PEG-peptide Conjugates Research Articles

Cyclodextrin-Induced Suppression of PEG Crystallization from the Melt in a PEG-Peptide Conjugate.

The influence of alpha-cyclodextrin (αCD) on PEG crystallization is examined for a peptide-PEG conjugate, YYKLVFF-PEG3k comprising an amyloid peptide YYKLVFF linked to PEG with molar mass 3 kg mol-1. Remarkably, differential scanning calorimetry (DSC) and simultaneous synchrotron small-angle/wide-angle X-ray scattering (SAXS/WAXS) show that crystallization of PEG is suppressed by αCD, provided that the cyclodextrin content is sufficient. A hexagonal mesophase is formed instead. The αCD threading reduces the conformational flexibility of PEG, and hence suppresses crystallization. These results show that addition of cyclodextrins can be used to tune the crystallization of peptide-polymer conjugates and potentially other polymer/biomolecular hybrids.

In Situ Synthesis and Self-Assembly of Peptide-PEG Conjugates: A Facile Method for the Construction of Fibrous Hydrogels.

Peptide-based hydrogels have gained considerable attention as a compelling platform for various biomedical applications in recent years. Their attractiveness stems from their ability to seamlessly integrate diverse properties, such as biocompatibility, biodegradability, easily adjustable hydrophilicity/hydrophobicity, and other functionalities. However, a significant drawback is that most of the functional self-assembling peptides cannot form robust hydrogels suitable for biological applications. In this study, we present the synthesis of novel peptide-PEG conjugates and explore their comprehensive hydrogel properties. The hydrogel comprises double networks, with the first network formed through the self-assembly of peptides to create a β-sheet secondary structure. The second network is established through covalent bond formation via N-hydroxysuccinimide chemistry between peptides and a 4-arm PEG to form a covalently linked network. Importantly, our findings reveal that this hydrogel formation method can be applied to other peptides containing lysine-rich sequences. Upon encapsulation of the hydrogel with antimicrobial peptides, the hydrogel retained high bacterial killing efficiency while showing minimum cytotoxicity toward mammalian cells. We hope that this method opens new avenues for the development of a novel class of peptide-polymer hydrogel materials with enhanced performance in biomedical contexts, particularly in reducing the potential for infection in applications of tissue regeneration and drug delivery.

Tuning the Solution Self-Assembly of a Peptide-PEG (Polyethylene Glycol) Conjugate with alpha-Cyclodextrin.

Cyclodextrins are saccharide ring molecules which act as host cavities that can encapsulate small guest molecules or thread polymer chains. We investigate the influence of alpha-cyclodextrin (αCD) on the aqueous solution self-assembly of a peptide-polymer conjugate YYKLVFF-PEG3K previously studied by our group [Castelletto et al., Polym. Chem., 2010, 1, 453-459]. This conjugate comprises a designed amyloid-forming peptide YYKLVFF that contains the KLVFF sequence from Amyloid β peptide, Aβ16-20, along with two aromatic tyrosine residues to enhance hydrophobicity as well as polyethylene glycol PEG with molar mass 3000 g mol-1. The conjugate self-assembles into β-sheet fibrils in aqueous solution. Here we show that complexation with αCD leads instead can produce free-floating nanosheets in aqueous solution (with a β-sheet structure). The transition from fibrils to nanosheets is driven by an increase in the number of αCD molecules threaded on the PEG chains, as determined by 1H NMR spectroscopy.These findings point to the use of cyclodextrin additives as a powerful means to tune the solution self-assembly in peptide-polymer conjugates and potentially other polymer/biomolecular hybrids.

