Super-hydrophilic zwitterionic poly(carboxybetaine) and amphiphilic non-ionic poly(ethylene glycol) for stealth nanoparticles

Nano Today (2012) 7, 404—413

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/nanotoday

REVIEW

Super-hydrophilic zwitterionic poly(carboxybetaine) and amphiphilic non-ionic poly(ethylene glycol) for stealth nanoparticles Zhiqiang Cao, Shaoyi Jiang ∗ Department of Chemical Engineering, University of Washington, Seattle, WA 98195, USA Received 7 May 2012; received in revised form 7 July 2012; accepted 6 August 2012 Available online 29 August 2012

KEYWORDS Polyethylene glycol; Zwitterions; Non-fouling; Protein conjugation; Liposome; Drug delivery

Summary This review compares two types of non-fouling polymers, the widely used nonionic poly(ethylene glycol) (PEG) and the recently established zwitterionic poly(carboxybetaine) (PCB), for their use in creating stealth nanoparticles (NPs) for drug delivery and protein protection. While both types of polymers exhibit reasonable non-fouling properties, such as good protein and colloidal stability and extended blood circulation time in vivo, amphiphilic PEG has negative effects on proteins and NPs due to its hydrophobic nature, including reduced protein bioactivity, instability of assembled NPs, and lipid bilayer destabilization. These problems can be overcome by super-hydrophilic PCB. © 2012 Elsevier Ltd. All rights reserved.

Introduction Avoiding non-specific interactions (or possessing non-fouling properties) [1—7] is one of the most important characteristics required by nanoparticles (NPs) in a wide range of applications from drug delivery to diagnostics [8,9], especially for use in complex media. To achieve this stealth property, non-fouling polymeric materials are used to modify the surface of particles. The treated particles exhibit improved stability and blood circulation time, promoting

∗ Corresponding author. Tel.: +1 206 616 6509; fax: +1 206 543 3778. E-mail address: [email protected] (S. Jiang). URL: http://depts.washington.edu/jgroup/Index.htm (S. Jiang).

their diagnostic or therapeutic functions. There are generally two distinct types of non-fouling polymers; non-ionic polymers, among which the most notable is polyethylene glycol (PEG) [10], and zwitterionic polymers such as poly(carboxybetaine) (PCB) [7,11—13] (Fig. 1).

Amphiphilic non-ionic poly(ethylene glycol) PEG has been the most popular for modifying particles because of its historical use since the 1970s and PEGylation has become a standard surface and particle modification method for biological applications [14—23]. The non-fouling property of PEG was attributed to its steric exclusion effect [24,25]. More recently, it is generally agreed upon that its hydration ability or hydrophilic nature plays a key role in its non-fouling property [26,27]. Molecular simulation studies of oligo(ethylene glycol) (OEG) self-assembled

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Zwitterionic and non-ionic polymers for stealth nanoparticles EG Headgroup (Amphiphilic)

405 CB Headgroup (Superhydrophilic)

CH3

O

CH2

CH2

CH2

N

O CH2

C

O

CH3

Figure 1 Molecular structures of non-ionic EG and zwitterionic CB headgroups. PEG is amphiphilic with both hydrophilic and hydrophobic properties. PCB is super-hydrophilic. The number of carbon groups (n) between the positive quaternary amine group and the negative carboxylate group of each PCB unit can be varied. N = 1 is shown here, which is glycine betaine.

monolayers (SAMs) have shown a tightly bound water layer around OEG, generating large repulsive forces to repel proteins when they are approaching the surface [28—30]. PEGylation on particles results in increased water solubility and colloid stability, decreased interactions with blood components (opsonization), and improved blood residence time in vivo [15,16,31—33]. Because of these favorable features, several PEGylated products have been launched to the market, such as PEGylated proteins and liposomes [15,16,18,19,31—33]. It should be pointed out that PEG is, in fact, amphiphilic. In addition to its hydrophilic property, PEG also has a hydrophobic character [13,34,35] although PEG is often considered as a hydrophilic polymer. Because of this amphiphilic feature, PEG is able to dissolve in many organic solvents in addition to water. This makes it convenient to use PEG in both organic and aqueous solvents to modify both hydrophobic and hydrophilic particles. This accounts for its popularity as a modifying material. Unfortunately, due to its hydrophobic nature, the ease in its application results in negative effects when it is attached to hydrophilic proteins or it is used with a hydrophobic polymer to form core—shell particles. For protein protection, PEG is the standard material to be conjugated to a protein, increasing protein stability (e.g. against heat, salts, and proteolysis), water solubility, and circulation time in the body [13,15,36]. The resulting PEGylated protein, however, often significantly loses its biological activity [31,37—40]. Taking PEG-interferon ␣2a (PEGASYS® , to treat hepatitis C) as one example, PEGylation results in a 93% loss of its original bioactivity [31]. This has been attributed to PEG chains interfering with the zwitterionic nature of protein surfaces and the functional pocket of the protein (mostly hydrophobic domains) through hydrophobic—hydrophobic interactions [12,13]. For carrier-based drug delivery systems, PEG is so far solely used to modify liposomes in commercial drug formulations for long blood circulating purposes. For example, LIPO-DOX® and DOXIL® , both PEGylated liposomes with doxorubicin encapsulated, have been clinically used to treat AIDS-related Kaposi’s sarcoma, breast cancer, ovarian cancer, and other solid tumors [18,19,22,32,41,42]. Nevertheless, the inclusion of PEG tends to destabilize the lipid bilayer because of its hydrophobicity, leading to rapid drug leakage [43]. To compensate these destabilizing issues arising from PEG, a large amount of cholesterol (i.e., 39 mol%) is required in PEGylated liposomes to enhance drug retention [43]. In addition to these hydrophobic issues, PEG also suffers oxidation damage in biological media for long-term applications [5,26,34,44—46]. Furthermore, PEG antibodies occur in animals [15,34,47,48] and

even humans [48], complicating clinical trials involving this material.

