RNA plays a myriad of roles in the body including the coding, decoding, regulation, and expression of genes. RNA oligonucleotides have garnered significant interest as therapeutics via antisense oligonucleotides or small interfering RNA strategies for the treatment of diseases ranging from hyperlipidemia, HCV, and others. Additionally, the recently developed CRISPR-Cas9 mediated gene editing strategy also relies on Cas9-associated RNA strands. However, RNA presents numerous challenges as both a synthetic target and a potential therapeutic. RNA is inherently unstable, difficult to deliver into cells, and potentially immunogenic by itself or upon modification. Despite these challenges, with the help of chemically modified oligonucleotides, multiple RNA-based drugs have been approved by the FDA. The progress is made possible due to the nature of chemically modified oligonucleotides bearing advantages of nuclease stability, stronger binding affinity, and some other unique properties. This review will focus on the chemical synthesis of RNA and its modified versions. How chemical modifications of the ribose units and of the phosphatediester backbone address the inherent issues with using native RNA for biological applications will be discussed along the way.

Nucleic acid therapy is and will continue to be of great interest in cancer treatment. The development of nanocarriers with high nucleic acid loading capacity, low toxicity, and specific targeting, with excellent pharmacokinetic/pharmacodynamic profiles, will enable us to realize safe and effective nucleic acid therapy. Tremendous efforts have been directed toward the production of optimized theranostic nanocarriers that can simultaneously provide treatment and real-time monitoring to aid researchers and physicians in making adaptation strategies during early drug development and patient’s treatment, respectively. In this review, several nanomaterials with inherent optical and magnetic properties, developed for bioimaging and imaging-guided nucleic acid therapies, are introduced and discussed. In each subsection, the unique characteristics of the corresponding theranostic nanomaterials are reviewed and discussed with examples. Finally, we present the remaining challenges that must be addressed and provide our opinions on the future of nanomaterial medicines for bioimaging and imaging-guided nucleic acid therapy.

As a classical nanocatalyst-based therapeutic modality, chemodynamic therapy (CDT) has received more and more attention. To improve the therapeutic efficacy of CDT, various metal-based nanocatalysts have been designed and constructed to catalyze the Fenton or Fenton-like reaction in the past few years. However, the therapeutic efficacy of certain CDT is still restricted by the tumor microenvironment, such as limited concentration of intracellular H₂O₂, inappropriate pH condition, as well as overexpressed glutathione (GSH). Therefore, many other therapeutic modalities, such as photodynamic therapy (PDT), photothermal therapy (PTT), starvation therapy, chemotherapy, and gas therapy, have been utilized to combine with CDT for increasing the tumor treatment performance. In this review, we summarized the development of combinatory therapeutic modalities based on CDT in recent years.

Molecular chaperones play critical roles in biological functions. They are closely involved in the maintenance of cell homeostasis, proper folding of proteins and nucleic acids, and inhibition of irreversible aggregation in denatured proteins. In addition to protein production, molecular chaperone function is widely recognized as important for peptide and protein drug delivery systems. Therefore, much effort has been made in recent decades to develop chaperone-mimetic molecules that have similar structures and biological functions to natural chaperones. These artificial molecular chaperone systems have been demonstrated to facilitate proper protein and nucleic acid folding, in addition to the formation of higher-order structures of synthetic molecules. Furthermore, the functions of these artificial systems show promising clinical applications in drug delivery and biomolecule detection. This topical review focuses on recent advances in the design, construction, characterization, and potential applications of different artificial molecular systems with distinct functional roles, such as the folding of water-soluble and membrane proteins, nucleic acids, and the self-assembly of synthetic molecules. Strategies used in the construction of some artificial molecule chaperone systems for proteins (such as pairs of amphiphilic molecules or self-assembled nanogels) and their applications as biomaterials are described. Specific examples from each design strategy are also highlighted to demonstrate the mechanisms, challenges, and limitations of the different artificial molecular systems. By highlighting the many new developments that have expanded the applications of the artificial chaperones beyond protein folding, this review aims to stimulate further studies on their design and applications.

The tumor microenvironment significantly influences cancer progression and therapeutic response. Reprogramming of tumor microenvironment has emerged as a strategy to assist conventional cancer treatment. In recent years, photothermal therapy has received considerable attention owing to its noninvasiveness, high temporal–spatial resolution, and minimal drug resistance. Apart from ablating cancer cells by generating heat upon light irradiation, photothermal therapy can also affect the tumor microenvironment, such as disrupting the tumor extracellular matrix and tumor vasculature. Moreover, cancer cell death by hyperthermia could potentially activate the immune system to fight against tumor. In this topical review, we focus on the recent progress of photothermal therapy based on tumor microenvironment remodeling, aiming to better guide the design of nanoparticles for cancer photoimmunotherapy.

The conjugation of metal-based scaffolds to peptides, proteins, or antibodies allows the systemic targeting of these payloads to specific locations in the body, such as target cells/tissues (e.g., cancer) and subcellular compartments, for either therapy or imaging. This Topical Review includes an overview of the available chemical strategies to achieve metal-peptidic bioconjugates for biomedical applications, focusing on the types of chemical functionalities used to tether the drug to the peptide directly or indirectly. Central to all the possible approaches is the development of highly efficient and selective bioconjugation reactions that operate under mild, peptide-compatible conditions. For each strategy, selected examples are highlighted with particular emphasis to the studies reporting the therapeutic effects of the metal-peptidic conjugates in the treatment of cancer. Overall, some of the herewith discussed cases clearly hold promise for translation into clinically meaningful applications in the field of targeted therapeutics. Nevertheless, novel chemical approaches enabling the chemoselective metalation of specific residues in peptides under biologically friendly conditions, as well as the design of stimuli-responsive bioconjugates, are still expected to emerge. Certainly, the peculiar biorthogonal reactivity of metallodrugs provides an enlarged toolbox of opportunities for bioconjugation. Therefore, we outline a number of possible future directions and applications.