C-Terminal PEGylation Improves SAAP-148 Peptide’s Immunomodulatory Activities

Synthetic antibacterial and anti-biofilm peptide (SAAP)-148 was developed to combat bacterial infections not effectively treatable with current antibiotics. SAAP-148 is highly effective against antimicrobial-resistant bacteria without inducing resistance; however, challenges for further development of SAAP-148 include its cytotoxicity and short circulation half-life. To circumvent these drawbacks, a library of SAAP-148 linked to polyethylene glycol (PEG) groups of various lengths was synthesized and screened for in vitro antibacterial activity and hemolytic activity. Results indicated that PEGylated SAAP-148 variants combine antibacterial activities with reduced hemolysis compared to SAAP-148. Interestingly, proinflammatory immunomodulatory activities of SAAP-148 were enhanced upon C-terminal PEGylation, with SAAP-148-PEG27 showing the most effect. SAAP-148-PEG27 enhanced SAAP-148’s capacity to chemoattract human neutrophils and was able to more efficiently (re)direct M-CSF-induced monocyte-macrophage differentiation toward type 1 macrophages as opposed to SAAP-148. Furthermore, dendritic cells with a stronger mature expression profile were produced if monocytes were exposed to SAAP-148-PEG27 during monocyte-immature dendritic cell differentiation in comparison to SAAP-148. Parameters that influenced the immunomodulatory activities of the peptide-PEG conjugate include (i) the length of the PEG group, (ii) the position of PEG conjugation, and (iii) the peptide sequence. Together, these results indicate that SAAP-148-PEG27 is highly effective in redirecting monocyte-macrophage differentiation toward a proinflammatory phenotype and promoting monocyte-mature dendritic cell development. Therefore, SAAP-148-PEG27 may be a promising agent to modulate inadequate immune responses in case of tumors and chronically infected wounds.

Investigation on the Interactions between Self-Assembled β-Sheet Peptide Nanofibers and Model Cell Membranes.

Self-assembled peptide nanofibers (NFs) obtained from β-sheet peptides conjugated with drugs, including antigenic peptides, have recently attracted significant attention. However, extensive studies on the interactions of β-sheet peptide NFs with model cell membranes have not been reported. In this study, we investigated the interactions between three types of NFs, composed of PEG-peptide conjugates with different ethylene glycol (EG) lengths (6-, 12- and 24-mer), and dipalmitoylphosphatidylcholine (DPPC) Langmuir membranes. When increasing the EG chain length, those interactions significantly decreased considering measurements in the presence of the NFs of: (i) changes in surface pressure of the DPPC Langmuir monolayers and (ii) surface pressure–area (π–A) compression isotherms of DPPC. Because the observed trend was similar to the EG length dependency with regard to cellular association and cytotoxicity of the NFs that was reported previously, the interaction of NFs with phospholipid membranes represented a crucial factor to determine the cellular association and toxicity of the NFs. In contrast to NFs, no changes were observed with varying EG chain length on the interaction of the building block peptide with the DPPC membrane. The results obtained herein can provide a design guideline on the formulation of β-sheet peptide NFs, which may broaden its potential.

Information-Based Design of Polymeric Drug Formulation Additives.

Tailor-made copolymers are designed based on a peptide-poly(ethylene glycol) (QFFLFFQ-PEG) conjugate as a blueprint, to solubilize the photosensitizer meta-tetra(hydroxyphenyl)chlorin (m-THPC). The relevant functionalities of the parent peptide-PEG are mimicked by employing monomer pairs that copolymerize in a strictly alternating manner. While styrene (S) or 4-vinylbenzyl-phthalimide (VBP) provide aromatic moieties like Phe, the aliphatic isobutyl side chain of Leu4 is mimicked by maleic anhydride (MA) that reacts after polymerization with isobutylamine to give the isobutylamide-carboxyl functional unit (iBuMA). A set of copolymer-PEG solubilizers is synthesized by controlled radical polymerization, systematically altering the length of the functional segment (DPn = 2, 4, 6) and the side chain functionalization (iBuMA, iPrMA, MeMA). The m-THPC hosting and release properties of P[S-alt-iBuMA]6-PEG reached higher payload capacities and more favored release rates than the parent peptide-PEG conjugate. Interestingly, P[S-alt-RMA]n-PEG mimics the sensitivity of the peptide-PEG solubilizer well, where the exchange of Leu4 residue by Val and Ala significantly reduces the drug loading by 92%. A similar trend is found with P[S-alt-RMA]n-PEG as the exchange of iBu → iPr → Me reduces the payload capacity up to 78%.

