Peptide-polymer Conjugates Research Articles

Atomistic Molecular Dynamics Simulations of ABA-Type Polymer Peptide Conjugates: Insights into Supramolecular Structures and their Circular Dichroism Spectra.

A combination of atomistic molecular dynamics (aMD) simulations and circular dichroism (CD) analysis is used to explore supramolecular structures of amphiphilic ABA-type triblock polymer peptide conjugates (PPC). Using the example of a recently introduced PPC with pH- and temperature responsive self-assembling behavior [Otter etal., Macromolecular Rapid Communications 2018, 39, 1800459], this study shows how molecular dynamics simulations of simplified fragment molecules can add crucial information to CD data, which helps to correctly identify the self-assembled structures and monitor the folding/unfolding pathways of the molecules. The findings offer insights into the nature of structural transitions induced by external stimuli, thus contributing to the understanding of the connection of microscopic structures with macroscopicproperties.

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.

Supramolecular Assembly and Thermogelation Strategies Using Peptide-Polymer Conjugates.

Peptide-polymer conjugates (PPCs) are of particular interest in the development of responsive, adaptive, and interactive materials due to the benefits offered by combining both building blocks and components. This review presents pioneering work as well as recent advances in the design of peptide-polymer conjugates, with a specific focus on their thermoresponsive behavior. This unique class of materials has shown great promise in the development of supramolecular structures with physicochemical properties that are modulated using soft and biorthogonal external stimuli. The temperature-induced self-assembly of PPCs into various supramolecular architectures, gelation processes, and tuning of accessible processing parameters to biologically relevant temperature windows are described. The discussion covers the chemical design of the conjugates, the supramolecular driving forces involved, and the mutual influence of the polymer and peptide segments. Additionally, some selected examples for potential biomedical applications of thermoresponsive PPCs in tissue engineering, delivery systems, tumor therapy, and biosensing are highlighted, as well as perspectives on future challenges.

Project RACCOON: Automated construction of PDB files for polymers and polymer peptide conjugates

Project RACCOON: Automated construction of PDB files for polymers and polymer peptide conjugates

Precise Engineering of Growth Factor Presentation Using Extracellular Microenvironment-Mimicking Microfluidic Microparticles.

One of the main challenges in tissue engineering is finding a way to deliver specific growth factors (GFs) with precise spatiotemporal control over their presentation. Here, we report a novel strategy for generating microscale carriers with enhanced affinity for high content loading suitable for the sustained and localized delivery of GFs. Our developed microparticles can be injected locally and sustainably release encapsulated growth factors for up to 28 days. Fine-tuning of particles' size, affinity, microstructures, and release kinetics is achieved using a microfluidic system along with bioconjugation techniques. We also describe an innovative 3D micromixer platform to control the formation of core-shell particles based on superaffinity using a polymer-peptide conjugate for further tuning of release kinetics and delayed degradation. Chitosan shells block the burst release of encapsulated GFs and enable their sustained delivery for up to 10 days. The matched release profiles and degradation provide the local tissues with biomimetic, developmental-biologic-compatible signals to maximize regenerative effects. The versatility of this approach is verified using three different therapeutic proteins, including human bone morphogenetic protein-2 (rhBMP-2), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1α). As in vivo morphogenesis is typically driven by the combined action of several growth factors, the proposed technique can be developed to generate a library of GF-loaded particles with designated release profiles.

Modular design of cyclic peptide – polymer conjugate nanotubes for delivery and tunable release of anti-cancer drug compounds

High aspect-ratio nanomaterials have recently emerged as promising drug delivery vehicles due to evidence of strong cellular association and prolonged in vivo circulation times. Cyclic peptide - polymer conjugate nanotubes are excellent candidates due to their elongated morphology, their supramolecular composition and high degree of pliability due to the versatility in manipulating amino acid sequence and polymer type. In this work, we explore the use of a nanotube structure on which a potent anti-cancer drug, camptothecin, is attached alongside hydrophilic or amphiphilic RAFT polymers, which shield the cargo. We show that subtle modifications to the cleavable linker type and polymer architecture have a dramatic influence over the rate of drug release in biological conditions. In vitro studies revealed that multiple cancer cell lines in 2D and 3D models responded effectively to the nanotube treatment, and analogous fluorescently labelled materials revealed key mechanistic information regarding the degree of cellular uptake and intracellular fate. Importantly, the ability to instruct specific drug release profiles indicates a potential for these nanomaterials as vectors which can provide sustained drug concentrations for a maximal therapeutic effect.