Super-hydrophilic zwitterionic poly(carboxybetaine) As alternatives to non-ionic PEG, zwitterionic nonfouling polymers, namely poly(phosphobetaine), poly(sulfobetaine), and PCB are promising [7,49—55]. While PEG and other non-ionic materials (e.g., tetraglyme [56], dextran [57], mannitol [58], polyamines functionalized with acetyl chloride [59], PEG-mimetic peptoid [60], and serine-rich peptides [61]) bind water molecules via hydrogen-bonding hydration, zwitterionic polymers achieve stronger hydration that is electrostatically induced [62,63]. It has been shown that surfaces modified with zwitterionic polymers have excellent non-fouling properties against proteins, cells, and bacteria [7,64,65]. Take PCB as an example, derived from naturally occurring betaines abundant in the animal kingdom including humans as well as plants [66]. We have shown that PCB polymer brush coatings resist non-specific protein adsorption from undiluted blood plasma and serum to an undetectable level (<0.3 ng/cm2 ) using a surface plasmon resonance (SPR) sensor, and resist bacterial adhesion and delay biofilm formation for 10 days at room temperature [7,67—69]. Although both PEG and PCB-coated gold NPs are stable in 10% blood plasma and serum, PCB-coated NPs are stable in undiluted blood plasma and serum for 3 days while PEG-coated NPs aggregate [70]. This review focuses on PCB as one example of zwitterionic polymers and compares the pros and cons of PEG and PCB for creating stealth NPs. Unlike amphiphilic PEG, zwitterionic polymers are superhydrophilic, and insoluble in most organic solvents. Such super-hydrophilic character circumvents the hydrophobic character of PEG mentioned above. Three examples are given below to illustrate the use of zwitterionic PCB and its advantage over the non-ionic PEG counterpart in constructing polymer—protein conjugates, self-assembled polymeric NPs, and liposomes.

Three examples Polymer—protein conjugates PEGylation has been the benchmark for stabilizing proteins and increasing their blood circulation time [15,36]. However, PEG reduces the bioactivity of conjugated therapeutic proteins, antibodies, and enzymes [31,37—40]. Our recent work has shown the stabilization of a protein by attaching

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Figure 2 (a) Super-hydrophilic PCB increases enzyme—substrate hydrophobic—hydrophobic interactions, thereby increasing the affinity of the substrate for the binding pocket [12]. (b) Amphiphilic PEG reduces enzyme—substrate hydrophobic—hydrophobic interactions, and thus the bioactivity of the enzyme [12].

zwitterionic PCB to the protein without sacrificing its bioactivity. Such a combination of high stability and bioactivity has never been achieved before (Fig. 2) [12]. Specifically, 5 kDa PEG was attached to ␣-chymotrypsin, a proteolytic digestive enzyme, to various degrees with different numbers of polymer chains per protein. For fair comparison, PCB with an equal molecular weight of 5 kDa (determined by NMR) and

PCB with an equal hydrodynamic size of 5 kDa PEG (determined by size exclusion chromatography) were synthesized and conjugated to the same enzyme with similar degrees of conjugation as PEG. Results show that enzymes can be better stabilized by PCB than PEG, maintaining enzyme activity at high urea concentrations and at elevated temperatures. Fig. 3a shows the binding affinities between the substrate

Figure 3 (a) Binding affinities (Michaelis constant, Km ) of ␣-chymotrypsin (CT) conjugated with PEG (5 kDa molecular weight), pCB Mn (the same molecular weight as PEG, 5 kDa), and pCB Rh (the same hydrodynamic size as 5 kDa PEG). Km increased as more PEG conjugated to CT, showing the inhibitory effects of PEG. Unlike PEG, PCB showed either no significant effect on binding affinity (i.e., pCB Mn conjugates) or even an increase in substrate affinity (i.e., pCB Rh conjugates) [12]. (b) Km of native CT in the presence of 650 Mn PEG and ammonium acetate solutions. The effects of these small molecular weight solutes on Km are similar to their respective high molecular weight versions (PEG 5 kDa and pCB Rh respectively). Error bars in (a) and (b) are values of standard error (or standard error of the mean) [12].

