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Cancer nanomedicines require different, even opposite, properties to voyage the cascade drug delivery process involving a series of biological barriers. Currently-approved nanomedicines can only alleviate adverse effects but cannot improve patient survival because they fail to meet all the requirements. Therefore, nanocarriers with synchronized functions are highly requisite to capacitate efficient drug delivery and enhanced therapeutic efficacies. This perspective article summarizes recent advances in the two main strategies for nanomedicine design, the All-in-One approach (integration of all the functions in one system) and the One-for-All approach (one functional group with proper affinity enables all the functions), and presents our views on future nanomedicine development.

Cancer drug delivery systems often suffer from premature drug leakage during transportation and/or inefficient drug release within cancer cells. We present here a polymeric prodrug nanoassembly that addresses these problems simultaneously. This nanoassembly comprises a polymeric prodrug with novel trivalent phenylboronate moieties for drug conjugation via ether linkages, as well as β-lapachone (Lapa). While the ether linkage enables nearly no drug release under physiological conditions, the Lapa molecules can induce the reactive oxygen species (ROS) burst specifically in cancer cells via NAD(P)H: quinone oxidoreductase-1 catalysis, which triggers the cleavage of the ether bonds and thus cascade amplification drug release in cancer cells. As a result, the nanoassemblies exhibit much higher cytotoxicity against cancer cells than normal cells, and also increased therapeutic efficacy and reduced side effects compared to the clinically used irinotecan. We anticipate that this strategy can be applied to other drug delivery platforms to enable more precise drug release.

Subcutaneous injection of insulin is the current standard medication for many diabetic patients. Convenient and painless noninvasive transdermal insulin delivery has long been pursued but yet succeeded due to no such technologies for large biomacromolecules. We find a tertiary amine oxide-based polyzwitterion, OPDMA, that can efficiently penetrate both the stratum corneum (SC) and viable epidermis into circulation. So its conjugate with insulin, OPDMA-I, applied on the skin can exhibit hypoglycemic effects as efficiently as subcutaneously injected insulin in type-1 diabetic mice and minipigs. The unique pH-dependent cationic-to-zwitterionic transition of OPDMA in the characteristic acidic-to-neutral pH gradient from the skin surface to deep SC enables fast transdermal delivery of OPDMA and its conjugate. On the skin, OPDMA binds to carboxylic acids in the acidic sebum layer, enriching OPDMA-I on the SC. As pH increases in deeper SC layers, binding between OPDMA-I and the skin weakens gradually, allowing for diffusion through inter-corneocyte gaps and penetration into viable epidermis and finally entering the systemic circulation via dermal lymphatic vessels. This process does not alter SC microstructures or cause any physiological changes in the skin. This study represents a groundbreaking example of noninvasive transdermal protein delivery.

Multiplex protein imaging technologies enable deep phenotyping and provide rich spatial information about biological samples. Existing methods have shown great success but also harbored trade-offs between various pros and cons, underscoring the persisting necessity to expand the imaging toolkits. Here we present PACIFIC: photoactive immunofluorescence with iterative cleavage, a new modality of multiplex protein imaging methods. PACIFIC achieves iterative multiplexing by implementing photocleavable fluorophores for antibody labeling with one-step spin-column purification. PACIFIC requires no specialized instrument, no DNA encoding, or chemical treatments. We demonstrate that PACIFIC can resolve cellular heterogeneity in both formalin-fixed paraffin-embedded (FFPE) samples and fixed cells. To further highlight how PACIFIC assists discovery, we integrate PACIFIC with live-cell tracking and identify phosphor-p70S6K as a critical driver that governs U87 cell mobility. Considering the cost, flexibility, and compatibility, we foresee that PACIFIC can confer deep phenotyping capabilities to anyone with access to traditional immunofluorescence platforms.