Correlative Analysis of Specific Compatibilization in Composites by Coupling in situ X-Ray Scattering and Mechanical Tensile Testing

In this study, a bio-inspired hybrid material is investigated by in situ X-ray scattering experiments in combination with mechanical tensile testing. The material is composed of nanometer-sized spherical magnesium fluoride particles which are linked via material-specific peptide-poly(ethylene glycol) (PEG) conjugates to a semi-crystalline poly(ethylene oxide) (PEO) matrix. Mechanically relevant changes in crystal size and orientation in the PEO matrix are followed by wide angle X-ray scattering during the application of tensile stress. The amorphous phase of PEO is stabilized by the surface-engineered MgF2 nanoparticles, leading to increased Young´s modulus and tensile strength. Furthermore, small angle X-ray scattering experiments allowed the identification of a layer on the MgF2 particle surfaces, which increases in thickness with the conjugate amount and leads to suppression of the agglomeration of MgF2 nanoparticles. In conclusion, the use of selected peptide-PEG conjugates tailored to link MgF2 particles to a PEO matrix successfully mimics the biological principle of interface polymers and suggests new directions for material fabrication for bio-applications.

Plasmin-Cleavable Nanoparticles for On-Demand Release of Morphogens in Vascularized Osteogenesis.

The objective of this work was to engineer self-assembled nanoparticles (NPs) for on-demand release of bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) in response to enzymes secreted by the migrating human mesenchymal stem cells (hMSCs) and human endothelial colony forming cells (ECFCs) to induce osteogenesis and vasculogenesis. Gene expression profiling experiments revealed that hMSCs and ECFCs, encapsulated in osteogenic/vasculogenic hydrogels, expressed considerable levels of plasminogen, urokinase plasminogen activator and its receptor uPAR, and tissue plasminogen activator. Therefore, the plasmin-cleavable lysine-phenylalanine-lysine-threonine (KFKT) was used to generate enzymatically cleavable NPs. The acetyl-terminated, self-assembling peptide glycine-(phenylalanine)3GFFF-ac and the plasmin-cleavable GGKFKTGG were reacted with the cysteine-terminated CGGK(Fmoc/MTT) peptide through the MTT and Fmoc termini, respectively. The difunctional peptide was conjugated to polyethylene glycol diacrylate (PEGDA) with molecular weights (MW) ranging from 0.5 to 7.5 kDa, and the chain ends of the PEG-peptide conjugate were terminated with succinimide groups. After self-assembly in aqueous solution, BMP2 was grafted to the self-assembled, plasmin-cleavable PEG-based (PxSPCP) NPs for on-demand release. The NPs' stability in aqueous solution and that of the grafted BMP2 were strongly dependent on PEG MW. P2SPCP NPs showed high particle size stability, BMP2 grafting efficiency, grafted protein stability, and high extent of osteogenic differentiation of hMSCs. The localized and on-demand release of BMP2 from PxSPCP NPs coencapsulated with hMSCs in the linear polyethylene glycol-co-lactide acrylate patterned hydrogel with microchannels encapsulating hMSCs + ECFCs and VEGF-conjugated nanogels resulted in the highest extent of osteogenic and vasculogenic differentiation of the encapsulated cells compared to directly added BMP2/VEGF. The on-demand release of BMP2 from PxSPCP NPs not only enhances osteogenesis and vasculogenesis but also potentially reduces many undesired side effects of BMP2 therapy in bone regeneration.

Dynamic control of hydrogel crosslinking via sortase-mediated reversible transpeptidation