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Figure 4 Structures of OEG (four EG segments), CB (1 carbon group between the positive quaternary amine group and the negative carboxylate group), and chymotrypsin inhibitor 2 (CI2). The bar graph represents the SASAs of the hydrophobic domains of CI2 in pure water, OEG solution, or CB solution, from molecular dynamics simulations [72].

and the polymer—protein conjugate as reflected by their Michaelis constants (Km ) or the concentration of substrate required to achieve 50% full activity. A lower Km indicates higher binding affinity. It was shown that the attachment of PEG chains decreases the enzyme binding affinity, while PCB conjugates of similar hydrodynamic size, in contrast, have no impact on or even increase binding affinity. The reason why the binding affinity of a protein is influenced differently by PEG and PCB has been attributed to the hydrophobicity of these polymers [12]. Fig. 3b illustrates the Km values of bare enzymes dissolved in solutions of 650 Mn PEG and ammonium acetate (NH4 OAC). It should be noted that NH4 OAC is composed of the most protein-stabilizing and bioactive monovalent ions (kosmotropic anion, OAC− , and chaotropic cation, NH4 + ) in the Hofmeister series [71], and PCB can be considered the polymeric version of these ions. The impact on Km or binding affinity by 650 Mn PEG and free NH4 OAC molecules was in a good agreement with that of the respective high-molecular-weight versions of PEG and PCB polymers attached to the proteins as shown in Fig. 3a. Results show that polymers, whether chemically attached to the protein or not, will interfere with hydrophobic—hydrophobic attractions between the substrate and the enzyme binding site, depending on the degree of their hydrophobicity. As further exploration, the partitioning of the tested substrate into free polymer solutions of PEG and PCB through a semi-permeable membrane was also measured [12]. It was found that the substrate had higher solubility in PEG and lower solubility in PCB than that in buffer control. Thus, the hydrophobic nature of PEG increases the local hydrophobicity around the surface of a protein and near its binding pocket, and results in higher solubility of the substrate. As a result, the hydrophobic nature of PEG reduces the substrate—enzyme hydrophobic—hydrophobic interaction (Fig. 2). In contrast, the super-hydrophilic nature of PCB facilitates the substrate—enzyme hydrophobic—hydrophobic interaction in the reverse manner (Fig. 2). Recently, further evidence was obtained, showing that the amphiphilic PEG segment has more favorable interactions with the hydrophobic domains of a protein than

super-hydrophilic PCB. The hydrophobic domains are generally recognized to play an essential role in the bioactivity of a protein. We studied the effects of CB and ethylene glycol (OEG) solutes [(EG)4 in this case] on chymotrypsin inhibitor 2 (CI2) using molecular dynamics simulations (Fig. 4) [72]. It was found that CI2 has much less solvent access surface area (SASA) for the hydrophobic domains in the OEG solution than in the CB solution. With EG4 , nearly 1/3 of the hydrophobic domains originally accessible in the water solvent become inaccessible, whereas the corresponding loss for CB is only around 1/10. For the hydrophilic domains, CI2 has identical SASA in both OEG and CB solutions.

Self-assembled polymeric NPs PEG has been widely used to modify a hydrophobic polymer block to form amphiphilic block copolymers, which can be further assembled into various nanostructures [17,73—78]. The assembled nanostructures have been explored for many applications such as in drug delivery [17,73,78—81]. Some of these NPs have been commercialized as chemo-drug formulations such as Genexol-PM in a phase IIa trial, a poly(D,L-lactide-b-ethylene glycol) (PLA-PEG) NP with paclitaxel [82] and BIND-014 in a preclinical trial, a poly(lactic acid-co-glycolic acid-b-ethylene glycol) PLGA—PEG NP with docetaxel that possesses cell targeting ability [83]. The potential of these systems comes from the PEG block which can be dissolved in aqueous media via hydrogen bonding with water molecules, stabilizing the hydrophobic particle cores of PLA or PLGA from aggregation in complete biological media and improving their blood circulation time [78—81]. However, PEGylated NPs suffer limited stability under harsh conditions such as freeze-drying, which is a common way to prevent the degradation of polymers such as PLA and PLGA, and to avoid drug leakage in aqueous storage media. To the best of our knowledge, few polymer-based NPs can survive lyophilization without cryoprotectant additives [17,84—86]. Even for PEGylated NPs, cryoprotectants such as 10% sucrose are required to help stabilize these NPs [17,86]. By replacing PEG with PCB, we synthesized

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Figure 5 PCB—PLGA and PEG—PLGA assembled NPs. It is expected that all PCB chains migrate to the hydrated shell region of the NP due to the super-hydrophilic nature. Amphiphilic PEG has dual solubility in both oil and water phases. PEG chains can exist simultaneously in both the hydrated shell and hydrophobic core regions.

PLGA—PCB block copolymers (Fig. 5) and found that their assembled NPs were remarkably stable after freeze-drying, without any cryoprotectant additives (Fig. 6a) [11]. With brief re-suspension with a pipette and without the need of sonication, the freeze-dried PLGA—PCB NPs with or without drug loaded have the same mean diameter and low polydispersity index (PDI) number as before freeze-drying (Fig. 6a). Fig. 6b and c shows the morphology of PLGA—PCB NPs before and after lyophilization in water under scanning electron microscopy (SEM). It should be noted that both PEG and PCB protected polymeric NPs show reasonably good colloidal stability

in aqueous media (e.g., biological or physiological environments) [11,78,80,81], since both polymers are fully solvated. When NPs are dehydrated (e.g., freeze-dried), the amphiphilic PEG crystallizes and loses its function to prevent NP aggregation [85]. The super-hydrophilic PCB, in contrast, has stronger hydration ability and binds a greater number of water molecules more strongly to prevent crystallization during lyophilization. Strong hydration ability has also been used to explain few cases of NP systems requiring no cryoprotectant through the lyophilization processes, such as micelles protected by ionic polysialic acid [87].