Bispecific aptamer–drug conjugates (BsApDC) may improve the efficacy of drugs by enhancing cellular internalization and targeted delivery. Nevertheless, the synthesis of single-molecular BsApDC has not yet been reported, and it could be thwarted by synthetic challenges. Herein we report a general approach to synthesize a BsApDC hybridized chemical and biological method. Primers incorporated with 5-Fluorouracil (5-FU), 10-Hydroxycamptothecin, and Maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethyl auristatin E(vcMMAE) were prepared by chemical synthesis, which were converted to corresponding ApDCs efficiently by enzymatic reaction. Biological studies revealed that BsApDC binds with target cells with enhanced internalization and better inhibitory activity, demonstrating the potential of BsApDCs for targeted tumor therapy.

Chemically modified antigen-binding proteins are widely applied for their targeting abilities in the fields of biotechnology, medicine, and diagnostics. However, the production of site-selectively modified proteins remains a challenge. Here, we have designed a chemical probe for the introduction of a reactive aldehyde on nanobodies by metal-complex-guided conjugation. The probe design allows for purification of the conjugates, and the aldehyde constitutes an efficient handle for further modification of the nanobodies. In vitro experiments confirmed the binding activity and selectivity of fluorescent conjugates toward the native antigen. Furthermore, the modification strategy allowed for production of a nanobody–drug conjugate that was active in vitro.

Herein, conjugation of the amyloid-β (Aβ) peptide fragment, Lys-Leu-Val-Phe-Phe (KLVFF, fragment of Aβ_16–20), with an unsymmetrical near-infrared (NIR) cyanine-5 (Cy-5) chromophore is achieved using microwave-assisted solid phase synthesis on 2-chlorotrityl chloride resin. Selective mitochondria tracking and staining in human carcinoma cells are accomplished by the KLVFF/Cy-5 conjugate containing triphenylphosphonium functionality, and this is compared to a control molecule KLVFF/Cy-5c. Mitochondrial target specificity of KLVFF/Cy-5 is established by the colocalization assay using mitochondria selective probe MitoTracker Red, which is monitored by confocal laser scanning microscope and shows a high Pearson’s correlation coefficient. The KLVFF/Cy-5 conjugate has high photostability, NIR absorption/emission, high molar extinction coefficient, narrow absorption/emission band, high fluorescence lifetime, and high fluorescence quantum yield. Moreover, mitochondria targeting KLVFF/Cy-5 conjugate reaches the critical aggregation concentration inside the mitochondria of cancer cells due to the strong negative inner mitochondrial membrane potential [(ΔΨ_m)_cancer −220 mV] and self-assembles to form amyloid fibrils at the target site, which is responsible for the mitochondrial dysfunction and cytotoxicity. Annexin V-FITC/PI apoptosis detection assay is used to determine the signal pathway of mitochondria targeted cellular dysfunction.

Silk fibroin is a semicrystalline protein used as a renewable polymer source and as a biomaterial platform, but existing methods to synthetically modify fibroin suffer from low efficiencies that can limit the protein’s utility. This work reports on a mild synthesis that results in a 2-fold increase in carboxylation through the disruption of noncovalent interactions during the reaction. Importantly, silk fibroin maintains its ability to form β-sheets that are critical for tailoring mechanical and degradation properties, as well as for rendering solid constructs (e.g., films and scaffolds) insoluble in water. Increasing carboxyl functionalization affords control over protein charge, which permits tailoring the loading and release of small molecules using electrostatic interactions. Disruption of noncovalent interactions during aqueous carbodiimide coupling also significantly enhances conjugation efficiency of molecules containing primary amine groups, thus enabling high degrees of functionalization with biological molecules, such as proteins and peptides, for biomaterial applications.

Since the asialoglycoprotein receptor (also known as the “Ashwell–Morell receptor” or ASGPR) was discovered as the first cellular mammalian lectin, numerous drug delivery systems have been developed and several gene delivery systems associated with multivalent ligands for liver disease targeting are undergoing clinical trials. The success of these systems has facilitated the further study of new ligands with comparable or higher affinity and less synthetic complexity. Herein, we designed two novel trivalent ligands based on the esterification of tris(hydroxymethyl) aminomethane (TRIS) followed by the azide–alkyne Huisgen cycloaddition with azido N-acetyl-d-galactosamine. The presented triazolyl glycoconjugates exhibited good binding to ASGPR, which was predicted using in silico molecular docking and assessed by a surface plasmon resonance (SPR) technique. Moreover, we demonstrated the low level of in vitro cytotoxicity, as well as the optimal spatial geometry and the required amphiphilic balance, for new, easily accessible ligands. The conjugate of a new ligand with Cy5 dye exhibited selective penetration into HepG2 cells in contrast to the ASGPR-negative PC3 cell line.

