘‘Polymerization-reactive’’ groups include vinyl groups and free radical initiator or chain transfer groups.

amino-reactive groups and the protein has multiple lysine sites (or similarly, when the polymer has multiple thiol-reactive groups and the protein has multiple cysteine sites). When that occurs, there are three situations that may occur: (1) more than one pendant group from the same polymer chain may be conjugated to the same protein, (2) pendant groups from different polymer chains may be conjugated to the same protein, and (3) one polymer chain with multiple pendant groups may link together two or more protein molecules [10,62]. To avoid some of these situations it is possible to carefully synthesize and purify a polymer with only one reactive pendant group per chain [82,83]. One common feature of smart polymers is that they are usually very soluble in aqueous solutions under certain conditions, such as above a critical pH or below their critical temperature, called the LCST. These polymers typically contain polar and/or ionic groups that provide for their water solubility, plus hydrophobic groups that confer the balance needed for their smart behavior. The polar or ionic groups are also the groups used to conjugate the polymer to the protein; thus, in order to retain a desired stimuliresponsive behavior, it may be necessary to ‘‘molecularly engineer’’ the final composition of the smart polymer to compensate for the substitution of one

small polar group by a large, polar protein molecule [84]. The attachment of the protein can not only affect the environmental conditions for collapse of the polymer conjugate, but also lessen the sharpness of the stimuli response of the conjugate. 2.2. Conjugation reactions between smart polymers and proteins There are many different chemical reactions that can be used to derivatize or ‘‘activate’’ polymerreactive groups for subsequent conjugation to proteins [85]. Many of these reactions were originally developed in the 1980s for PEGylation of proteins [86–88]. Sometimes spacer segments (e.g., usually short chain PEGs) may be incorporated between the reactive group(s) on the polymer backbone and the protein, in order to provide more steric freedom to the protein. The spacer may also contain biodegradable linkages (e.g., susceptible to enzymatic or hydrolytic breakdown), or disulfide linkages (e.g., susceptible to glutathione reduction in the cytosol of cells) for release of the protein, especially when it is a drug. Smart polymers may be conjugated to many different types of proteins, including enzymes, antibodies, hormones, anti-inflammatory cytokines,

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and many others. Essentially, all proteins will have lysine residues on their surfaces. Usually, the lysine amino groups are the easiest and preferred reactive sites for conjugation of polymers to proteins, but other possible sites include –COOH groups of aspartic or glutamic acid, –OH groups of serine or tyrosine and, especially, –SH groups of cysteine residues (see site-specific conjugation below). Because the conjugations usually are designed to occur ‘‘randomly’’ at lysine sites, the polymer may react with and conjugate to several different lysine sites on the same protein surface. Different lysine sites vary in reactivity, which is mainly due to their different accessibilities; usually only the most exposed lysine sites will react readily with the polymer. Cell membrane ligands may also be conjugated to the polymer molecule when it has two reactive end groups or multiple reactive pendant groups, for targeting a conjugated protein drug to a specific cell. Antibody targeting ligands have been linked to a pH-sensitive smart polymer by biotinylating both the antibody and the smart polymer and using streptavidin to link the two together [54,55]. In this interesting example, a biotinylated monoclonal antibody to a CD-3 lymphoma cell receptor and a biotinylated pH-sensitive, lipid membrane-disruptive smart polymer, poly(propylacrylic acid) (PPAA) were linked together by streptavidin [54,55]; see also Stayton et al. [66,89]. The PPAA phase separates sharply as the pH in the endosome drops through the polymer’s pK of about 6.8, and in so doing PPAA disrupts the endosomal vesicle membrane. Thus, when the antibody–streptavidin–PPAA conjugate was endocytosed after binding to the CD-3 receptor in Jurkat lymphoma cells, the conjugate was observed (by a dye label on the streptavidin) to escape from the endosome to the cytosol [55]. This is a desirable event for a protein drug (streptavidin can be viewed as a model for a protein drug) since otherwise it would be trafficked to the lysosome where lysosomal enzymes would degrade it. Targeted drug delivery with PNIPAAm–drug conjugates has been studied [90]. Despite the well-known possibility that conjugation of a polymer to a protein could reduce the activity of the protein, there have been a few surprising examples in the literature of increased biological activity of the protein after polymer conjugation [31]. Such increased activity could be due to allosteric modifications in the protein active site after conjugation, and/or to selective partition-