Figure 6 Stability of PLGA—PCB NPs throughout lyophilization. (a) NP sizes (mean ± SD, N = 3) for PLGA NPs, PLGA—PCB NPs, and PLGA—PCB/Dtxl NPs with 1 wt% drug loading (shown as NP suspended), and those after lyophilization without any addition of a cryoprotectant. Polydispersity indexes (PDIs, mean ± SD, N = 3) are indicated accompanying each size point. Dtxl = Docetaxel. (b) SEM image for PLGA—PCB NPs before lyophilization. (c) SEM image for PLGA—PCB NPs after lyophilization and brief re-suspension in water by pipettes without sonication. The scale bar for (b) and (c) is 1 ␮m [11].

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Figure 7 PEGylated and poly(zwitterionic) liposomes. For PEGylated liposome, amphiphilic PEG provides a stealth layer by itself via hydration due to its hydrophilic nature, but dehydrates the polar head group region of the liposome due to its hydrophobic nature, leading to negative consequences such as a rapid leakage of hydrophilic drugs. The addition of cholesterol is the most typical way to neutralize these impacts from PEGylation by increasing the fluidity of the membranes. For poly(zwitterionic) liposome, superhydrophilic zwitterionic PCB provides a stealth layer via hydration, similar to PEG. Unlike PEG, PCB further hydrates the polar head group region due to its super-hydrophilic nature, rearranging the membrane structure in a way opposite to PEG, leading to good drug retention without the need for cholesterol [95].

Liposomes PEG is often conjugated to a lipid for liposome protection. The resulting PEGylated liposomes have achieved great success in delivering chemo-drugs intravenously, such as Doxil® (known as Caelyx® in Canada and Europe). The nonfouling PEG reduces the interaction of plasma proteins with liposomes and thus their uptake by the reticuloendothelial system (RES) [22,88]. As a consequence, PEGylated liposomes show prolonged blood circulation time, and target tumor tissues through the enhanced permeability and retention (EPR) effect [18,19,22,32,88]. These positive effects all come from the hydrophilic nature of PEG. However, being also hydrophobic, PEG tends to destabilize the lipid bilayer (Fig. 7). Specifically, PEG lowers the polarity of the aqueous phase [89] and decreases the hydration level of the lipid polar group region [90]. This further enhances the lateral packing of the phospholipid acyl chains, inhibits the diffusional motion of PEG, strengthens PEG chain—chain interactions, and tends to induce bilayer demixing [43]. This series of consequences cause the rapid leakage of hydrophilic drugs [43], and are enhanced by PEG with a higher molecular weight such as ≥5 kDa, and phospholipids with a higher phase transition temperature (Tm ) such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Tm = 55 ◦ C) [43,91]. To compensate for the destabilizing effects of PEG, cholesterol is frequently required in these

PEGylated liposomes [43]. Cholesterol plays the opposite role of PEG by increasing the hydration of the lipid polar region and membrane fluidity, reducing PEG chain—chain interactions and bilayer demixing, and improving drug retention [43,92—94]. The necessity of cholesterol in PEGylated liposomes has been shown in LIPO-DOX® and DOXIL® , both containing 39 mol% cholesterol of the total lipids. It has also been shown in Fig. 8a, that PEGylation makes DSPC liposomes leak more encapsulated carboxyfluorescein (CF, a hydrophilic fluorescent dye) in the absence of cholesterol, while CF retention was maintained with cholesterol present. By replacing PEG with PCB, we synthesized DSPE-PCB (DSPE: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine) and formulated PCB-protected liposomes (Fig. 7) [95]. PCB-protected liposomes exhibit long blood circulating characteristics in vivo, comparable to PEGylated liposomes [95]. More importantly, they achieve drug retention without cholesterol, which is otherwise required by PEGylated liposomal formulations (shown in Fig. 8a). It was hypothesized that unlike how the hydrophobic nature of PEG destabilizes the lipid bilayer, super-hydrophilic PCB would behave the opposite. This is supported in a differential scanning calorimetry (DSC) study by measuring the major Tm of DSPC in both PEG- and PCB-protected liposomes. As shown in Fig. 8b, the addition of DSPE-PEG into DSPC liposomes gradually increases the Tm from 55 ◦ C by less than 1 ◦ C. This phenomenon has been related to the dehydration of the

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(a)

(b) 58 DSPC/DSPE-PEG 5K DSPC/DSPE-PCB 5K

DSPC DSPC/DSPE-PEG 5K 5% DSPC/DSPE-PEG 2K 5% DSPC/DSPE-PEG 2K 5%/Chol 39% DSPC/DSPE-PCB 5K 10%

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Figure 8 (a) Carboxyfluorescein (CF) was encapsulated into different liposome formulations. CF leakage profile was recorded in PBS at 37 ◦ C (mean ± SD, N = 3). The percentage value in the formulation represents the molar composition of the specified component. 2K or 5K represents the molecular weight of PCB or PEG. Chol, cholesterol; SD, standard deviation [95]. (b) Phase transition temperature (Tm , mean ± SD, N = 3) of DSPC with the addition of DSPE-PCB 5K or DSPE-PEG 5K into the liposomes [95].

lipid polar region and a compact lateral packing of lipid acyl chains [90]. The incorporation of DSPE-PCB, however, decreases Tm , indicating an enhanced hydration around the lipid polar region and a more fluidic membrane. It should be noted that the addition of cholesterol will have the same effect as PCB in lowing Tm [96,97].