Whereas small siRNA nanocarriers with a size of 10–20 nm exert high tissue-permeability, they encounter the challenge of inefficient adsorption on the cell surface, resulting in poor cellular uptake of siRNA. To solve this dilemma, this study aims to control the hydrophobicity of a small siRNA nanocarrier, unimer polyion complex (uPIC), with a size of ∼10 nm. The uPICs are fabricated to consist of a single pair between siRNA and a smart triblock copolymer comprising hydrophilic poly(2-ethyl-2-oxazoline) (PEtOx), thermoswitchable poly(2-n-propyl-2-oxazoline) (PnPrOx), and cationic poly(l-lysine) (PLL). The PnPrOx segment is dehydrated at 37 °C (>lower critical solution temperature) to enhance the hydrophobicity of uPICs. The uPICs with a hydrophobic domain facilitates cellular uptake of the siRNA payload through stronger binding to the cell surface, compared with control uPICs without a PnPrOx segment, leading to a significantly enhanced gene silencing effect in cultured cancer cells.

Two NIR-emitting platinum [Pt(N^N^C)(phosphine)] and iridium [Ir(N^C)2(N^N)]⁺ complexes containing reactive succinimide groups were synthesized and characterized with spectroscopic methods (N^N^C, 1-phenyl-3-(pyridin-2-yl)benzo[4,5]imidazo[1,2-a]pyrazine, N^C, 6-(2-benzothienyl)phenanthridine, phosphine-3-(diphenylphosphaneyl)propanoic acid N-hydroxysuccinimide ether, and N^N, 4-oxo-4-((1-(pyridin-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)butanoic acid N-hydroxysuccinimide ether). Their photophysics were carefully studied and analyzed using time-dependent density functional theory calculations. These complexes were used to prepare luminescent micro- and nanoparticles with the “core–shell” morphology, where the core consisted of biodegradable polymers of different hydrophobicity, namely, poly(d,l-lactic acid), poly(ε-caprolactone), and poly(ω-pentadecalactone), whereas the shell was formed by covalent conjugation with poly(l-lysine) covalently labeled with the platinum and iridium emitters. The surface of the species was further modified with heparin to reverse their charge from positive to negative values. The microparticles’ size determined with dynamic laser scanning varies considerably from 720 to 1480 nm, but the nanoparticles’ diameter falls in a rather narrow range, 210–230 nm. The species with a poly(l-lysine) shell display a high positive (>30 mV) zeta-potential that makes them essentially stable in aqueous media. Inversion of the surface charge to a negative value with the heparin cover did not deteriorate the species’ stability. The iridium- and platinum-containing particles displayed emissions the spectral patterns of which were essentially similar to those of unconjugated complexes, which indicate retention of the chromophore nature upon binding to the polymer and further immobilization onto polyester micro- and nanoparticles for drug delivery. The obtained particles were tested to determine their ability to penetrate into different cells types: cancer cells, stem cells, and fibroblasts. It was found that all types of particles could effectively penetrate into all cells types under investigation. Nanoparticles were shown to penetrate into the cells more effectively than microparticles. However, positively charged nanoparticles covered with poly(l-lysine) seem to interact with negatively charged proteins in the medium and enter the inner part of the cells less effectively than nanoparticles covered with poly(l-lysine)/heparin. In the case of microparticles, the species with positive zeta-potentials were more readily up-taken by the cells than those with negative values.

Mitochondria are therapeutic targets in many diseases including cancer, metabolic disorders, and neurodegenerative diseases. Therefore, strategies to deliver therapeutics of interest to mitochondria are important for therapeutic development. As delocalized lipophilic cations (DLCs) preferentially accumulate in mitochondria, DLC-conjugation has been utilized to facilitate therapeutic delivery systems with mitochondrial targeting capability. Here we report that upon DLC-conjugation, anionic polymers exhibit significantly improved mitochondrial targeting when compared to cationic polymers and charge-neutral polymers. Considering that the cell membrane generally bears a net negative charge, the observed phenomenon is unexpected. Notably, the DLC-conjugated anionic polymers circumvent endosomal entrapment. The rapid mitochondrial accumulation of DLC-conjugated anionic polymers is likely a membrane-potential-driven process, along with the involvement of the mitochondrial pyruvate carrier. Moreover, the structural variations on the side chain of DLC-conjugated anionic polymers do not compromise the overall mitochondrial targeting capability, widely extending the applicability of anionic macromolecules in therapeutic delivery systems.

Titanium dioxide (TiO₂) nanoparticles are used on a massive scale in commercial and industrial products. Of specific concern is how the inhalation of these nanoparticles in a manufacturing setting may affect human health. We examine the cellular response to TiO₂ nanoparticles using a combination of cell-free spectroscopic assays, fluorescence microscopy, Western blotting, and TiO₂ nanoparticle surface modifications. These experiments show that TiO₂ nanoparticles generate superoxide, both in solution and in cells, and this intracellular superoxide decreases expression of histone deacetylase 9 (HDAC9), an epigenetic modifier. We use protein coronas formed from superoxide dismutase (SOD) and catalase, enzymes that scavenge reactive oxygen species (ROS), to probe the relationship between TiO₂ nanoparticles, ROS, and the subsequent cellular response. These protein coronas provide nanoparticle-localized scavengers that demonstrate that the nanoparticles are the source of the intracellular superoxide. Importantly, the use of a SOD corona or surface passivated TiO₂ nanoparticles prevents the decrease of HDAC9. These experiments elucidate the underlying mechanism of TiO₂ nanoparticle-mediated cellular responses including oxidative stress and changes in gene expression. They also provide the first demonstration of a protein corona as a tool for probing cellular responses to nanoparticles. Overall, this research shows that low, nontoxic concentrations of TiO₂ nanoparticles alter an enzyme responsible for epigenetic modifications, which points to concerns regarding long-term exposures in manufacturing settings.