Cell-laden hydrogels whose crosslinking density can be dynamically and reversibly tuned are highly sought-after for studying pathophysiological cellular fate processes, including embryogenesis, fibrosis, and tumorigenesis. Special efforts have focused on controlling network crosslinking in poly(ethylene glycol) (PEG) based hydrogels to evaluate the impact of matrix mechanics on cell proliferation, morphogenesis, and differentiation. In this study, we sought to design dynamic PEG-peptide hydrogels that permit cyclic/reversible stiffening and softening. This was achieved by utilizing reversible enzymatic reactions that afford specificity, biorthogonality, and predictable reaction kinetics. To that end, we prepared PEG-peptide conjugates to enable sortase A (SrtA) induced tunable hydrogel crosslinking independent of macromer contents. Uniquely, these hydrogels can be completely degraded by the same enzymatic reactions and the degradation rate can be tuned from hours to days. We further synthesized SrtA-sensitive peptide linker (i.e., KCLPRTGCK) for crosslinking with 8-arm PEG-norbornene (PEG8NB) via thiol-norbornene photocrosslinking. These hydrogels afford diverse softening paradigms through control of network structures during crosslinking or by adjusting enzymatic parameters during on-demand softening. Importantly, user-controlled hydrogel softening promoted spreading of human mesenchymal stem cells (hMSCs) in 3D. Finally, we designed a bis-cysteine-bearing linear peptide flanked with SrtA substrates at the peptide’s N- and C-termini (i.e., NH2-GGGCKGGGKCLPRTG-CONH2) to enable cyclic/reversible hydrogel stiffening/softening. We show that matrix stiffening and softening play a crucial role in growth and chemoresistance in pancreatic cancer cells. These results represent the first dynamic hydrogel platform that affords cyclic gel stiffening/softening based on reversible enzymatic reactions. More importantly, the chemical motifs that affords such reversible crosslinking were built-in on the linear peptide crosslinker without any post-synthesis modification. Statement of SignificanceCell-laden ‘dynamic’ hydrogels are typically designed to enable externally stimulated stiffening or softening of the hydrogel network. However, no enzymatic reaction has been used to reversibly control matrix crosslinking. The application of SrtA-mediated transpeptidation in crosslinking and post-gelation modification of biomimetic hydrogels is innovative because of the specificity of the reaction and reversible tunability of crosslinking kinetics. While SrtA has been previously used to crosslink and fully degrade hydrogels, matrix softening and reversible stiffening of cell-laden hydrogels has not been reported. By designing simple peptide substrates, this unique enzymatic reaction can be employed to form a primary network, to gradually soften hydrogels, or to reversibly stiffen hydrogels. As a result, this dynamic hydrogel platform can be used to answer important matrix-related biological questions that are otherwise difficult to address.

On the way to precision formulation additives: 2D-screening to select solubilizers with tailored host and release capabilities

A 2-dimensional high-throughput screening method is presented to select peptide sequences from large peptide libraries for precision formulation additives, having a high capacity to specifically host a drug of interest and provide tailored drug release properties. The identified sequences are conjugated with poly(ethylene glycol) (PEG) to obtain peptide-PEG conjugates that proved to be valuable as solubilizers for small organic molecule drugs to overcome limitations of poor water-solubility and low bio-availability. The 2D-screening method selects peptide sequences on both (i) high loading capacities and (ii) preferred drug-release capabilities as demonstrated on an experimental Tau-protein aggregation inhibitor/Tau- deaggregator with potentials for an anti-Alzheimer disease drug (BB17). To enable 2D-screening, a one-bead one-compound (OBOC) peptide library was immobilized on a glass slide, allocating individual beads to permanent positions. While the first screening step involved incubation of the supported OBOC library with BB17 to identify beads with high drug binding capacities by fluorescence scanner readouts, the second step reveals release properties of the high capacity binders by incubation with blood plasma protein model solutions. Efficiently peptides with high BB17 capacities and either keeper or medium or fast releaser properties can be identified by direct sequence readouts from the glass slide supported resin beads via matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Four peptides are synthesized as peptide-PEG solubilizers representing strong, medium, weak releasers and non-binders. Loading capacities reached up to 1:3.4 (mol drug per mol carrier) and release kinetics (fast/medium/slow) are in agreement with the selection process as investigated by fluorescence anisotropy and fluorescence correlation spectroscopy. The ability of BB17/conjugate complexes to inhibit the aggregation of Tau4RDΔK (four repeat Tau ((M)Q244-E372 with deletion of K280), 129 residues) in N2a cells is studied by a Tau-pelleting assay showing the modulation of cellular Tau aggregation. Promising effects such as the reduction of 55% of total Tau load are observed for the strong releaser additive. Studies of in vitro Thioflavin S Tau-aggregation assays show half-maximal inhibitory activities (IC50 values) of BB17/conjugates in the low micro-molar range.

PEG-peptide conjugate containing cathepsin B degradation unit as a doxorubicin carrier system

A drug delivery system (DDS) containing a cathepsin B degradable sequence and pH-responsive histidines was prepared by methoxypolyethylene glycol and peptide conjugation. Doxorubicin was attached to the carrier system using amide linkage to give the final form of the DDS, denoted as mPEG-AT3-DOX. mPEG-AT3-DOX exhibited a bimodal size distribution at about 15 and 30 nm independent of pH, whereas the size of the control DDS containing no peptide sequence, mPEG-DOX, was measured as $\sim$ 15-20 nm. At the end of 72 h, % doxorubicin release from both of the DDSs was observed to be below 8.5 $\pm$ 3% in the absence of cathepsin B, and it increased to 17 $\pm$ 2% in the presence of cathepsin B for mPEG-AT3-DOX. Complete degradation of AT3 peptide within 3 h upon incubation with cathepsin B suggests that lower than expected doxorubicin release is likely due to the aggregation tendency of mPEG-AT3-DOX. Absolute IC$_{50}$ values indicated that the cytotoxicity trend of the samples is in the order of free DOX $\ge $ mPEG-AT3-DOX \textgreater mPEG-DOX. Considering these results, PEG-peptide-doxorubicin conjugates can be promising candidates in cancer therapy if they are designed to have more pronounced pH-responsive behavior to increase the drug release rate.