How to connect super-hydrophilic PCB to a hydrophobic segment As discussed, super-hydrophilic PCB overcomes a number of issues with amphiphilic PEG. However, linking PCB to a

hydrophobic segment, particularly a super-hydrophobic one such as polydimethylsiloxane (PDMS), and a hydrolysable one such as PLGA, is not as straightforward as PEG. Amphiphilic PEG is soluble in a wide spectrum of solvents, including aqueous and organic solvents, enabling the reaction with both hydrophilic and hydrophobic moieties. The PCB can dissolve mostly in aqueous solvents due to its superhydrophilicity. Thus, it is challenging to find a common solvent in which a reaction of PCB with hydrophobic moieties can occur. This solubility issue can be partially addressed by using a mixed solvent system [70]. To fully resolve the solubility issue, a hydrophobic precursor (i.e., CB—tBu in Fig. 9) of the super-hydrophilic CB was introduced [11]. CB—tBu and

Figure 9 Synthesis of PLGA—PCB copolymers. DMF = N,N-dimethylformamide, HMTETA = 1,1,4,7,10,10-hexamethyltriethylenetetramine, TFA = trifluoroacetic acid. (1) CB-tBu monomers are polymerized through ATRP initiated by an initiator, 2-aminoethyl 2-bromoisobutyrate. (2) TFA− is removed to generate PCB—tBu—NH2 . (3) PCB—tBu—NH2 is conjugated with PLGA—NHS in anhydrous acetonitrile to form PLGA—PCB—tBu block copolymers. (4) PLGA—PCB is obtained by treating PLGA—PCB—tBu with TFA to generate the zwitterionic CB structure while the PLGA remains intact [11].

Zwitterionic and non-ionic polymers for stealth nanoparticles its polymer (PCB—tBu) have good solubility in many organic solvents such as acetonitrile and dimethylformamide, making it possible to incorporate PCB into hydrophobic blocks such as PLGA (Fig. 9) [11] and lipids [95] in organic solvents. After covalent binding of PCB—tBu to the hydrophobic moieties, zwitterionic PCB can be readily regenerated by hydrolysis of the tBu ester groups under mild acid conditions such as trifluoroacetic acid (TFA). TFA does not degrade typical ester bonds as observed in PLA [98—100], PLGA [11,101], or DSPE [95]. In this way, amphiphilic diblocks with a sharp polarity contrast between super-hydrophilic PCB and hydrophobic PLGA or lipid can be prepared.

Conclusions and future directions This review aims to compare non-ionic PEG with zwitterionic PCB in creating stealth NPs for protein protection and drug delivery. Although both polymers render non-fouling properties to NPs due to their respective hydrophilic natures, they are quite different in their physical and chemical properties, leading to different effects from NP stability to protein protection. PEG is amphiphilic in nature and is robust for applications since it can dissolve in a wide range of polar and non-polar solvents. However, the amphiphilic nature of PEG will alter the bioactivity of PEGylated proteins and the stability of assembled PEGylated polymeric particles and liposomes. Some of these PEG issues may be overcome, for example, by inserting cholesterol into PEGylated liposomes to rescue their stability lost or using a cyroprotectant to maintain the stability of PLGA—PEG NPs during lyophilization. PCB, in contrast, solves all these PEG issues due to its super-hydrophilic nature. Although PCB and PEG are used to illustrate the difference between zwitterionic and non-ionic polymers, general conclusions are applicable to other zwitterionic materials such as poly(sulfobetaine) and poly(phosphobetaine), and other non-ionic polymers. Among zwitterionic materials, PCB is unique in several aspects desirable for medical applications. PCB is derived from naturally occurring betaines abundant in the animal kingdom including humans as well as plants [66]. It is generally considered to be biocompatible. For example, results show that PCB-protected liposome or PLGA NPs have no observable cytotoxicity even at high concentrations [11,95]. When PCB-protected liposome with doxorubicin loaded was injected intravenously to mice at the same recommended dose as DOXIL® , no severe toxicity was found such as >15% weight loss, symptom of illness or death [95]. PCB is not only non-fouling, but also functionalizable for the convenient immobilization of bio-recognition elements for targeted drug delivery or diagnostics via conventional coupling chemistry such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and Nhydroxysuccinimide (EDC/NHS) [11,67,68,102]. After functionalization, unreacted moieties will be hydrolyzed back to the zwitterionic CB structure, enabling the excellent nonfouling properties of post-functionalized surfaces [67,70]. PCB can also be prepared as its hydrolysable cationic precursor, which not only alters its solubility for use in the synthesis of block copolymers with a sharp contrast in hydrophobicity, but also to condense and release genes [103,104]. The nonfouling, biocompatible, super-hydrophilic, functionalizable,

411 and hydrolysable properties make PCB and its derivatives a unique class of materials for constructing next-generation NPs for medical applications.

Acknowledgments This work has been supported by the Office of Naval Research (N000140910137, N000141010600, and N000141210441), National Science Foundation (DMR-1005699 and CBET 0854298), Defense Threat Reduction Agency (HDTRA1-10-10074), and Boeing-Roundhill Professorship.