Immobilizing a signaling protein to guide cell behavior has been employed in a wide variety of studies. This approach draws inspiration from biology, where specific, affinity-based interactions between membrane receptors and immobilized proteins in the extracellular matrix guide many developmental and homeostatic processes. Synthetic immobilization approaches, however, do not necessarily recapitulate the in vivo signaling system and potentially lead to artificial receptor–ligand interactions. To investigate the effects of one example of engineered receptor–ligand interactions, we focus on the immobilization of interferon-γ (IFN-γ), which has been used to drive differentiation of neural stem cells (NSCs). To isolate the effect of ligand immobilization, we transfected Cos-7 cells with only interferon-γ receptor 1 (IFNγR1), not IFNγR2, so that the cells could bind IFN-γ but were incapable of canonical signal transduction. We then exposed the cells to surfaces containing covalently immobilized IFN-γ and studied membrane morphology, receptor–ligand dynamics, and receptor activation. We found that exposing cells to immobilized but not soluble IFN-γ drove the formation of filopodia in both NSCs and Cos-7, showing that covalently immobilizing IFN-γ is enough to affect cell behavior, independently of canonical downstream signaling. Overall, this work suggests that synthetic growth factor immobilization can influence cell morphology beyond enhancing canonical cell responses through the prolonged signaling duration or spatial patterning enabled by protein immobilization. This suggests that differentiation of NSCs could be driven by canonical and non-canonical pathways when IFN-γ is covalently immobilized. This finding has broad implications for bioengineering approaches to guide cell behavior, as one ligand has the potential to impact multiple pathways even when cells lack the canonical signal transduction machinery.

Labeling a protein of interest (POI) with a fluorescent reporter is a powerful strategy for studying protein structures and dynamics in their native environments. Compared to fluorescent proteins, synthetic dyes provide more choices in photophysical or photochemical attributes to microscopic characterizations. The specificity of bioorthogonal reactions in conjunction with the fidelity of subcellular destinations of genetically encoded protein tags can be employed to label POIs in live and fixed cells in a two-step process. In the present study the orthogonality of the strain-promoted azide–alkyne cycloaddition (SPAAC) and the inverse electron demand Diels–Alder (IEDDA) reaction is corroborated in concurrent labeling of two different intracellular targets. An azido group and a strained alkene are first installed at specific subcellular locations via orthogonal enzymatic reactions of the genetically incorporated SNAP- and CLIP-tags. The subsequent bioorthogonal reactions with fluorophores carrying matching reactive functionalities result in simultaneous dual labeling. The two-step “orthogonal-bioorthogonal” labeling process would increase the utilities of SNAP/CLIP-tags and, as a consequence, would expand the capability of decorating biological specimens with functionalities beyond fluorophores to potentially include spin labels, radioactive tracers, or catalysts.

Functional hydrogels that can be obtained through facile fabrication procedures and subsequently modified using straightforward reagent-free methods are indispensable materials for biomedical applications such as sensing and diagnostics. Herein a novel hydrogel platform is obtained using polymeric precursors containing the maleimide functional group as a side chain. The maleimide groups play a dual role in fabrication of functional hydrogels. They enable photochemical cross-linking of the polymers to yield bulk and patterned hydrogels. Moreover, the maleimide group can be used as a handle for efficient functionalization using the thiol–maleimide conjugation and Diels–Alder cycloaddition click reactions. Obtained hydrogels are characterized in terms of their morphology, water uptake capacity, and functionalization. Micropatterned hydrogels are obtained under UV-irradiation using a photomask to obtain reactive micropatterns, which undergo facile functionalization upon treatment with thiol-containing functional molecules such as fluorescent dyes and bioactive ligands. The maleimide group also undergoes conjugation through the Diels–Alder reaction, where the attached molecule can be released through thermal treatment via the retro Diels–Alder reaction. The antibiofouling nature of these hydrogel micropatterns enables efficient ligand-directed biomolecular immobilization, as demonstrated by attachment of streptavidin-coated quantum dots.

Recently we have reported that the ortho-hydroxy-protected aryl sulfate (OHPAS) system can be exploited as a new self-immolative group (SIG) for phenolic payloads. We extended the system to nonphenolic payloads by simply introducing a para-hydroxy benzyl (PHB) spacer. As an additional variation of the system, we explored a benzylsulfonate version of the OHPAS system and found that it has two distinct breakdown pathways, cyclization and 1,4-elimination, the latter of which implies that para-hydroxy-protected (PHP) benzylsulfonate (BS) can also be used as an alternative SIG. The PHP-BS system was found to be stable chemically and in mouse and human plasma, having payload release rates comparable to those of the original OHPAS conjugates.

Explosive compounds, such as 2,4,6-trinitrotoluene (TNT), pose a great concern in terms of both global public security and environmental protection. There are estimated to be hundreds of TNT contaminated sites all over the world, which will affect the health of humans, wildlife, and the ecosystem. Clearly, the ability to detect TNT in soils, water supplies, and wastewater is important for environmental studies but also important for security, such as in ports and boarders. However, conventional spectroscopic detection is not practical for on-site sensing because it requires sophisticated equipment and trained personnel. We report a rapid and simple chemical sensor for TNT by using TNT binding peptides which are conjugated to fluorescent CdTe/CdS quantum dots (QDs). QDs were synthesized in the aqueous phase, and the peptide was attached directly to the surface of the QDs by using thiol groups. The fluorescent emission from the QDs was quenched in response to the addition of TNT. The response could even be observed by the naked eye. The limit of detection from fluorescence spectroscopic measurement was estimated to be approximately 375 nM. In addition to the rapid response (within a few seconds), selective detection was demonstrated. We believe this label-free chemical sensor contributes to progress for the on-site explosive sensing.