Oral Environment-Adaptive Peptide–Polymer Conjugate for Caries Prevention with Targeting, Antibacterial, and Antifouling Abilities

Oral Environment-Adaptive Peptide–Polymer Conjugate for Caries Prevention with Targeting, Antibacterial, and Antifouling Abilities

Spectroscopic characterization of europium binding to a calmodulin-EF4 hand peptide-polymer conjugate.

The emergence of biological ligand as an alternative to chemical ligands enables a sustainable lanthanide extraction route. In this study, a peptide originating from the loop of domain 4 calmodulin (EF4) was synthesized and the interaction with europium ions was monitored using time resolved laser fluorescence spectroscopy (TRLFS). Despite being retracted from its full protein structure, the twelve amino acids of calmodulin-EF4 showed binding to europium. Europium-peptide complex formation was evident by an increase in decay time from 110 to 187 μs. The spectra of europium bound to peptide can be easily distinguished from the free europium ion as the 5D0 → 7F2 peak intensifies. When europium bound to the peptide-polymer conjugate, the decay time was further increased to 259 μs. This suggests that lanthanide binding can be enhanced by immobilizing the short peptide into a polymer matrix. The europium-peptide/conjugate bond was reversible, triggered by pH, promoting peptide reusability. Due to the fact that the study was conducted exclusively in water, it suggests minimal use of chemicals is possible while maintaining peptide affinity. This makes the calmodulin-EF4 peptide an ideal candidate as biological ligand. This study lays the groundwork for developing a peptide-based filter material for lanthanide separation.

Fluorophore-Tagged Poly-Lysine RAFT Agents: Controlled Synthesis of Trackable Cell-Penetrating Peptide-Polymers.

The conjugation of a fluorophore and a variety of cell-penetrating peptides onto a RAFT agent allowed for the synthesis of polymers of defined sizes with quantifiable cell-uptake. Each peptide-RAFT agent was used to polymerize acrylamide, acrylate, and styrene monomers to form high or low molecular weight polymers (here 50 or 7.5 kDa) with the peptide having no influence on the RAFT agent's control. The incorporation of a single fluorophore per polymer chain allowed cellular analysis of the uptake of the size-specific peptide-polymers via flow cytometry and confocal microscopy. The cell-penetrating peptides had a direct effect on the efficiency of polymer uptake for both high and low molecular weight polymers, demonstrating the versatility of the strategy. These "all-in-one", synthetically accessible RAFT agents allow highly controlled preparation of synthetic peptide-polymer conjugates and subsequent quantification of their delivery into cells.

Investigating the Impact of Hydrophobic Polymer Segments on the Self-Assembly Behavior of Supramolecular Cyclic Peptide Systems via Asymmetric-Flow Field Flow Fractionation.

The present study examines the behavior of cyclic peptide polymer conjugates that have been designed to combine their self-assembling ability via H-bonding with the properties of amphiphilic diblock copolymers. Using a combination of asymmetric flow-field flow fractionation (AF4) and small-angle neutron scattering (SANS), we have uncovered unique insight based on the population of structures established at a 24 h equilibrium profile. Our results determine that by introducing a small quantity of hydrophobicity into the conjugated polymer corona, the resulting nanotube structures exhibit low unimer dissociation which signifies enhanced stability. Furthermore, as the hydrophobicity of the polymer corona is increased, the elongation of the nanotubes is observed due to an increase in the association of unimers. This encompasses not only the H-bonding of unimers into nanotubes but also the self-assembly of single nanotubes into segmented-nanotube structures with high aspect ratios. However, this influence relies on a subtle balance between the hydrophobicity and hydrophilicity of the polymer corona. This balance is proposed to determine the solvent entropic penalty of hydrating the system, whereby the cost scales with the hydrophobic quantity. Consequently, it has been suggested that at a critical hydrophobic quantity, the solvation penalty becomes high enough such that the self-assembly of the system deviates from ordered hydrogen bonding. The association behavior is instead dominated by the hydrophobic effect which results in the undesirable formation of disordered aggregates.