Nanocarriers have been employed extensively to enhance drug delivery efficacy and reduce the side effect. However, carrier materials for drug delivery have challenging aspects, including safety concerns, low drug content, complexity in preparation, and low reproducibility. Herein, we propose a facile, universal, and green preparation way to use natural polyphenols to build platinum nanocomplex with stable structure, proper size, and high Pt content. The nanocomplexes are constructed by metal-polyphenol coordination using natural polyphenols and 1,2-diaminocyclohexane-Pt (II), enabling dual-responsive drug release behavior. For proof of concept, we demonstrate the antitumor activity of the Pt nanocomplex using a representative tannic acid-Pt nanocomplex (denoted as PTI). PTI can induce intensive tumor cell apoptosis, trigger immunogenic cell death (ICD), remarkably promote cytotoxic T lymphocytes (CTLs) infiltration in tumors, and significantly reduce immunosuppression of the tumor microenvironments, thus stimulating potent antitumor immune responses and showing effective antitumor activity by synergizing immune checkpoint blockade (ICB) therapy.

Biomedical polymers have been extensively developed for promising applications in a lot of biomedical fields, such as therapeutic medicine delivery, disease detection and diagnosis, biosensing, regenerative medicine, and disease treatment. In this review, we summarize the most recent advances in the synthesis and application of biomedical polymers, and discuss the comprehensive understanding of their property-function relationship for corresponding biomedical applications. In particular, a few burgeoning bioactive polymers, such as peptide/biomembrane/microorganism/cell-based biomedical polymers, are also introduced and highlighted as the emerging biomaterials for cancer precision therapy. Furthermore, the foreseeable challenges and outlook of the development of more efficient, healthier and safer biomedical polymers are discussed. We wish this systemic and comprehensive review on highlighting frontier progress of biomedical polymers could inspire and promote new breakthrough in fundamental research and clinical translation.

Tumor enzyme-responsive charge-reversal carriers can induce efficient transcytosis and lead to efficient tumor infiltration and potent anticancer efficacy. However, the correlations of molecular structure with charge-reversal property, tumor penetration, and drug delivery efficiency are unknown. Herein, aminopeptidase N (APN)-responsive conjugates were synthesized to investigate these correlations. We found that the monomeric unit structure and the polymer chain structure determined the enzymatic hydrolysis and charge-reversal rates, and accordingly, the transcytosis and tumor accumulation and penetration of the APN-responsive conjugates. The conjugate with moderate APN responsiveness balanced the in vitro transcytosis and in vivo overall drug delivery process and achieved the best tumor delivery efficiency, giving potent antitumor efficacy. This work provides new insight into the design of tumor enzyme-responsive charge-reversal nanomedicines for efficient cancer drug delivery.

Delivering cargo molecules across the plasma membrane is critical for biomedical research, and the need to develop molecularly well-defined tags that enable cargo transportation is ever-increasing. We report here a hydrophilic endocytosis-promoting peptide (EPP6) rich in hydroxyl groups with no positive charge. EPP6 can transport a wide array of small-molecule cargos into a diverse panel of animal cells. Mechanistic studies revealed that it entered the cells through a caveolin- and dynamin-dependent endocytosis pathway, mediated by the surface receptor fibrinogen C domain-containing protein 1. After endocytosis, EPP6 trafficked through early and late endosomes within 30 min. Over time, EPP6 partitioned among cytosol, lysosomes, and some long-lived compartments. It also demonstrated prominent transcytosis abilities in both in vitro and in vivo models. Our study proves that positive charge is not an indispensable feature for hydrophilic cell-penetrating peptides and provides a new category of molecularly well-defined delivery tags for biomedical applications.

We present a chemical approach to profile fatty acid uptake in single cells. We use azide-modified analogues to probe the fatty acid influx and surface-immobilized dendrimers with dibenzocyclooctyne (DBCO) groups for detection. A competition between the fatty acid probes and BHQ2-azide quencher molecules generates fluorescence signals in a concentration-dependent manner. By integrating this method onto a microfluidics-based multiplex protein analysis platform, we resolved the relationships between fatty acid influx, oncogenic signaling activities, and cell proliferation in single glioblastoma cells. We found that p70S6K and 4EBP1 differentially correlated with fatty acid uptake. We validated that cotargeting p70S6K and fatty acid metabolism synergistically inhibited cell proliferation. Our work provided the first example of studying fatty acid metabolism in the context of protein signaling at single-cell resolution and generated new insights into cancer biology.