Amine/thiol-reactive chemistries are commonly used to conjugate antibodies to pharmaceuticals or nanoparticles. Yet, these conjugation strategies often result in unfavorable outcomes such as heterogeneous antibody display with hindered biological activity or aggregation due to multivalent interactions of the antibody and nanoparticles. Here, we report the application of a site-specific and enzymatically driven antibody conjugation strategy to functionalize virus-based nanoparticles (VNPs). Specifically, an azide-handle was introduced into the Fc region of a set of immunoglobulins using a two-step enzymatic reaction: (1) cleavage of N-linked glycan in the Fc region by a glycosidase and (2) conjugation of a chemically reactive linker (containing an azide functional handle) using a microbial transglutaminase. Conjugation of the azide-functional antibodies to several VNPs was achieved by making use of strain-promoted azide–alkyne cycloaddition. We report the conjugation of three immunoglobulin (IgG) isotypes (human IgG from sera, anti-CD47 Rat IgG2a, κ, and Trastuzumab recombinant humanized IgG1, κ) to the plant virus cowpea mosaic virus (CPMV) and the lysine mutant of tobacco mosaic virus (TMVlys) as well as bacteriophage Qβ. Site-specific conjugation resulted in stable and functional antibody-VNP conjugates. In stark contrast, the use of heterobifunctional linkers targeting thiols and amines on the antibodies and VNPs, respectively, led to aggregation due to nonspecific and multivalent coupling between the antibodies and VNPs. We demonstrate that antibody-VNP conjugates were functional, and Trastuzumab-displaying VNPs targeted HER2-positive SKOV-3 human ovarian cancer cells. This bioconjugation strategy adds to the portfolio of methods that can be used for designing functional antibody-VNP conjugates.

Targeting less abundant amino acid residues on the protein surface may realize site-selective protein modification of natural proteins. The relative hydrophobicity of tyrosine combined with the π–π stacking tendency of the aromatic rings results in generally low accessibility. In this study, site-selective protein modification was achieved by targeting surface-exposed tyrosine residues without using a genetic encoding system. Tyrosine residues were modified with N-methylated luminol derivative under single-electron transfer (SET) reaction conditions. Horseradish peroxidase (HRP)-catalyzed SET and electrochemically activated SET modified surface-exposed tyrosine residues selectively. N-Methylated luminol derivative modified tyrosine residues more efficiently than 4-arylurazole under tyrosine click conditions using HRP and electrochemistry. Tyrosine residues that are evolutionarily exposed only in the complementarity-determining region (CDR) of an antibody were selectively modified by tyrosine click reactions. CDR-modified antibodies were applied to in vivo imaging and antibody–drug conjugated (ADC).

Serious bacterial infections by multi-drug-resistant pathogens lead to human losses and endanger public health. The discovery of antibiotics with new modes of action, in combination with nanotechnology, might offer a promising route to combat multi-drug-resistant pathogens. Platensimycin (PTM), a potent inhibitor of FabB/FabF for bacterial fatty acid biosynthesis, is a promising drug lead against many drug-resistant bacteria. However, the clinical development of PTM is hampered by its poor pharmacokinetics. Herein, we report a nanostrategy that encapsulated PTM in two types of nanoparticles (NPs) poly(lactic-co-glycolic acid) (PLGA) and poly(amidoamine) (PAMAM) dendrimer to enhance its antibacterial activity in vitro and in vivo. The PTM-encapsulated NPs were effective to inhibit Staphylococcus aureus biofilm formation, and killed more S. aureus in a macrophage cell infection model over free PTM. The pharmacokinetic studies showed that PTM-loaded PLGA and PAMAM NPs exhibited increased AUC_0-t (area under the curve) (∼4- and 2-fold) over free PTM. In a mouse peritonitis model, treatment of methicillin-resistant S. aureus infected mice using both PTM-loaded NPs (10 mg/kg) by intraperitoneal injection led to their full survival, while all infected mice died when treated by free PTM (10 mg/kg). These results not only suggest that PTM-loaded NPs may hold great potential to improve the poor pharmacokinetic properties of PTM, but support the rationale to develop bacterial fatty acid synthase inhibitors as promising antibiotics against drug-resistant pathogens.

Fabrication of a multifunctional near-infrared (NIR) theranostic nanoplatform has attracted increasing attention. Indocyanine green (ICG), a clinic-approved NIR fluorescence-imaging agent, is an excellent photothermal agent candidate. However, the stability and tumor targeting are still great obstacles for its wide application. In this work, C-phycocyanin (CPC) as a tumor-associated macrophages (TAMs) targeted vehicle was used to fabricate noncovalent ICG conjugate of CPC (ICG@CPC) via self-assembly in aqueous media. Compared to free ICG, ICG@CPC displays improved stabilities in aqueous solutions and under light irradiation and threefold increase in photothermal conversion efficiency. The in vitro results indicated that ICG@CPC could be selectively internalized into J774A.1 cells via SR-A-mediated endocytosis and lead to enhanced photocytotoxicity against J774A.1 cells. In vivo results showed that ICG@CPC had significantly improved drug accumulation in the tumor and photothermal therapeutic efficacy relative to ICG alone. This study for the first time utilizes CPC as a TAMs-targeted nanocarrier for ICG and may promote further rational design of ICG-based photothermal nanodrugs for precise and efficient cancer theranosis.