Table 1 Important micro-environmental effects of the conjugated smart polymer on the protein activity

 Effect of the conjugated smart polymer on the

   

microenvironment of the active site (e.g., local pH, ionic strength, concentration of specific ions, concentration of metabolites, bound vs. free water). MW, length, and volume of the conjugated polymer and its location relative to the protein active site. Size of active site reactants such as affinity ligands, antigens, receptors, or enzyme substrates. Presence of a spacer arm between the polymer and the protein. Allosteric changes in the conformation of the protein active site.

ing of an enzyme substrate in the micro-environment of the polymer. The polymer can also affect the water structure, pH, ionic strength, etc., near or within the active site, any of which could enhance protein activity. Table 1 summarizes the important variables affecting the micro-environment of the protein active site after polymer conjugation. 3. Synthesis of site-specific smart polymer–protein conjugates If the amino acid sequence of a protein is known, site-specific mutagenesis (‘‘genetic engineering’’ of the protein) may be used to substitute one amino acid at a specific site with another. For example, a cysteine residue can be introduced by such techniques to yield a mutant protein with an exposed thiol group. Then, polymers with terminal or pendant maleimide, vinyl, or vinyl sulfone groups can be conjugated to the protein. These groups react preferentially with thiol groups rather than with lysine amino groups [51,91]. Furthermore, pendant or terminal thiol or disulfide groups on smart polymers may be used to form disulfide linkages of the polymer to the protein [84]. Kochendoerfer et al. [98] have used site-specific mutagenesis plus specific chemistries to conjugate branched PEGs with negatively charged terminal groups via oxime linkages to two lysine sites that are known to be glycosylated in native erythropoietin. In another approach, Tirrell has shown that non-natural amino acids may be inserted into a protein structure by genetic engineering of the protein expression process within cells, combined with appropriate cell culture conditions [92]. Such non-natural amino acids could permit different chemistries, such as

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‘‘Click Chemistry’’ [93] to be used to conjugate polymers at those sites. Such site-specific conjugation of smart polymers to proteins can be carried out either near the active site or far away from the active site. In the former case, the protein activity can be directly affected. Thus, by stimulating the polymer first to collapse and then to re-hydrate, the collapsed smart polymer coil can first ‘‘block’’ (turn ‘‘off’’) the protein active site, and then ‘‘unblock’’ (turn back ‘‘on’’) the active site [67,91]. However, it is expected that both the location of the conjugation site and the volume (or MW) of the smart polymer coil, either before or after collapse, can control protein activity (Krishnamurthy et al. [94] have demonstrated the strong influence on enzyme inhibition of both the conjugation site and the MW of a PEG-inhibitor molecule conjugated at various sites on an enzyme). Stayton et al. were the first to do this, and the first to demonstrate the versatility of site-specific conjugation by conjugating polyNIPAAm far away from the active site of Cytochrome-b5 [95] and close to the active site of streptavidin [51,67,91]. Polymers with terminal or pendant thiol (or disulfide) groups may also be conjugated to such mutant protein thiol groups via disulfide linkages. It should be noted that thiol groups on the surface of proteins are usually rare, since they would otherwise lead to dimerization of the protein. Albumin is one exception, having several thiol groups on its surface; because of this, a small fraction of circulating albumin is a dimer. It is also possible to derivatize the thiol group with a polymerization initiator group via a disulfide linkage, and then polymerize the smart polymer directly from that particular site. Maynard and coworkers have done this with two proteins, streptavidin and albumin, linking ATRP initiator groups to the protein via biotin in the former case, and via disulfide groups in the latter case. They then used ATRP polymerization chemistry to polymerize NIPAAm onto those two proteins at those specific thiol sites [59–61]. The protein thiol group might also serve as a chain transfer group in a free radical polymerization, which could lead to polymerization from the protein thio-radical group. However, it is possible that the chain transfer reaction and subsequent monomer addition would be inefficient because the protein is so dilute. Another important factor in site-specific conjugation near the active site is the size of the ligand or substrate that reacts at the active site. A large ligand or substrate reactant may be sterically blocked by