Amphiphilic block copolymer prodrugs, which can self-assemble into stable core–shell micelles, have been widely used for anticancer drug delivery. However, a major challenge is to design drug-conjugating linkers stable in blood but selectively cleavable inside tumor cells for drug release. Hydrogen sulfide (H2S) is an important gaseous signaling molecule involved in tumor formation, development, and metastasis and is highly upregulated in multiple cancer types. Accordingly, we designed an H2S-sensitive azide-based linker and used it to conjugate paclitaxel (PTX) to a prodrug copolymer (PEG-PAMPTX). The amphiphilic PEG-PAMPTX self-assembled into micelles with a diameter of about 30 nm, stable under normal physiological conditions for long blood circulation. Once in tumors, the intratumoral H2S selectively reduced the azide group into an amino group, triggering self-cyclization to release PTX rapidly. As a result, the micelles showed remarkably enhanced antitumor activity compared to Taxol in a triple-negative breast cancer orthotopic model.

We present here a cyclic peptide ligand, cy(WQETR), that binds to terbium ion (Tb(3+)) and enhances Tb(3+) luminescence intensity through the antenna effect. This peptide was identified through screening a cyclic peptide library against Tb(3+) with an apparent EC(50) of 540 μM. The tryptophan residue from the peptide directly interacts with the Tb(3+) ion, which provides access to a low-lying triplet excited state of the tryptophan. Direct excitation of this triplet state enables energy transfer to the Tb(3+) ion and enhances Tb(3+) luminescence intensity by 150 fold. We further showcase the application of this cy(WQETR)-Tb(3+) system by demonstrating the detection of tromethamine with a detection limit of 0.5 mM.

Extravasation is the first step for nanomedicines in circulation to reach targeted solid tumors. Traditional nanomedicines have been designed to extravasate into tumor interstitium through the interendothelial gaps previously assumed rich in tumor blood vessels, i.e., the enhanced permeability and retention (EPR) effect. While the EPR effect has been validated in animal xenograft tumor models, accumulating evidence implies that the EPR effect is very limited and highly heterogeneous in human tumors, leading to highly unpredictable and inefficient extravasation and thus limited therapeutic efficacy of nanomedicines, including those approved in clinics. Enabling EPR-independent extravasation is the key to develop new generation of nanomedicine with enhanced efficacy. Transcytosis of tumor endothelial cells can confer nanomedicines to actively extravasate into solid tumors without relying on the EPR effect. Here, we review and prospectthe development of transcytosis-inducing nanomedicines, in hope of providing instructive insights for design of nanomedicines that can undergo selective transcellular transport across tumor endothelial cells, and thus inspiring the development of next-generation nanomedicines for clinical translation.

Here we present a protocol for interrogating AKT signaling activities in living single cells, using a pair of cyclic peptide-based fluorescent probes. These probes are encapsulated in liposomes and delivered into cells, where they continuously report on AKT signaling activities through a Föster resonance energy transfer mechanism. We describe the use of a microwell chip to achieve single-cell resolution and demonstrate the procedure for on-chip immunostaining. Finally, we provide a method for data extraction, correction, and processing.

Over the past three decades, the enhanced permeability and retention (EPR) effect has been considered the basis of tumor-targeted drug delivery. Various cancer nanomedicines, including macromolecular drugs, have been designed to utilize this mechanism for preferential extravasation and accumulation in solid tumors. However, such nanomedicines have not yet achieved convincing therapeutic benefits in clinics. Increasing evidence suggests that the EPR effect is over-represented in human tumors, especially in metastatic tumors. This review covers the evolution of the concept, the heterogeneity and limitation of the EPR effect in clinical realities, and prospects for alternative strategies independent of the EPR effect.

Celastrol, a natural triterpene extracted from traditional Chinese medicine, shows anticancer effects on various cancer cells. However, its poor water-solubility, short plasma half-life, and high systemic toxicity impede its applications in vivo, necessitating a stable drug delivery system to overcome these critical drawbacks. We present here a block copolymer, poly(2-(N-oxide-N,N-dimethylamino)ethyl methacrylate)-block-poly(2-hydroxyethyl methacrylate) (OPDMA-HEMA), as the carrier for celastrol delivery. The amphiphilic polymer-celastrol conjugate can self-assemble into nanoparticles in aqueous solutions. The OPDMA outer shell confers the nanoparticles with improved pharmacokinetics and efficient mitochondria targeting capacity, and profoundly potentiates celastrol's induction of immunogenic cell death, which collectively contribute to the enhanced therapeutic effects of celastrol in vivo. This mitochondria-targeted polymer-celastrol conjugate may promise the applications of celastrol in cancer treatment.