Advances in bioconjugation, the ability to link biomolecules to each other, small molecules, surfaces, and more, can spur the development of advanced materials and therapeutics. We have discovered that pyrocinchonimide, the dimethylated analogue of maleimide, undergoes a surprising transformation with biomolecules. The reaction targets amines and involves an imide transfer, which has not been previously reported for bioconjugation purposes. Despite their similarity to maleimides, pyrocinchonimides do not react with free thiols. Though both lysine residues and the N-termini of proteins can receive the transferred imide, the reaction also exhibits a marked preference for certain amines that cannot solely be ascribed to solvent accessibility. This property is peculiar among amine-targeting reactions and can reduce combinatorial diversity when many available reactive amines are available, such as in the formation of antibody–drug conjugates. Unlike amides, the modification undergoes very slow reversion under high pH conditions. The reaction offers a thermodynamically controlled route to single or multiple modifications of proteins for a wide range of applications.

Sortase-mediated ligation is a powerful method for generating site-specifically modified proteins. However, this process is limited by the inherent reversibility of the ligation reaction. To address this, here we report the continued development and optimization of an experimentally facile strategy for blocking reaction reversibility. This approach, which we have termed metal-assisted sortase-mediated ligation (MA-SML), relies on the use of a solution additive (Ni²⁺) and a C-terminal tag (LPXTGGHH₅) that is widely used for converting protein targets into sortase substrates. In a series of model systems utilizing a 1:1 molar ratio of sortase substrate and glycine amine nucleophile, we find that MA-SML consistently improves the extent of ligation. This enables the modification of proteins with fluorophores, PEG, and a bioorthogonal cyclooctyne moiety without the need to use precious reagents in excess. Overall, these results demonstrate the potential of MA-SML as a general strategy for improving reaction efficiency in a broad range of sortase-based protein engineering applications.

Exploring a combined phototherapeutic strategy to overcome the limitations of a single mode therapy and inducing high anticancer efficiency is highly promising for precision cancer nanomedicine. However, a single-wavelength laser activates dual photothermal/photodynamic therapy (PTT/PDT) treatment is still a formidable challenge. Herein, we strategically design and fabricate a multifunctional theranostic nanosystem based on chlorin e6-functionalized polydopamine (PDA) coated prussian blue/manganese dioxide nanoparticles (PB-MnO₂@PDA-Ce6 NPs). Interestingly, the obtained PB-MnO₂@PDA NPs not only offer an effective delivery system for Ce6 but also provide strong optical absorption in the near-infrared range, endowing high antitumor efficacy of PTT. More importantly, the as-prepared PB-MnO₂@PDA-Ce6 nanoagents exhibit an effective oxygen generation, superior reactive oxygen species (ROS), and outstanding photothermal conversion ability to greatly improve PTT and PDT treatments. As a result, both in vitro and in vivo treatments guided by MR imaging on liver cancer cells reveal the complete cell/tumor eradication under a single wavelength of 660 nm laser irradiation, implying the simultaneous synergistic PDT/PTT effects triggered by PB-MnO₂@PDA-Ce6 nanoplatform, which are much higher than individual treatment. Taken together, our phototherapeutic nanoagents exhibit an excellent therapeutic performance, which may act as a nanoplatform to find safe and clinically translatable routes to accelerate cancer therapeutics.

Polyamine and polyammonium ion conjugates are often used to direct reagents to nucleic acids based on their strong electrostatic attraction to the phosphoribose backbone. Such nonspecific interactions do not typically alter the specificity of the attached reagent, but polyammonium ions dramatically redirected the specificity of a series of quinone methide precursors. Replacement of a relatively nonspecific intercalator based on acridine with a series of polyammonium ions resulted in a surprising change of DNA products. Piperidine stable adducts were generated in duplex DNA that lacked the ability to support a dynamic cross-linking observed previously with acridine conjugates. Minor reaction at guanine N7, the site of reversible reaction, was retained by a monofunctional quinone methide-polyammonium ion conjugate, but a bisfunctional analogue designed for tandem quinone methide formation modified guanine N7 in only single-stranded DNA. The resulting intrastrand cross-links were sufficiently dynamic to rearrange to interstrand cross-links. However, no further transfer of adducts was observed in duplex DNA. An alternative design that spatially and temporally decoupled the two quinone methide equivalents neither restored the dynamic reaction nor cross-linked DNA efficiently. While di- and triammonium ion conjugates successfully enhanced the yields of cross-linking by a bisquinone methide relative to a monoammonium equivalent, alternative ligands will be necessary to facilitate the migration of cross-linking and its potential application to disrupt DNA repair.