References [1] B.D. Ratner, A.S. Hoffman, F.J. Schoen, J.E. Lemons, Biomaterials Science, an Introduction to Materials in Medicine, 2nd ed., Elsevier, Amsterdam, 2004. [2] T.A. Horbett, Coll. Surf. B 2 (1994) 225. [3] B.D. Ratner, S.J. Bryant, Annu. Rev. Biomed. Eng. 6 (2004) 41. [4] K.L. Prime, G.M. Whitesides, Science 252 (1991) 1164. [5] E. Ostuni, R.G. Chapman, R.E. Holmlin, S. Takayama, G.M. Whitesides, Langmuir 17 (2001) 5605. [6] R.E. Holmlin, X.X. Chen, R.G. Chapman, S. Takayama, G.M. Whitesides, Langmuir 17 (2001) 2841. [7] S. Jiang, Z.Q. Cao, Adv. Mater. 22 (2010) 920. [8] R. Langer, Science 293 (2001) 58. [9] R. Liu, B.K. Kay, S. Jiang, S. Chen, MRS Bull. 34 (2009) 432. [10] J.M. Harris, Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications, Plenum Press, New York and London, 1992. [11] Z.Q. Cao, Q.M. Yu, H. Xue, G. Cheng, S.Y. Jiang, Angew. Chem. Int. Ed. 49 (2010) 3771. [12] A.J. Keefe, S. Jiang, Nat. Chem. 4 (2012) 59. [13] M.C. Parrott, J.M. DeSimone, Nat. Chem. 4 (2012) 13. [14] M.J. Roberts, M.D. Bentley, J.M. Harris, Adv. Drug Deliv. Rev. 54 (2002) 459. [15] J.M. Harris, R.B. Chess, Nat. Rev. Drug Discov. 2 (2003) 214. [16] K. Knop, R. Hoogenboom, D. Fischer, U.S. Schubert, Angew. Chem. Int. Ed. 49 (2010) 6288. [17] J. Cheng, B.A. Teply, I. Sherifi, J. Sung, G. Luther, F.X. Gu, et al., Biomaterials 28 (2007) 869. [18] M.E. Davis, Z. Chen, D.M. Shin, Nat. Rev. Drug Discov. 7 (2008) 771. [19] R.A. Petros, J.M. DeSimone, Nat. Rev. Drug Discov. 9 (2010) 615. [20] J.M. Chan, L.F. Zhang, R. Tong, D. Ghosh, W.W. Gao, G. Liao, et al., Proc. Natl. Acad. Sci. U.S.A. 107 (2010) 2213. [21] C.M.J. Hu, L. Zhang, S. Aryal, C. Cheung, R.H. Fang, L.F. Zhang, Proc. Natl. Acad. Sci. U.S.A. 108 (2011) 10980. [22] V.P. Torchilin, Nat. Rev. Drug Discov. 4 (2005) 145. [23] M. Elsabahy, K.L. Wooley, Chem. Soc. Rev. 41 (2012) 2545. [24] S.I. Jeon, J.H. Lee, J.D. Andrade, P.G. Degennes, J. Colloid Interf. Sci. 142 (1991) 149. [25] T. McPherson, A. Kidane, I. Szleifer, K. Park, Langmuir 14 (1998) 176. [26] P. Harder, M. Grunze, R. Dahint, G.M. Whitesides, P.E. Laibinis, J. Phys. Chem. B 102 (1998) 426. [27] L. Li, S. Chen, J. Zheng, B.D. Ratner, S. Jiang, J. Phys. Chem. B 109 (2005) 2934. [28] J. Zheng, L.Y. Li, S.F. Chen, S.Y. Jiang, Langmuir 20 (2004) 8931. [29] J. Zheng, L.Y. Li, H.K. Tsao, Y.J. Sheng, S.F. Chen, S.Y. Jiang, Biophys. J. 89 (2005) 158. [30] Y. He, Y. Chang, J.C. Hower, J. Zheng, S.F. Chen, S. Jiang, Phys. Chem. Chem. Phys. 10 (2008) 5539.