Analyzing intracellular signalling protein activities in living cells promises a better understanding of the signalling cascade and related biological processes. We have previously developed cyclic peptide-based probes for analyzing intracellular AKT signalling activities, but these peptide probes were not cell-permeable. Implementing fusogenic liposomes as delivery vehicles could circumvent the problem when analyzing adherent cells, but it remained challenging to study suspension cells using similar approaches. Here, we present a method for delivering these imaging probes into suspension cells using digitonin, which could transiently perforate the cell membrane. Using U87, THP-1, and Jurkat cells as model systems representing suspended adherent cells, myeloid cells, and lymphoid cells, we demonstrated that low concentrations of digitonin enabled a sufficient amount of probes to enter the cytosol without affecting cell viability. We further combined this delivery method with a microwell single-cell chip and interrogated the AKT signalling dynamics in THP-1 and Jurkat cells, followed by immunofluorescence-based quantitation of AKT expression levels. We resolved the cellular heterogeneity in AKT signalling activities and showed that the kinetic patterns of AKT signalling and the AKT expression levels were related in THP-1 cells, but decoupled in Jurkat cells. We expect that our approach can be adapted to study other suspension cells.

D-2-hydroxyglutarate (D2HG) is over-produced as an oncometabolite due to mutations in isocitrate dehydrogenases (IDHs). Accumulation of D2HG can cause the dysfunction of many enzymes and genome-wide epigenetic alterations, which can promote oncogenesis. Quantification of D2HG at single-cell resolution can help understand the phenotypic signatures of IDH-mutant cancers and identify effective therapeutics. In this study, we developed an analytical method to detect D2HG levels in single cancer cells by adapting cascade enzymatic reactions on a resazurin-based fluorescence reporter. The resazurin probe was immobilized to the sensing surface via biotin-streptavidin interaction. This surface chemistry was rationally optimized to translate the D2HG levels to sensitive fluorescence readouts efficiently. This D2HG assay demonstrated good selectivity and high sensitivity toward D2HG, and it was compatible with the previously developed single-cell barcode chip (SCBC) technology. Using the SCBC platform, we performed simultaneous single-cell profiling of D2HG, glucose uptake, and critical oncogenic signaling proteins in single IDH-mutant glioma cells. The results unveiled the complex interplays between metabolic and oncogenic signaling and led to the identification of effective combination targeted therapy against these IDH-mutant glioma cells.

We report on a cyclic peptide that inhibits matrix metalloproteinase-2 (MMP2) activation with a low-nM-level potency. This inhibitor specifically binds to the D(570)-A(583) epitope on proMMP2 and interferes with the protein–protein interaction (PPI) between proMMP2 and tissue inhibitor of metalloproteinases-2 (TIMP2), thereby preventing the TIMP2-assisted proMMP2 activation process. We developed this cyclic peptide inhibitor through an epitope-targeted library screening process and validated its binding to proMMP2. Using a human melanoma cell line, we demonstrated the cyclic peptide’s ability to modulate cellular MMP2 activities and inhibit cell migration. These results provide the first successful example of targeting the PPI between proMMP2 and TIMP2, confirming the feasibility of an MMP2 inhibition strategy that has been sought after for 2 decades.

Orally administrable anticancer nanomedicines are highly desirable due to their easy and repeatable administration, but are not yet feasible because the current nanomedicine cannot simultaneously overcome the strong mucus and villi barriers and thus have very low bioavailability (BA). Herein, this work presents the first polymeric micelle capable of fast mucus permeation and villi absorption and delivering paclitaxel (PTX) efficiently to tumors with therapeutic efficacy even better than intravenously administered polyethylene glycol based counterpart or free PTX. Poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate] (OPDEA), a water-soluble polyzwitterion, is highly nonfouling to proteins and other biomacromolecules such as mucin but can weakly bind to phospholipids. Therefore, the micelle of its block copolymer with poly(ε-caprolactone) (OPDEA-PCL) can efficiently permeate through the viscous mucus and bind to villi, which triggers transcytosis-mediated transepithelial transport into blood circulation for tumor accumulation. The orally administered micelles deliver PTX to tumors, efficiently inhibiting the growth of HepG2 and patient-derived hepatocellular carcinoma xenografts and triple-negative breast tumors. These results demonstrate that OPDEA-based micelles may serve as an efficient oral nanomedicine for delivering other small molecules or even large molecules.