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.

Characterizing properties of scaffolds 3D printed with peptide-polymer conjugates

Three-dimensional (3D) printing is a popular biomaterials fabrication technique because it enables scaffold composition and architecture to be tuned for different applications. Modifying these properties can also alter mechanical properties, making it challenging to decouple biochemical and physical properties. In this study, inks containing peptide-poly(caprolactone) (PCL) conjugates were solvent-cast 3D printed to create peptide-functionalized scaffolds. We characterized how different concentrations of hyaluronic acid-binding (HAbind-PCL) or mineralizing (E3-PCL) conjugates influenced properties of the resulting 3D-printed constructs. The peptide sequences CGGGRYPISRPRKR (HAbind-PCL; positively charged) and CGGGAAAEEE (E3-PCL; negatively charged) enabled us to evaluate how conjugate chemistry, charge, and concentration affected 3D-printed architecture, conjugate location, and mechanical properties. For both HAbind-PCL and E3-PCL, conjugate addition did not affect ink viscosity, filament diameter, scaffold architecture, or scaffold compressive modulus. Increasing conjugate concentration in the ink prior to printing correlated with an increase in peptide concentration on the scaffold surface. Interestingly, conjugate type affected final conjugate location within the 3D-printed filament cross-section. HAbind-PCL conjugates remained within the filament bulk while E3-PCL conjugates were located closer to the filament surface. E3-PCL at all concentrations did not affect mechanical properties, but an intermediate HAbind-PCL concentration resulted in a moderate decrease in filament tensile modulus. These data suggest final conjugate location within the filament bulk may influence mechanical properties. However, no significant differences were observed between PCL filaments printed without conjugates and filaments printed with higher HAbind-PCL concentrations. These results demonstrate that this 3D printing platform can be used to functionalize the surface without significant changes to the physical properties of the scaffold. The downstream potential of this strategy will enable decoupling of biochemical and physical properties to fine-tune cellular responses and support functional tissue regeneration.

A self-assembled dynamic extracellular matrix-like hydrogel system with multi-scale structures for cell bioengineering applications

Extracellular matrix (ECM) provides various types of direct interactions with cells and a dynamic environment, which can be remodeled through different assembly/degradation mechanisms to adapt to different biological processes. Herein, through introducing polyphosphate-modified hyaluronic acid and bioactive glass (BG) nano-fibril into a self-assembled hydrogel system with peptide-polymer conjugate, we can realize many new ECM-like functions in a synthetic polymer network. The hydrogel network formation is mediated by coacervation, followed by a gradual transition of peptide structure from α-helix to β-sheet. The ECM-like hydrogels can be degraded through a number of orthogonal mechanisms, including treatments with protease, hyaluronidase, alkaline phosphatase, and calcium ion. As 2D coating, the ECM-like hydrogels can be used to modify the planar surface to promote the adhesion of mesenchymal stromal cells, or to coat the cell surface in a layer-by-layer fashion to shield the interaction with the substrate. As ECM-like hydrogels for 3D cell culture, the system is compatible with injection and cell encapsulation. Upon incorporating fragmented electrospun bioactive glass nano-fibril into the hydrogels, the synergetic effects of soft hydrogel and stiff reinforcement nanofibers on recapitulating ECM functions result in reduced cell circularity in 3D. Finally, by injecting the ECM-like hydrogels into mice, gradual degradations over a time period of one month and high biocompatibility have been shown in vivo. The contribution of complex network dynamics and hierarchical structures to cell-biomatrix interaction can be investigated multi-dimensionally, as many mechanisms are orthogonal to each other and can be regulated individually. STATEMENT OF SIGNIFICANCE: A list of native ECM features has attracted the most interest and attention in the research of synthetic biomaterials. In this research, we have described a simple ECM-like hydrogel system in which the complex and elegant functions of native ECM can be recapitulated in a chemically defined synthetic system. The ECM-like hydrogel systems were developed to avoid undesired features of biological substances (e.g., ethical concerns, batch-to-batch variation, immunogenicity, and potential risk of contamination), as well as gaining new functions to facilitate bioengineering applications (e.g., 3D cell culture, injection, and high stability). To this end, we have developed an ECM-like hydrogel system and provide evidence that this purely synthetic biomaterial is a promising candidate for cell bioengineering applications.