We detail the preparation of highly fluorescent quantum dots (QDs), surface-engineered with multifunctional polymer ligands that are compact and readily compatible with strain-promoted click conjugation, and the use of these nanocrystals in immunofluorescence and in vivo imaging. The ligand design combines the benefits of mixed coordination (i.e., thiol and imidazole) with zwitterion motifs, yielding sterically-stabilized QDs that present a controllable number of azide groups, for easy conjugation to biomolecules via the selective click chemistry. The polymer coating was characterized using NMR spectroscopy to extract estimates of the diffusion coefficient, hydrodynamic size, and ligand density. The azide-functionalized QDs were conjugated to anti-tropomyosin receptor kinase B antibody (α-TrkB) or to the brain-derived neurotrophic factor (BDNF). These conjugates were highly effective for labeling the tropomyosin receptor kinase B (TrkB) in pyramidal neurons within cortical tissue and for monitoring the BDNF induced activation of TrkB signaling in live neuronal cells. Finally, the polymer-coated QDs were applied for in vivo imaging of Drosophila melanogaster embryos, where the QDs remained highly fluorescent and colloidally stable, with no measurable cytotoxicity. These materials would be of great use in various imaging applications, where a small size, ease of conjugation, and great colloidal stability for in vivo studies are needed.

In this study, we reported a ^99mTc-labeled integrin α₆-targeted peptide as the molecular imaging probe for tumor imaging by single-photon emission computed tomography (SPECT). We found that replacing Cys–Cys cyclized RWY peptide (sequence: cCRWYDENAC) with lactam-bridged cyclic cKiE peptide (sequence: cKRWYDENAisoE) did not sacrifice the integrin α₆-binding affinity and specificity of cKiE radiotracer. To further improve the radiotracer’s tumor targeting capability, the dimerized cKiE peptide (termed cKiE2) was designed, and the corresponding radiotracer ^99mTc-cKiE2 was evaluated for tumor uptake and in vivo pharmacokinetics properties in tumor models. We found that cKiE2 showed higher binding affinity to integrin α₆ than did monomeric RWY or cKiE peptide. The biodistribution results showed that the tumor uptake of ^99mTc-cKiE2 was twice higher than that of ^99mTc-RWY (3.20 ± 0.12 vs 1.26 ± 0.06 %ID/g, P < 0.001) at 0.5 h postinjection. The tumor to nontargeting tissue ratios were also enhanced in most normal organs. Specificity of ^99mTc-cKiE2 for integrin α₆ was demonstrated by competitive blocking of tumor uptake with excess cold peptide (3.20 ± 0.24 to 1.38 ± 0.23 %ID/g, P < 0.001). The integrin α₆-positive tumors were clearly visualized by ^99mTc-cKiE2/SPECT with low background except with a relatively high kidney uptake. The tumor uptake of ^99mTc-cKiE2 correlates well with the tumor integrin α₆ expression levels in a linear fashion (R² = 0.9623). We also compared ^99mTc-cKiE2 with an integrin α_vβ₃-targeted radiotracer ^99mTc-3PRGD₂ in the orthotopic hepatocellular carcinoma tumor models. We found that the orthotopic tumor was clearly visualized with ^99mTc-cKiE2. ^99mTc-3PRGD₂ imaging did not show tumor contours in situ as clearly as ^99mTc-cKiE2. The tumor-to-liver ratios of ^99mTc-cKiE2 and ^99mTc-3PRGD₂ were 2.20 ± 0.17 and 0.85 ± 0.20. In conclusion, ^99mTc-cKiE2 is an improved SPECT radiotracer for imaging integrin α₆-positive tumors and has great potential for further clinical application.

The coming era of precision nanomedicine demands engineered nanoparticles that can be readily translated into the clinic, like that of molecular agents, without being hindered by intrinsic size heterogeneity and long-term body retention. Herein we report that conjugation of indocyanine green (ICG), an FDA-approved near-infrared (NIR) dye, onto an atomically precise glutathione-coated Au25 (GS-Au25) nanocluster led to a molecular-like photothermal nanoparticle (ICG₄–GS-Au25) with significantly enhanced ICG photostability and tumor targeting. Under weak NIR light irradiation conditions, free ICG failed to suppress tumor growth but the original tumors were completely eradicated with ICG₄–GS-Au25. In the meantime, “off-target” ICG₄–GS-Au25 was effectively cleared out from the body like small-molecule drugs after glutathione-mediated biotransformation in the liver. These findings highlight the merits of molecular-like nanomedicines, offering a new pathway to meet FDA’s criteria for the clinical translation of nanomedicines.

Icosahedral virus-like particles (VLPs) derived from bacteriophages Qβ and PP7 encapsulating small-ultrared fluorescent protein (smURFP) were produced using a versatile supramolecular capsid disassemble–reassemble approach. The generated fluorescent VLPs display identical structural properties to their nonfluorescent analogs. Encapsulated smURFP shows indistinguishable photochemical properties to its unencapsulated counterpart, exhibits outstanding stability toward pH, and produces bright in vitro images following phagocytosis by macrophages. In vivo imaging allows the biodistribution to be imaged at different time points. Ex vivo imaging of intravenously administered encapsulated smURFP reveals a localization in the liver and kidneys after 2 h blood circulation and substantial elimination after 16 h of imaging, highlighting the potential application of these constructs as noninvasive in vivo imaging agents.