We present here a library of protein mimetic bicyclic peptides. These nanosized structures exhibit rigid backbones and spatially diversifiable side chains. They present modular amino acids on all three linkages, providing access to a true 3D diversifiable chemical space. These peptides are synthesized through a Cu-catalyzed click reaction and a Ru-catalyzed ring-closing metathesis reaction. Their bicyclic topology can be reduced to a linear one, using Edman degradation and Pd-catalyzed deallylation reactions. The linearization approaches allow de novo sequencing through mass spectrometry methods. We demonstrate the function of a particular peptide that was identified through a high throughput screening against the E363-R378 epitope on the intrinsically disordered c-Myc oncoprotein. Intracellular delivery of this peptide could interfere with the c-Myc-mediated transcription and inhibit proliferation in a human glioblastoma cell line.

We describe an analytical method for profiling lactate production in single cells, via the use of coupled enzyme reactions on surface-grafted resazurin molecules. The immobilization of the redox-labile probes was achieved through chemical modifications on resazurin, followed by bio-orthogonal click reactions. The lactate detection scheme was demonstrated to be sensitive and specific. The method was incorporated into the single cell barcode chip platform for simultaneous quantification of aerobic glycolysis activities and oncogenic signaling phosphoproteins in cancer. We interrogated the interplay between glycolysis and oncogenic signaling activities on a glioblastoma cell line. Results revealed a drug-induced oncogenic signaling reliance accompanying shifted metabolic paradigms. A drug combination that exploits this induced reliance exhibited synergistic effects in growth inhibition.

Taking advantage of the highly permeable vasculature and lack of lymphatic drainage in solid tumors (EPR effect), nanosized drug delivery systems or nanomedicines have been extensively explored for tumor-targeted drug delivery. However, in most clinical cases tumors such as the early stage tumors and post-surgery microscopic residual tumors have not yet developed such pathological EPR features, i.e., EPR-deficient. Therefore, nanomedicines may not be applicable for such these tumors. Macrophages by nature can actively home and extravasate through the tight vascular wall into tumors and migrate to their hypoxic regions, and possess perfect stealth ability for long blood circulation and impressive phagocytosis for drug loadings. Thus, nanomedicines loaded in macrophages would harness both merits and gain the active tumor homing capability independent of the EPR effect for treatments of the EPR-deficient tumors. Herein, the critical considerations, current progress, challenges and future prospects of macrophages as carriers for nanomedicines are summarized, aiming at rational design of EPR-independent tumor-targeting active nanomedicines for targeted early and adjuvant cancer chemotherapy.

We present here a novel chemical method to continuously analyze intracellular AKT signaling activities at single-cell resolution, without genetic manipulations. A pair of cyclic peptide-based fluorescent probes were developed to recognize the phosphorylated Ser474 site and a distal epitope on AKT. A Förster resonance energy transfer signal is generated upon concurrent binding of the two probes onto the same AKT protein, which is contingent upon the Ser474 phosphorylation. Intracellular delivery of the probes enabled dynamic measurements of the AKT signaling activities. We further implemented this detection strategy on a microwell single-cell platform, and interrogated the AKT signaling dynamics in a human glioblastoma cell line. We resolved unique features of the single-cell signaling dynamics following different perturbations. Our study provided the first example of monitoring the temporal evolution of cellular signaling heterogeneities and unveiled biological information that was inaccessible to other methods.

Supramolecular interactions, such as those observed between antibodies and antigens, have been employed in developing analytical methods for several decades. One major area of interest concerns cancer research, where intricate supramolecular designs have emerged to tackle difficult analytes in complex tumor systems. Our increasing knowledge toward supramolecular systems have elicited profound interest in creating more efficient analytical approaches, evidenced by the ever-growing body of literature in the field. Some of the novel tools have indeed facilitated our understanding of cancer biology, through providing previously inaccessible information. In this review, we describe common strategies of developing supramolecular analytical methods and their implementations in cancer research. We provide an overview for each of the approaches and discuss representative examples in recent literature.