Monomer Choice Influences N-Acryloyl Amino Acid Grafter Conversion via Protease Catalysis.

End-capped peptides modified with reactive functional groups on the N-terminus provide a route to prepare peptide-polymer conjugates for a broad range of applications. Unfortunately, current chemical methods to construct modified peptides rely largely on solid-phase peptide synthesis (SPPS), which lacks green preparative characteristics and is costly, thus limiting its applicability to specialty applications such as regenerative medicine. This work evaluates N-terminally modified N-acryloyl-glutamic acid diethyl ester, N-acryloyl-leucine ethyl ester, and N-acryloyl-alanine ethyl ester as grafters and papain as the protease for the direct addition of amino acid ethyl ester (AA-OEt) monomers via protease-catalyzed peptide synthesis (PCPS) and the corresponding formation of N-acryloyl-functionalized oligopeptides in a one-pot aqueous reaction. It was hypothesized that by building N-acryloyl grafters from AA-OEt monomers that are known to be good substrates for papain in PCPS, the corresponding grafters would yield high grafter conversions, high ratio of grafter-oligopeptide to free NH2-oligopeptide, and high overall yield. However, this work demonstrates based on the grafter/monomers studied herein that the dominant factor in N-acryloyl-AA-OEt grafter conversion is the co-monomer used in co-oligomerizations. Computational modeling using Rosetta qualitatively recapitulates the results and provides insight into the structural and energetic bases underlying substrate selectivity. The findings herein expand our knowledge of factors that determine the efficiency of preparing N-acryloyl-terminated oligopeptides by PCPS that could provide practical routes to peptide macromers for conjugation to polymers and surfaces for a broad range of applications.

A bispecific peptide-polymer conjugate bridging target-effector cells to enhance immunotherapy.

Peptide-based immune checkpoint inhibitors exhibited remarkable therapeutic benefits although their application was hindered by quick blood clearance and low affinity with receptors. The modification of the peptides into artificial antibodies was an ideal platform to solve these problems, and one of the optional pathways was the conjugation of peptides with a polymer. More importantly, the bridging effect, mediated by bispecific artificial antibodies, could promote the interaction of cancer cells and T cells, which will benefit cancer immunotherapy. Herein, we prepared a bispecific peptide-polymer conjugate (octa PEG-PD1-PDL1) by simultaneously conjugating PD1-binding and PDL1-binding peptides onto 8-arm-PEG. octa PEG-PD1-PDL1 bridged T cells and cancer cells and thus enhanced T cell-mediated cytotoxicity against cancer cells. Meanwhile, the tumor-targeting octa PEG-PD1-PDL1 increased the infiltration of cytotoxic T lymphocytes in tumors and reduced their exhaustion. It effectively activated the tumor immune microenvironment and exerted a potent antitumor effect against CT26 tumor models with a tumor inhibition rate of 88.9%. This work provided a novel strategy to enhance tumor immunotherapy through conjugating bispecific peptides onto a hyperbranched polymer to effectively engage target-effector cells. This article is protected by copyright. All rights reserved.

Biophysical and molecular characterization of the immobilization of biomimetic peptide-polymer conjugates as building blocks for antimicrobial catheter surfaces to prevent infectious diseases.

