Protein immobilization and modification are widely used techniques in the fields of chemical biology and biomaterials science. While covalent strategies based on small molecules are traditionally used, supramolecular chemistry offers numerous useful opportunities for guiding the modification locations on complex protein landscapes and introducing different degrees of reversibility into the products. In this opinion, we highlight recent advances in using supramolecular interactions, particularly host-guest chemistry, for controlling protein modification and immobilization. We discuss supramolecular strategies for protein-conjugate purification and capture, as well as for protein modification via host-guest interactions and metal coordination. Lastly, we address recent advances in utilizing supramolecular interactions to direct covalent protein modification. These examples of supramolecular chemical biology present opportunities to advance a wide range of applications, including proteomics and drug delivery.

Injectable colloids that self-assemble into three-dimensional networks are promising materials for applications in regenerative engineering, as they create open systems for cellular infiltration, interaction, and activation. However, most injectable colloids have spherical morphologies, which lack the high material-biology contact areas afforded by higher aspect ratio materials. To address this need, injectable high aspect ratio particles (HARPs) were developed that form three-dimensional networks to enhance scaffold assembly dynamics and cellular interactions. HARPs were functionalized for tunable surface charge through layer-by-layer electrostatic assembly. Positively charged Chitosan-HARPs had improved particle suspension dynamics when compared to spherical particles or negatively charged HARPs. Chit-HARPs were used to improve the suspension dynamics and viability of MIN6 cells in three-dimensional networks. When combined with negatively charged gelatin microsphere (GelMS) porogens, Chit-HARPs reduced GelMS sedimentation and increased overall network suspension, due to a combination of HARP network formation and electrostatic interactions. Lastly, HARPs were functionalized with fibroblast growth factor 2 (FGF2) to highlight their use for growth factor delivery. FGF2-HARPs increased fibroblast proliferation through a combination of 3D scaffold assembly and growth factor delivery. Taken together, these studies demonstrate the development and diverse uses of high aspect ratio particles as tunable injectable scaffolds for applications in regenerative engineering.

Bacterial biofilm infections, particularly those of Pseudomonas aeruginosa (PA), have high rates of antimicrobial tolerance and are commonly found in chronic wound and cystic fibrosis lung infections. Combination therapeutics that act synergistically can overcome antimicrobial tolerance; however, the delivery of multiple therapeutics at relevant dosages remains a challenge. We therefore developed a nanoscale drug carrier for antimicrobial codelivery by combining approaches from polyelectrolyte nanocomplex (NC) formation and layer-by-layer electrostatic self-assembly. This strategy led to NC drug carriers loaded with tobramycin antibiotics and antimicrobial silver nanoparticles (AgTob-NCs). AgTob-NCs displayed synergistic enhancements in antimicrobial activity against both planktonic and biofilm PA cultures, with positively charged NCs outperforming negatively charged formulations. NCs were evaluated in mouse models of lung infection, leading to reduced bacterial burden and improved survival outcomes. This approach therefore shows promise for nanoscale therapeutic codelivery to treat recalcitrant bacterial infections.

Neutrophil extracellular traps (NETs) are networks of decondensed chromatin, histones, and antimicrobial proteins released by neutrophils in response to an infection. NET overproduction can cause an exacerbated hyperinflammatory response in a variety of diseases and can lead to host tissue damage without clearance of infection. Nanoparticle drug delivery is a promising avenue for creating materials that can both target NETs and deliver sustained amounts of NET-degrading drugs to alleviate hyperinflammation. Here, we study how particle physicochemical properties can influence NET interaction and leverage our findings to create NET-interfacing and NET-degrading particles. We fabricated a panel of particles of varying sizes (200 to 1000 nm) and charges (positive, neutral, negative) and found that positive charge is the main driver of NET-particle interaction, with smaller 200 nm positive particles having a 10-fold increase in binding compared to larger 1000 nm positive particles. Negative and neutral particles were mostly noninteracting, except for small negatively charged particles that exhibited very low levels of NET localization. Interaction strength of particles with NETs was quantified via shear flow assays and atomic force microscopy. This information was leveraged to create DNase-loaded particles that could adhere to NETs at varying degrees and therefore degrade NETs at different rates in vitro. Positively charged, 200 nm DNase-loaded particles showed the highest degree of interaction with NETs and therefore led to faster degradation compared with larger sizes, underscoring the importance of physicochemical design for NET-targeting drug delivery. Overall, this work provides fundamental knowledge of the drivers of particle-NET interaction and a basis for designing NET-targeting particles for various disease states.