A tumour microenvironment imposes barriers to the passive diffusion of molecules, which renders tumour penetration an unresolved obstacle to an effective anticancer drug delivery. Here, we present a γ-glutamyl transpeptidase-responsive camptothecin–polymer conjugate that actively infiltrates throughout the tumour tissue through transcytosis. When the conjugate passes on the luminal endothelial cells of the tumour blood vessels or extravasates into the tumour interstitium, the overexpressed γ-glutamyl transpeptidase on the cell membrane cleaves the γ-glutamyl moieties of the conjugate to generate positively charged primary amines. The resulting cationic conjugate undergoes caveolae-mediated endocytosis and transcytosis, which enables transendothelial and transcellular transport and a relatively uniform distribution throughout the tumour. The conjugate showed a potent antitumour activity in mouse models that led to the eradication of small solid tumours (~100 mm3) and regression of large established tumours with clinically relevant sizes (~500 mm3), and significantly extended the survival of orthotopic pancreatic tumour-bearing mice compared to that with the first-line chemotherapeutic drug gemcitabine. A zwitterionic camptothecin–polymer conjugate that is enzymatically transformed in a cationic molecule at the tumour periphery penetrates deep into tumours via caveolae-mediated endocytosis and transcytosis, resulting in high anticancer efficiency in vivo.

Supramolecular interactions, such as those observed between antibodies and antigens, have been employed in developing analytical methods for several decades. One major area of interest concerns cancer research, where intricate supramolecular designs have emerged to tackle difficult analytes in complex tumor systems. Our increasing knowledge toward supramolecular systems have elicited profound interest in creating more efficient analytical approaches, evidenced by the ever-growing body of literature in the field. Some of the novel tools have indeed facilitated our understanding of cancer biology, through providing previously inaccessible information. In this review, we describe common strategies of developing supramolecular analytical methods and their implementations in cancer research. We provide an overview for each of the approaches and discuss representative examples in recent literature.

Novel classes of jellyfish-shaped amphiphilic dendrimers composed of 7 hydrophilic poly(ethylene glycol) (PEG) arms and 14 hydrophobic polyester dendrons with β-cyclodextrin (βCD) as the core molecule were synthesized by a facile method. Seven PEG chains were first conjugated to the C-6 positions of native βCD. Subsequently, dendritic polyester architectures were constructed from the remaining 14 secondary hydroxyl groups at C-2 and C-3 positions of the βCD moiety, resulting in jellyfish-shaped amphiphilic dendrimers of different generations (7PEG-βCD-Gx) with well-defined molecular structures. The amphiphilic dendrimers self-assembled into different morphologies dependent upon the hydrophilic fraction of the dendrimers, and very surprisingly, the fourth-generation dendrimers consisting of only several percent of PEG could form aggregates with extremely narrow size distributions.

The structural perfection and multivalency of dendrimers have made them useful for biodelivery and bioactivity via peripheral functionalization and the modulation of core-forming structures and dendrimer generations. Yet only few dendrimers have shown inherent therapeutic activity arising from their inner repeating units. Here, we report the synthesis and characterization of a polyacylthiourea dendrimer with inherent potent anticancer activity and the absence of cytotoxicity in mice. The poly(ethylene glycol)-functionalized fourth generation of the dendrimer, which can be efficiently synthesized from sequential click reactions of orthogonal monomers, displays low in vivo acute and subacute toxicities yet potently inhibits tumour growth and metastasis. The dendrimer’s in vivo anticancer activity arises from the depletion of bioavailable copper and the subsequent inhibition of angiogenesis and cellular proliferation. When compared with some clinically used cytotoxin drugs, the dendrimer exerts inherent anticancer activity via non-cytotoxic pathways and leads to higher therapeutic efficacy, yet without cytotoxin-induced side effects. A dendrimer that depletes bioavailable copper and leads to the suppression of tumour angiogenesis acts as a potent and non-cytotoxic anticancer therapeutic.