Catheter-associated urinary tract infections (CAUTIs) are the most common type of healthcare-associated infection. The World Health Organization recently reported that patients with a catheter had a 5% greater probability of acquiring a urinary tract infection per day of being catheterized and in a month this probability increased up to a 100%. This work explores the catheter surface immobilization and characterization of the MH5C modified with acysteine (MH5C-Cys) and coupled to polyethylene glycol to serve as a surface protective coating. First, purification method by size exclusion chromatography (SEC) was performed followed by analytical SEC and SDS-PAGE and MALDI-ToF to confirm the reaction. Also, we immobilized on the catheter surface the MH5C-Cys-PEG and it was confirmed by the appearance of the characteristic peaks of 3,264 cm−1for N-H stretching, 1,532 cm−1for N-H bending and 1,157 cm−1for C-N stretching at the FTIR spectrum of the catheter with the immobilized peptide. Additionally, for this study, we have performed a Circular Dichroism (CD) technique for evaluating the secondary structure of the peptide-polymer conjugates. The CD spectra illustrated that conjugates possessed α-helical structures. Subsequently, we evaluated the efficacy of MH5C-Cys-PEG conjugate via scanning electron microscopy, minimum biofilm inhibition concentration (MBIC), assays to evaluate their ability to prevent the biofilms. Interestingly, this work demonstrated a critical PEG polymer weight of 5 kDa as ideal when coupled to the MH5C-Cysto achieve inhibition and eradication of the biofilm formation in Escherichia coli. According to theMBICs (300 μM), we can conclude that MH5C-Cys-PEG has an action that prevents the formation of biofilms. This type of study serves to improve progress toward new methods to counteract antimicrobial resistance of many of the bacteria known to cause CAUTIs.

Peptides-tethered vascular grafts enable blood vessel regeneration via endogenous cell recruitment and neovascularization

Cardiovascular injuries cause huge morbidity and mortality worldwide. Arterial reconstructions are generally performed either by using native grafts or synthetic grafts, both of which are limited by several complications. Synthetic biodegradable polymers offer a promising platform, which may also be modified to foster in situ tissue regeneration through the recruitment of host cells. Vascular endothelial growth factor (VEGF) promotes endothelialization and neovascularization in vascular grafts, however, an overdose of VEGF may induce tumor-like vasculature, which requires alternative strategies. The objective of this study was to exploit prominin-1-derived VEGF-binding peptide (BP) to improve neovascularization and endothelialization, while stromal cell-derived factor 1-alpha (SDF-1α) peptide to encourage endogenous stem/progenitor cells mobilization and complement BP-mediated vascular remodeling. The BP and SDF-1α peptides were covalently conjugated with low molecular weight poly (ε-caprolactone) (LPCL) to afford LPCL-BP and LPCL-SDF-1α, respectively. Chemical analysis revealed successful modification of LPCL with peptides, which also displayed good cytocompatibility in vitro once blended along with high molecular weight PCL (HPCL). The bioactived vascular grafts were fabricated by blending LPCL-BP, LPCL-SDF-1α or dual peptide-polymer conjugates with HPCL. The in vivo tests of vascular grafts through rat abdominal aorta implantation model revealed that, compared with HPCL grafts, the dual peptides modified grafts exhibited superior patency and tissue regeneration at 4-week post-implantation, including stem cell recruitment, rapid endothelialization and functional SMC layer formation. Taken together, these results may have implications for the in situ regeneration of artificial blood vessels through the orchestration of host's responses and endogenous cell recruitment.

Peptide–Polymer Conjugates: A Promising Therapeutic Solution for Drug-Resistant Bacteria

By 2050, it is estimated that 10 million people will die of drug-resistant bacterial infection caused by antibiotic abuse. Antimicrobial peptide (AMP) is widely used to prevent such circumstances, for the positively charged AMPs can kill drug-resistant bacteria by destroying negatively charged bacterial cell membrane, and has excellent antibacterial efficiency and low drug resistance. However, due to the defects in low in vivo stability, easy degradation, and certain cytotoxicity, its practical clinical application is limited. The emergence of peptide–polymer conjugates (PPC) helps AMPs overcome these shortcomings. By combining with functional polymers, the positive charge of AMPs is partially shielded, and its stability and water solubility are improved, so as to prolong the in vivo circulation time of AMPs and reduce its cytotoxicity. At the same time, the self-assembly ability of PPC enables it to assemble into different nanostructures to undertake specific antibacterial tasks. At present, PPC is mainly used in wound dressing, bone tissue repair, antibacterial coating of medical devices, nerve repair, tumor treatment, and oral health maintenance. In this study, we summarize the structure, synthesis methods, and the clinical applications of PPC, so as to present the current challenges and discuss the future prospects of antibacterial therapeutic materials.

Bioengineered Carboxymethylcellulose-Peptide Hybrid Nanozyme Cascade for Targeted Intracellular Biocatalytic-Magnetothermal Therapy of Brain Cancer Cells.