412 [31] F.M. Veronese, A. Mero, Biodrugs 22 (2008) 315. [32] D. Peer, J.M. Karp, S. Hong, O.C. FaroKHzad, R. Margalit, R. Langer, Nat. Nanotechnol. 2 (2007) 751. [33] L. Zhang, F.X. Gu, J.M. Chan, A.Z. Wang, R.S. Langer, O.C. Farokhzad, Clin. Pharm. Ther. 83 (2008) 761. [34] V. Gaberc-Porekar, I. Zore, B. Podobnik, V. Menart, Curr. Opin. Drug Discov. 11 (2008) 242. [35] G.G. Hammes, P.R. Schimmel, J. Am. Chem. Soc. 89 (1967) 442. [36] S. Jevsevar, M. Kunstelj, V.G. Porekar, Biotech. J. 5 (2010) 113. [37] C.S. Fishburn, J. Pharm. Sci. US 97 (2008) 4167. [38] F.M. Veronese, Biomaterials 22 (2001) 405. [39] A.P. Chapman, Adv. Drug Deliv. Rev. 54 (2002) 531. [40] J.A. Rodriguez-Martinez, I. Rivera-Rivera, R.J. Sola, K. Griebenow, Biotech. Lett. 31 (2009) 883. [41] F.M. Muggia, J.D. Hainsworth, S. Jeffers, P. Miller, S. Groshen, M. Tan, et al., J. Clin. Oncol. 15 (1997) 987. [42] T.C. Chang, H.H. Chou, K.L. Wang, C.A. Chen, L.H. Wei, C.H. Lai, et al., Gynecol. Oncol. 101 (2006) 423. [43] F.K. Beduaddo, L. Huang, Adv. Drug Deliv. Rev. 16 (1995) 235. [44] D.A. Herold, K. Keil, D.E. Bruns, Biochem. Pharmacol. 38 (1989) 73. [45] M.C. Shen, L. Martinson, M.S. Wagner, D.G. Castner, B.D. Ratner, T.A. Horbett, J. Biomater. Sci. Polym. E 13 (2002) 367. [46] L.Y. Li, S.F. Chen, S.Y. Jiang, J. Biomater. Sci. Polym. E 18 (2007) 1415. [47] T.L. Cheng, P.Y. Wu, M.F. Wu, J.W. Chern, S.R. Roffler, Bioconjug. Chem. 10 (1999) 520. [48] R.P. Garay, J.P. Labaune, Open Conf. Proc. J. 2 (2011) 104. [49] K. Ishihara, N.P. Ziats, B.P. Tierney, N. Nakabayashi, J.M. Anderson, J. Biomed. Mater. Res. 25 (1991) 1397. [50] T. Ueda, H. Oshida, K. Kurita, K. Ishihara, N. Nakabayashi, Polym. J. 24 (1992) 1259. [51] S. Kudaibergenov, W. Jaeger, A. Laschewsky, Adv. Polym. Sci. 201 (2006) 157. [52] T. Nakaya, Y.J. Li, Prog. Polym. Sci. 24 (1999) 143. [53] J.Z. Du, S.P. Armes, Langmuir 25 (2009) 9564. [54] A.B. Lowe, C.L. McCormick, Chem. Rev. 102 (2002) 4177. [55] S. Kudaibergenov, Polyampholytes: Synthesis, Characterization, and Application, Kluwer, New York, 2002. [56] L. Cao, M. Chang, C.-Y. Lee, D.G. Castner, S. Sukavaneshvar, B.D. Ratner, et al., J. Biomed. Mater. Res. A 81A (2007) 827. [57] S.L. McArthur, K.M. McLean, P. Kingshott, H.A.W. St John, R.C. Chatelier, H.J. Griesser, Coll. Surf. B 17 (2000) 37. [58] Y.Y. Luk, M. Kato, M. Mrksich, Langmuir 16 (2000) 9604. [59] R.G. Chapman, E. Ostuni, M.N. Liang, G. Meluleni, E. Kim, L. Yan, et al., Langmuir 17 (2001) 1225. [60] A.R. Statz, R.J. Meagher, A.E. Barron, P.B. Messersmith, J. Am. Chem. Soc. 127 (2005) 7972. [61] R. Chelmowski, S.D. Koster, A. Kerstan, A. Prekelt, C. Grunwald, T. Winkler, et al., J. Am. Chem. Soc. 130 (2008) 14952. [62] S.F. Chen, J. Zheng, L.Y. Li, S.Y. Jiang, J. Am. Chem. Soc. 127 (2005) 14473. [63] Y. He, J. Hower, S.F. Chen, M.T. Bernards, Y. Chang, S.Y. Jiang, Langmuir 24 (2008) 10358. [64] J.A. Callow, M.E. Callow, Nat. Commun. 2 (2011). [65] Z.Q. Cao, N. Brault, H. Xue, A. Keefe, S.Y. Jiang, Angew. Chem. Int. Ed. 50 (2011) 6102. [66] A. Nyyssölä, Pathways of Glycine Betaine Synthesis in Two Extremely Halophilic Bacteria, Actinopolyspora Halophila and Ectothiorhodospira Halochloris, PhD Thesis, Helsinki University of Technology, Espoo, Finland, 2001. [67] H. Vaisocherová, W. Yang, Z. Zhang, Z. Cao, G. Cheng, M. Piliarik, et al., Anal. Chem. 80 (2008) 7894.