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the hydrated volume of the smart polymer, without having to stimulate the collapse of the polymer [51]. It is also expected that the collapse of the polymer to block the active site will cause phase separation of the conjugate. However, RAFT or ATRP polymerization methods can produce smart block copolymers, such as a block copolymer of polyNIPAAm linked to a second block such as a pH-sensitive polymer or PEG, either of which can remain hydrated as temperature is raised to collapse the polyNIPAAm. If the polyNIPAAm block is conjugated near the active site and is stimulated to collapse while the second block remains soluble, the second block can prevent the conjugate from phaseseparating. Thus, in this case, the protein activity can be turned off and on while the protein remains in solution [79,80] (Fig. 4). Some important considerations involved in the synthesis of both random and site-specific smart polymer–protein conjugates are highlighted in Table 2, and Table 3 identifies some of the special features of smart polymer–protein conjugates. 4. Applications of stimuli-responsive or ‘‘smart’’ polymers in protein conjugates The environmental changes most relevant to in vivo therapeutic applications of conjugates are DpH and D(temperature) (DT). Other stimuli such as light (Dhn) may be more relevant to in vitro biotechnology applications. The following are three examples from Hoffman’s early work on potential uses of smart polymer– protein conjugates [10,62,64,68]: (1) A smart polymer–enzyme conjugate may be reacted with substrate to produce a desired product, and subsequent phase separation of the smart polymer can be used to selectively remove the enzyme and the product may then be recovered from the process solution. This is like an enzyme bioprocess carried out without having to immobilize the enzyme and use membrane separation techniques to remove it from the reaction mixture [10,29]. (2) A smart polymer–monoclonal antibody conjugate may bind by affinity recognition to a specific molecule in solution, which might be a recombinant protein product in a process stream, a target molecule in a complex mixture, or a toxin in an environmental stream, and subsequent stimulated phase separation of the

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+ Stimulus Inactive and precipitated Homopolymerprotein conjugate: Active and soluble

-Stimulus

+ Stimulus Inactive and still soluble Block copolymerprotein conjugate: Active and soluble

-Stimulus

Fig. 4. Schematic comparing the behavior of a site-specific conjugate of a smart homopolymer (e.g., PNIPAAm) and a smart block copolymer (e.g., poly[NIPAAm-co-AAc]), each conjugated near the active site of an enzyme. When the smart homo-PNIPAAm is stimulated to phase separate, the protein’s active site is blocked; in addition, the conjugate phase separates. Thus, the enzyme is both inactive and phase-separated. In the case of the block copolymer conjugate, when the PNIPAAm block, which is conjugated directly to the enzyme, is collapsed, the active site is similarly blocked, and the enzyme becomes inactive. However, the second segment of the block copolymer (e.g., PAAc) remains soluble and hydrated, and keeps the conjugate from phase-separating [57,80].

Table 2 Random and site-specific synthesis of smart polymer–protein conjugates

 A great variety of smart polymer compositions, structures,     



 

molecular weights (MWs), and MW distributions may be synthesized. Polymer end groups or pendant groups may be derivatized and conjugated to proteins. The number of reactive end or pendant groups on the polymer chain can be controlled. A wide range of chemistries can be used for conjugations. Specific sites such as thiol groups can be engineered into proteins by site-specific mutagenesis of the protein before conjugation of the smart polymer to those sites. Smart polymers may also be polymerized directly from such specific sites by derivatizing them with free radical polymerization initiator or chain transfer groups. When there are two or more reactive sites on the polymer chain, the polymer may be conjugated more than once to the same protein molecule or to more than one protein molecule. These possibilities add compositional diversity to the product. Spacer segments may be incorporated between the polymer and the protein. The spacer segments may contain linkages that are either hydrolytically or enzymatically biodegradable, or reducible disulfide groups, for controlled release of the conjugated protein, especially when it is to be delivered as a drug. There may be some loss of protein activity during the conjugation procedure. Cell receptor ligands may be conjugated to the smart polymer chain before it is conjugated to the protein.

smart polymer can be used to selectively remove the product, the target molecule, or the toxin. This is like affinity chromatography carried out without the packing. This process has been called ‘‘affinity precipitation’’ by some researchers [10,15]. (3) An extension of the affinity precipitation technique is when a smart polymer–monoclonal antibody conjugate binds by affinity recognition to a specific molecule in solution, which might be an analyte in a serum sample, and then, addition of a second, labeled antibody followed by phase separation of the polymer–immune complex sandwich conjugate can be used to assay the amount of the analyte in the sample. This is like ELISA carried out without the multi-well plate [11,13]. Diagnostic assays have been or are being developed that can be run in a fluorescence activated cell sorter (FACS), on a biochip or microarray, or in a microfluidic format [70]. 5. Some limitations of smart polymer–protein conjugates There are some limitations of synthetic polymers in protein conjugates, most of which relate either to