Glioblastoma remains the most lethal form of brain cancer, where hybrid nanomaterials biofunctionalized with polysaccharide peptides offer disruptive strategies relying on passive/active targeting and multimodal therapy for killing cancer cells. Thus, in this research, we report for the first time the rational design and synthesis of novel hybrid colloidal nanostructures composed of gold nanoparticles stabilized by trisodium citrate (AuNP@TSC) as the oxidase-like nanozyme, coupled with cobalt-doped superparamagnetic iron oxide nanoparticles stabilized by carboxymethylcellulose ligands (Co-MION@CMC) as the peroxidase-like nanozyme. They formed inorganic–inorganic dual-nanozyme systems functionalized by a carboxymethylcellulose biopolymer organic shell, which can trigger a biocatalytic cascade reaction in the cancer tumor microenvironment for the combination of magnetothermal–chemodynamic therapy. These nanoassemblies were produced through a green aqueous process under mild conditions and chemically biofunctionalized with integrin-targeting peptide (iRDG), creating bioengineered nanocarriers. The results demonstrated that the oxidase-like nanozyme (AuNP) was produced with a crystalline face-centered cubic nanostructure, spherical morphology (diameter = 16 ± 3 nm), zeta potential (ZP) of −50 ± 5 mV, and hydrodynamic diameter (DH) of 15 ± 1 nm. The peroxide-like nanostructure (POD, Co-MION@CMC) contained an inorganic crystalline core of magnetite and had a uniform spherical shape (2R = 7 ± 1 nm) which, summed to the contribution of the CMC shell, rendered a hydrodynamic diameter of 45 ± 4 nm and a negative surface charge (ZP = −41 ± 5 mV). Upon coupling both nanozymes, water-dispersible colloidal supramolecular vesicle-like organic–inorganic nanostructures were produced (AuNP//Co-MION@CMC, ZP = −45 ± 4 mV and DH = 28 ± 3 nm). They confirmed dual-nanozyme cascade biocatalytic activity targeted by polymer–peptide conjugates (AuNP//Co-MION@CMC_iRGD, ZP = −29 ± 3 mV and DH = 60 ± 4 nm) to kill brain cancer cells (i.e., bioenergy “starvation” by glucose deprivation and oxidative stress through reactive oxygen species generation), which was boosted by the magneto-hyperthermotherapy effect when submitted to the alternating magnetic field (i.e., induced local thermal stress by “nanoheaters”). This groundwork offers a wide avenue of opportunities to develop innovative theranostic nanoplatforms with multiple integrated functionalities for fighting cancer and reducing the harsh side effects of conventional chemotherapy.

Reproducible and controlled peptide functionalization of polymeric nanoparticles

Polymeric nanoparticles containing multiple amines and carboxylates have been frequently used in drug delivery research. Reproducible and controlled conjugation among these multifunctional biomaterials is necessary to achieve efficient drug delivery platforms. However, multiple functional groups increase the risk of unintended intramolecular/intermolecular reactions during conjugation. Herein, conjugation approaches and possible undesired reactions between multi-amine functionalized peptides, multi-carboxylate functionalized polymers, and anhydride-containing polymers [Poly(styrene-alt-maleic anhydride)-b-poly(styrene)] were investigated under different conjugation strategies (carbodiimide chemistry, anhydride ring-opening via nucleophilic addition elimination). Muti-amine peptides led to extensive crosslinking between polymers regardless of the conjugation chemistry. Results also indicate that conventional peptide quantification methods (i.e., o-phthalaldehyde assay, bicinchoninic acid assay) are unreliable. Gel permeation chromatography (GPC) provided more accurate qualitative and quantitative evidence for intermolecular crosslinking. Crosslinking densities were correlated with higher feed ratios of multifunctional peptides and carbodiimide coupling reagents. Selectively protected peptides (Lys-Alloc) exhibited no crosslinking and yielded peptide-polymer conjugates with controlled dispersity and molecular weight. Furthermore, anhydride ring-opening (ARO) nucleophilic addition elimination was successfully introduced as a facile yet robust peptide conjugation approach for cyclic anhydride-containing polymers.