Nanoplatform containing MMPs-sensitive peptide-PEG conjugate and tissue-penetrating moiety for enhanced cancer therapy

Nanoplatform containing MMPs-sensitive peptide-PEG conjugate and tissue-penetrating moiety for enhanced cancer therapy

Gaining Insights into Specific Drug Formulation Additives for Solubilizing a Potential Anti-Alzheimer Disease Drug B4A1.

The pharmacological profiles of small molecule drugs are often challenged by their poor water solubility. Sequence-defined peptides attached to poly(ethylene glycol) (PEG) offer opportunities to overcome these difficulties by acting as drug-specific formulation additives. The peptide-PEG conjugates enable specific, noncovalent drug binding via tailored peptide/drug interactions as well as provide water solubility and drug shielding by well-solvated PEG-blocks. A systematic set of specific solubilizers for B4A1 as a potential anti-Alzheimer disease drug is synthesized and variations involve the length of the PEG-blocks as well as the sequences of the peptidic drug-binding domain. The solubilizer/B4A1 complexes are studied in order to understand contributions of both PEG and peptide segments on drug payload capacities, drug/carrier aggregate sizes, and influences on inhibition of the Tau-protein aggregation in an in vitro assay.

Understanding Peptide Oligomeric State in Langmuir Monolayers of Amphiphilic 3-Helix Bundle-Forming Peptide-PEG Conjugates.

Coiled-coil peptide-polymer conjugates are an emerging class of biomaterials. Fundamental understanding of the coiled-coil oligomeric state and assembly process of these hybrid building blocks is necessary to exert control over their assembly into well-defined structures. Here, we studied the effect of peptide structure and PEGylation on the self-assembly process and oligomeric state of a Langmuir monolayer of amphiphilic coiled-coil peptide-polymer conjugates using X-ray reflectivity (XR) and grazing-incidence X-ray diffraction (GIXD). Our results show that the oligomeric state of PEGylated amphiphiles based on 3-helix bundle-forming peptide is surface pressure dependent, a mixture of dimers and trimers was formed at intermediate surface pressure but transitions into trimers completely upon increasing surface pressure. Moreover, the interhelical distance within the coiled-coil bundle of 3-helix peptide-PEG conjugate amphiphiles was not perturbed under high surface pressure. Present studies provide valuable insights into the self-assembly process of hybrid peptide-polymer conjugates and guidance to develop biomaterials with controlled multivalency of ligand presentation.

Self-assembly and drug release study of linear l,d-oligopeptide-poly(ethylene glycol) conjugates

The preparation and structural organisation of new bioinspired nanomaterials based on regular alternating enantiomeric sequence of tetra- and hexapeptides end-linked to poly(ethylene glycol) (PEG) is reported. The peptide moiety is composed of two or three repeats of l-Ala-d-Val units while the PEG has a molecular weight of 2kDa. The self-assembling properties of the two conjugates depend significantly on the length of the peptide. Nanoparticles with different sizes and morphologies are formed, the structural properties of which are compared with the previously studied l-Ala-d-Val octapeptide conjugate that self-assembles into rod-like nanoparticles. The aggregation properties were studied by NMR, circular dichroism, fluorescence spectroscopies and dynamic light scattering. The morphology and size of the nanoparticles were assessed by scanning electron microscopy and dynamic light scattering. The loading and release of a model drug were also investigated. This study demonstrates that, by changing the length of the peptide, it is possible to modulate the self-assembly and loading properties of peptide-PEG conjugates.

Generalizing the Concept of Specific Compound Formulation Additives towards Non-Fluorescent Drugs: A Solubilization Study on Potential Anti-Alzheimer-Active Small-Molecule Compounds.