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Table 4 Some limitations of the use of smart polymer–protein conjugates in vivo

 Protein activity may be turned ‘‘off’’ and ‘‘on’’ when smart    





  

polymers are conjugated near the active site and stimulated to collapse and rehydrate. When smart polymer–protein conjugates are stimulated to collapse, they may aggregate together and phase separate. Such phase-separating conjugates may also bind to nearby surfaces, especially when those surfaces are coated with the same smart polymer. Phase separation of an enzyme–polymer conjugate may be used to selectively remove the enzyme and recover the product from the process solution. A recognition protein (e.g., a monoclonal antibody)–polymer conjugate may be used to selectively bind a target molecule by affinity and then to remove it by phase separation. The target molecule could be a product from a process stream or a toxin in a process or environmental stream. A monoclonal antibody–polymer conjugate may be used to assay the amount of an analyte in a test sample by first binding the analyte by affinity recognition, then adding a second, labeled antibody, and phase-separating. Smart polymers usually collapse faster upon stimulation than they re-hydrate when the stimulus is reversed. This can result in a slow re-dissolution of the polymer–protein conjugates if aggregates have formed. The polymer–protein conjugate product should be purified to remove non-conjugated polymer molecules, non-conjugated protein molecules, and reaction byproducts. Sterilization of smart polymer–protein conjugates can be challenging. A new polymer–protein drug conjugate will probably be viewed by regulatory agencies as a new drug, even if it combines a GRAS polymer with an approved drug.

the synthesis process or to the resistance of the polymer molecule to biodegradation. When the application involves in vivo use of the polymer, the toxicity and immunogenicity of the polymer will become critical issues. If the polymer MW is above about 30,000, its elimination via the kidneys may be inefficient, and subsequent accumulation within the body can be a major barrier to its use in vivo. The very nature of the synthesis of polymers from small molecules produces a distribution of MWs, and the lower MWs may not be useful as smart polymer–protein conjugates, while the higher MW conjugates may be retained in body tissues or organs. Controlled MW polymerization methods like RAFT and ATRP have made these problems less of an issue. However, if the low MW conjugate does not provide the desired action (e.g., only higher MW conjugates are efficient at passive targeting to solid tumors, via the enhanced permeation and retention (EPR) effect [96]), then it may be

 The polymer should be non-toxic and non-immunogenic.  The polymer should have a controlled MW and MW  

distribution in order to avoid undesirable actions of low MW conjugates or chronic accumulation of high MW conjugates. The polymer should be purified to remove catalyst fragments or other undesirable impurities. The polymer should be eliminated from the body within a reasonable time after administration, i.e., it should not accumulate in any organs or tissues.

necessary to link together shorter MWs of the polymer with degradable linkages. In some cases, the polymerization catalysts (e.g., copper salts in ATRP) may need to be removed before the conjugate can be used for therapeutic or even diagnostic purposes. Other ‘‘impurities’’ that may need to be removed from the conjugation reaction solution are unreacted free smart polymer or protein. Sometimes the free smart polymer can enhance the stimuli-induced phase separation of the polymer–protein conjugate, so its removal may be unnecessary, and even undesirable. Another caveat is when smart copolymers are designed to have a specific content of a reactive, pendant functional group. In this case, the frequency and content of that group along the backbone can vary from one chain to the next, and the groups may not be randomly spaced due to differences in monomer reactivity ratios. When proteins are conjugated to pendant, reactive polymer groups, it may be difficult to avoid conjugating two or more groups on the same polymer molecule to one particular protein molecule, especially if the protein has many accessible lysine amino groups. It may also be difficult to avoid conjugating one polymer chain to more than one protein molecule. These issues have been discussed above. Finally, most polymer–protein conjugates will be difficult to sterilize. Table 4 summarizes some limitations of synthetic polymer–protein conjugates for in vivo applications.

References [1] Ringsdorf H. J Polym Sci Polym Symp 1975;51(135). [2] Duncan R, Kopecek J. Soluble synthetic polymers as potential drug carriers. Adv Polym Sci 1984;57:53–101.

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