Z. Cao, S. Jiang [68] W. Yang, H. Xue, W. Li, J. Zhang, S. Jiang, Langmuir 25 (2009) 11911. [69] G. Cheng, G.Z. Li, H. Xue, S.F. Chen, J.D. Bryers, S.Y. Jiang, Biomaterials 30 (2009) 5234. [70] W. Yang, L. Zhang, S. Wang, A.D. White, S. Jiang, Biomaterials 30 (2009) 5617. [71] M.G. Cacace, E.M. Landau, J.J. Ramsden, Q. Rev. Biophys. 30 (1997) 241. [72] Q. Shao, Y. He, A.D. White, S. Jiang, J. Chem. Phys. 136 (2012) 225101. [73] F. Ahmed, D.E. Discher, J. Control. Release 96 (2004) 37. [74] S. Jain, F.S. Bates, Science 300 (2003) 460. [75] D.E. Discher, A. Eisenberg, Science 297 (2002) 967. [76] H. Bermudez, A.K. Brannan, D.A. Hammer, F.S. Bates, D.E. Discher, Macromolecules 35 (2002) 8203. [77] B.M. Discher, Y.Y. Won, D.S. Ege, J.C.M. Lee, F.S. Bates, D.E. Discher, et al., Science 284 (1999) 1143. [78] R. Gref, Y. Minamitake, M.T. Peracchia, V. Trubetskoy, V. Torchilin, R. Langer, Science 263 (1994) 1600. [79] D.E. Discher, F. Ahmed, R.I. Pakunlu, A. Brannan, F. Bates, T. Minko, J. Control. Release 116 (2006) 150. [80] O.C. Farokhzad, J.J. Cheng, B.A. Teply, I. Sherifi, S. Jon, P.W. Kantoff, et al., Proc. Natl. Acad. Sci. U.S.A. 103 (2006) 6315. [81] F. Gu, L. Zhang, B.A. Teply, N. Mann, A. Wang, A.F. RadovicMoreno, et al., Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 2586. [82] http://www.samyangpharm.com/rnd/genexol pm.asp. [83] http://www.bindbio.com/content/pages/news/news detail. jsp/q/news-id/51. [84] C.E. Astete, C.M. Sabliov, J. Biomater. Sci. Polym. E 17 (2006) 247. [85] K. Avgoustakis, Curr. Drug Deliv. 1 (2004) 321. [86] J.M. Chan, L.F. Zhang, K.P. Yuet, G. Liao, J.W. Rhee, R. Langer, et al., Biomaterials 30 (2009) 1627. [87] R.A. Bader, A.L. Silvers, N. Zhang, Biomacromolecules 12 (2011) 314. [88] D.C. Drummond, O. Meyer, K.L. Hong, D.B. Kirpotin, D. Papahadjopoulos, Pharmacol. Rev. 51 (1999) 691. [89] A. Herrmann, L. Pratsch, K. Arnold, G. Lassmann, Biochim. Biophys. Acta 733 (1983) 87. [90] O. Tirosh, Y. Barenholz, J. Katzhendler, A. Priev, Biophys. J. 74 (1998) 1371. [91] S. Belsito, R. Bartucci, L. Sportelli, Biophys. Chem. 93 (2001) 11. [92] G.L. Jendrasiak, J. Nutr. Biochem. 7 (1996) 599. [93] D. Bach, I.R. Miller, Biochim. Biophys. Acta 1386 (1998) 216. [94] D. Bach, I.R. Miller, Chem. Phys. Lipids 136 (2005) 67. [95] Z.Q. Cao, L. Zhang, S. Jiang, Langmuir 28 (2012) 11625. [96] T.P.W. Mcmullen, R.N.A.H. Lewis, R.N. Mcelhaney, Biochemistry-US 32 (1993) 516. [97] R.J. Malcolmson, J. Higinbotham, P.H. Beswick, P.O. Privat, L. Saunier, J. Membr. Sci. 123 (1997) 243. [98] H. Arimura, Y. Ohya, T. Ouchi, Macromol. Rapid Commun. 25 (2004) 743. [99] S. Caillol, S. Lecommandoux, A.F. Mingotaud, M. Schappacher, A. Soum, N. Bryson, et al., Macromolecules 36 (2003) 1118. [100] M. Gotsche, H. Keul, H. Hocker, Macromol. Chem. Phys. 196 (1995) 3891. [101] E.B. Lavik, J.S. Hrkach, N. Lotan, R. Nazarov, R. Langer, J. Biomed. Mater. Res. 58 (2001) 291. [102] Z. Zhang, S. Chen, S. Jiang, Biomacromolecules 7 (2006) 3311. [103] Z. Zhang, G. Cheng, L.R. Carr, H. Vaisocherova, S.F. Chen, S.Y. Jiang, Biomaterials 29 (2008) 4719. [104] L.R. Carr, S.Y. Jiang, Biomaterials 31 (2010) 4186.

Zwitterionic and non-ionic polymers for stealth nanoparticles Shaoyi Jiang is the Boeing-Roundhill Professor of Chemical Engineering and Adjunct Professor of Bioengineering at the University of Washington, Seattle. He received his Ph.D. degree in chemical engineering from Cornell University in 1993. He was a postdoctoral fellow at the University of California, Berkeley between 1993 and 1994 and a research fellow at California Institute of Technology between 1994 and 1996 both in chemistry. Currently, he serves as a Senior Editor for Langmuir. He is a Fellow of the American Institute of Chemical Engineering, a Fellow of the American Institute of Medical and Biological Engineering and a Member of the Washington State Academy of Sciences. His current research focuses on the molecular understanding, design and development of zwitterionic-based functional materials for biomedical and engineering applications.

413 Zhiqiang Cao is currently a research fellow in the David H. Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology, and the Department of Anesthesiology at Children’s Hospital Boston and Harvard Medical School, under the direction of Prof. Robert Langer. He received his Ph.D. in Chemical Engineering from the University of Washington in 2011 under the guidance of Prof. Shaoyi Jiang. He received his B.Eng. in polymer materials and engineering and M.Eng. in biomedical engineering from Tianjin University, China in 2004 and 2007, respectively. He will be an assistant professor in the Department of Chemical Engineering and Materials Science at Wayne State University starting January 2013. His current research is focused on zwitterionic materials and diabetes research.