Nucleoside analogs have proven effective for the inhibition of viral polymerases and are the foundation of many antiviral therapies. In this work, the antiretroviral potential of 6-azauracil analogs was assessed using activity-based protein profiling techniques and functional assays. Probes based on the 6-azauracil scaffold were examined and found to bind to HCV polymerase and HIV-1 reverse transcriptase through covalent modification of residues near the active site. The modified sites on the HIV-1 RT were examined using a mass spectrometry approach, and it was discovered that the azauracil moieties modified the enzyme in proximity to its active site. However, these scaffolds gave little or no inhibition of enzyme activity. Instead, a bifunctional inhibitor was prepared using click chemistry to link the 6-azauracil moiety to azidothymidine (AzT) and the corresponding triphosphate (AzTTP). These bifunctional inhibitors were found to have potent inhibitory function through a mode of action that includes both alkylation and chain termination. An in vitro assay demonstrated that the bifunctional inhibitor was 23-fold more effective in inhibiting HIV-1 RT activity than the parent AzTTP. The bifunctional inhibitor was also tested in HIV-1 permissive T cells where it decreased Gag expression similarly to the front-line drug Efavirenz with no evidence of cytotoxicity. This new bifunctional scaffold represents an interesting tool for inhibiting HIV-1 by covalently anchoring a chain-terminating nucleoside analog in the active site of the reverse transcriptase, preventing its removal and abolishing enzymatic activity, and represents a novel mode of action for inhibiting polymerases including reverse transcriptases.

Herein, we report the use of two-photon fluorogenic probes using tetrazine-based bioorthogonal reactions with multicolor emissions that cover nearly all of the visible region. New fluorogenic probes were designed based on donor–acceptor-type naphthalene structures conjugated with a fluorescence-quenching tetrazine moiety for turn-on properties in one- and two-photon fluorescence. Our fluorescent probes showed a moderate to good turn-on ratio after bioorthogonal inverse electron demand Diels–Alder cycloaddition with trans-cyclooctenol in one- and two-photon fluorescence. We successfully applied our probes to mitochondria- and lysosome-selective bioorthogonal imaging in live cells with one-/two-photon and one-photon microscopy, respectively.

Human granulocyte-macrophage colony-stimulating factor (hGMCSF) is crucial in the immune system as it stimulates survival, proliferation, differentiation, and functional activation of myeloid hematopoietic cells. hGMCSF is integral to approved therapies, including monoclonal antibodies against checkpoint inhibitors, chimeric antigen receptors, and prevention of chemotherapy-induced neutropenia. Recombinant hGMCSF can be purified from Escherichia. coli; however, it forms inclusion bodies that require solubilization and refolding. Alternatively, this manuscript describes its fusion with an elastin-like polypeptide (ELP). Previously reported as purification tags and solubility enhancers, ELPs are recombinant polypeptides that undergo reversible temperature-dependent phase separation. This report is the first to show that fusion to an ELP enables direct purification of hGMCSF fusions from the soluble fraction of bacterial lysate. Surprisingly, these ELP-fusions assemble stable, small, spherical nanoparticles that maintain pro-mitotic activity of hGMCSF. These nanoparticles exhibit ELP-mediated phase separation; however, nanoparticle assembly significantly increases the entropic and enthalpic cost of phase separation compared to ELP alone. The attachment of a high molecular weight ELP to a difficult-to-express protein, like hGMCSF, appears to be a useful strategy to stabilize bioactive, protein-based nanoparticles, which may have broad applications in medicine and biology.

Exosomes are cellular components with promising uses in cancer diagnostics and therapeutics, and their imaging and tracking are essential to study their biological properties. Herein, we report on an in situ one-step fluorescence labeling strategy for exosomes via bioorthogonal click chemistry. First, exosome donor cancer cells were treated with tetraacetylated N-azidoacetyl-d-mannosamine (Ac₄ManNAz) to generate unnatural azide groups (−N₃) on their surface via metabolic glycoengineering. Then, the azide groups were labeled with near-infrared fluorescent dye-conjugated dibenzylcyclooctyne (DBCO-Cy5) via bioorthogonal click chemistry. After 2 days of incubation, the DBCO-Cy5-labeled exosomes (Cy5-Exo) were successfully secreted from the donor cancer cells and were isolated via classical ultracentrifugation, providing a high-yield of fluorescent dye-labeled exosomes. This in situ one-step bioorthogonal click chemistry offers improved labeling efficiency, biocompatibility, and imaging sensitivy compared to standard exosomes (ST-Exo), purified with classical ultracentrifugation or carbocyanine lipophilic dye (DiD)-labeled exosomes (DiD-Exo) in vitro. In particular, the Cy5-Exo were successfully taken up by A549 cells in a time-dependent manner, and they could escape from lysosome confinement, showing their possible use as a delivery carrier of therapeutic drugs or imaging agents. Finally, intraveneously injected Cy5-Exo were noninvasively tracked and imaged via near-infrared fluorescence (NIRF) imaging in tumor-bearing mice. This new fluorescence labeling strategy for natural exosomes may be useful to provide better understanding of their theranostic effects in many biomedical applications.

Protein corona formation has been regarded as an obstacle to developing diagnostic and therapeutic nanoparticles for in vivo applications. Serum proteins that assemble around nanoparticles can hinder their targeting efficiency. Virus-based nanoparticles should be naturally predisposed to evade such barriers in host organisms. Here, we demonstrate that virus-like particles derived from mouse polyomavirus do not form a rich protein corona. These particles can be efficiently targeted to cells that overproduce transferrin receptors, e.g., cancer cells, by conjugating transferrin to the particle surface. In this study, we provide evidence that the interaction of virus-like particles with their newly assigned target receptor is not obstructed by serum proteins. The particles enter target cells via a clathrin-dependent endocytic pathway that is not naturally used by the virus. Our results support the notion that the natural properties of virus-like particles make them well-suited for development of nanosized theranostic tools resistant to detargeting by protein coronas.