Tailor-made compound formulation additives enable the testing of potential drugs with undesirable pharmacological profiles. A combinatorial approach using Raman microscopy as the readout method is presented to select peptide sequences from large one-bead-one-compound libraries. The resulting peptide-PEG conjugates solubilize potential prophylactic and therapeutic anti-Alzheimer compounds and can be used as specific additives not only for fluorescent but also for non-fluorescent compounds.

Tailoring Supramolecular Peptide-Poly(ethylene glycol) Hydrogels by Coiled Coil Self-Assembly and Self-Sorting.

Physical hydrogels are extensively used in a wide range of biomedical applications. However, different applications require hydrogels with different mechanical and structural properties. Tailoring these properties demands exquisite control over the supramolecular interactions involved. Here we show that it is possible to control the mechanical properties of hydrogels using de novo designed coiled coil peptides with different affinities for dimerization. Four different nonorthogonal peptides, designed to fold into four different coiled coil heterodimers with dissociation constants spanning from μM to pM, were conjugated to star-shaped 4-arm poly(ethylene glycol) (PEG). The different PEG-coiled coil conjugates self-assemble as a result of peptide heterodimerization. Different combinations of PEG-peptide conjugates assemble into PEG-peptide networks and hydrogels with distinctly different thermal stabilities, supramolecular, and rheological properties, reflecting the peptide dimer affinities. We also demonstrate that it is possible to rationally modulate the self-assembly process by means of thermodynamic self-sorting by sequential additions of nonpegylated peptides. The specific interactions involved in peptide dimerization thus provides means for programmable and reversible self-assembly of hydrogels with precise control over rheological properties, which can significantly facilitate optimization of their overall performance and adaption to different processing requirements and applications.

Enzyme-Triggered Antifouling Coatings: Switching Bioconjugate Adsorption via Proteolytically Cleavable Interfering Domains.

Protease activable antifouling coatings based on peptide-poly(ethylene glycol) conjugates are shown. The material-specific adsorption of a bioconjugate is temporarily suppressed by extending a titanium binding sequence with a proteolytically cleavable epitope and a suitable interfering domain. The adsorption of the PEG-peptide conjugates onto titanium substrates can be regained by cleaving the interfering domain with Tobacco Etch Virus protease. This activates peptide-mediated PEGylation of titanium surfaces and results in coatings that are stable against dilution and suppress nonreversible adsorption of blood protein models. Effects of branched and linear peptidic binding domains on coating stability and antifouling properties are elucidated.

Specific Drug Formulation Additives: Revealing the Impact of Architecture and Block Length Ratio.

Combining poly(ethylene glycol) (PEG) with sequence-defined peptides in PEG-peptide conjugates offers opportunities to realize next-generation drug formulation additives for overcoming undesired pharmacological profiles of difficult small molecule drugs. The tailored peptide segments provide sequence-specific, noncovalent drug binding, and the hydrophilic PEG block renders the complexes water soluble. On the basis of a peptide sequence known to bind the photosensitizer m-tetra(hydroxyphenyl)chlorin (m-THPC) for photodynamic cancer therapy, a set of different conjugate architectures is synthesized and studied. Variations in PEG block length and amplification of the peptidic binding domain of PEG-peptide conjugates are used to fine tune critical parameters for hosting m-THPC, such as drug payload capacities, aggregation sizes, and drug release and activation kinetics.

Micelle stabilization via entropic repulsion: balance of force directionality and geometric packing of subunit.

Nanoparticles, 10-30 nm in size, have shown great prospects as nanocarriers for drug delivery. We designed amphiphiles based on 3-helix peptide-PEG conjugate forming 15 nm micelles (defined as "3-helix micelles") with good in vivo stability. Here, we investigated the effect of the site of PEG conjugation on the kinetic stability and showed that the conjugation site affects the PEG chain conformation and the overall molecular architecture of the subunit. Compared to the original design where the PEG chain is located in the middle of the 3-helix bundle, micelle kinetic stability was reduced when the PEG chain was attached near the N-terminus (t(1/2) = 35 h) but was enhanced when the PEG chain was attached near the C-terminus (t(1/2) = 80 h). Quantitative structural and kinetic analysis suggest that the kinetic stability was largely dictated by the combined effects of entropic repulsion associated with PEG chain conformation and the geometric packing of the trimeric subunits. The modular design approach coupled with a variety of well-defined protein stucture and functional polymers will significantly expand the utility of these materials as nanocarriers to meet current demands in nanomedine.