We have synthesized targeted, selective, and highly sensitive (129)Xe NMR nanoscale biosensors using a spherical MS2 viral capsid, Cryptophane A molecules, and DNA aptamers. The biosensors showed strong binding specificity toward targeted lymphoma cells (Ramos line). Hyperpolarized (129)Xe NMR signal contrast and hyper-CEST (129)Xe MRI image contrast indicated its promise as highly sensitive hyperpolarized (129)Xe NMR nanoscale biosensor for future applications in cancer detection in vivo.

We report a method for blocking interactions between (129)Xe and cucurbit[6]uril (CB6) until activation by a specific chemical event. We synthesized a CB6-rotaxane that allowed no (129)Xe interaction with the CB6 macrocycle component until a cleavage event released the CB6, which then produced a (129)Xe@CB6 NMR signal. This contrast-upon-activation (129)Xe NMR platform allows for modular synthesis and can be expanded to applications in detection and disease imaging.

We report a CB6 rotaxane for the ¹²⁹Xe hyperCEST NMR detection of matrix metalloprotease 2 (MMP-2) activity. MMP-2 is overexpressed in cancer tissue, and hence is a cancer marker. A peptide containing an MMP-2 recognition sequence was incorporated into the rotaxane, synthesized via CB6-promoted click chemistry. Upon cleavage of the rotaxane by MMP-2, CB6 became accessible for ¹²⁹Xe@CB6 interactions, leading to protease-responsive hyperCEST activation.

The development of sensitive and chemically selective MRI contrast agents is imperative for the early detection and diagnosis of many diseases. Conventional responsive contrast agents used in ¹ H MRI are impaired by the high abundance of protons in the body. ¹²⁹ Xe hyperCEST NMR/MRI comprises a highly sensitive complement to traditional ¹ H MRI because of its ability to report specific chemical environments. To date, the scope of responsive ¹²⁹ Xe NMR contrast agents lacks breadth in the specific detection of small molecules, which are often important markers of disease. Herein, we report the synthesis and characterization of a rotaxane-based ¹²⁹ Xe hyperCEST NMR contrast agent that can be turned on in response to H₂ O₂ , which is upregulated in several disease states. Added H₂ O₂ was detected by ¹²⁹ Xe hyperCEST NMR spectroscopy in the low micromolar range, as well as H₂ O₂ produced by HEK 293T cells activated with tumor necrosis factor.

Glioblastoma is a particularly challenging cancer, as there are currently limited options for treatment. New delivery routes are being explored, including direct intratumoral injection via convection-enhanced delivery (CED). While promising, convection-enhanced delivery of traditional chemotherapeutics such as doxorubicin (DOX) has seen limited success. Several studies have demonstrated that attaching a drug to polymeric nanoscale materials can improve drug delivery efficacy via CED. We therefore set out to evaluate a panel of morphologically distinct protein nanoparticles for their potential as CED drug delivery vehicles for glioblastoma treatment. The panel consisted of three different virus-like particles (VLPs), MS2 spheres, tobacco mosaic virus (TMV) disks and nanophage filamentous rods modified with DOX. While all three VLPs displayed adequate drug delivery and cell uptake in vitro, increased survival rates were only observed for glioma-bearing mice that were treated via CED with TMV disks and MS2 spheres conjugated to doxorubicin, with TMV-treated mice showing the best response. Importantly, these improved survival rates were observed after only a single VLP⁻DOX CED injection several orders of magnitude smaller than traditional IV doses. Overall, this study underscores the potential of nanoscale chemotherapeutic CED using virus-like particles and illustrates the need for further studies into how the overall morphology of VLPs influences their drug delivery properties.