The calcium carbonate drug delivery systems (DDSs) provide a very promising strategy for various types of treatments, including chemo- and radiotherapies. Effective design of this platforms including surface engineering and employing organic/inorganic additives has a significant influence on drug loading, stability and drug release. In this study, calcium carbonate microparticles (mCa) and nanoparticles (nCa) were synthesized for drug loading of novel therapeutic molecules and subjected to the so-called 'cold' labeling procedure, i.e. efficient non-radioactive synthetic routes that can be applied to radionuclide labeling. In this context, the effect of 'cold' labeling conditions on the morphology, size, drug loading, colloidal stability and drug release profiles of mCa and nCa was fully assessed. The in vitro cytotoxicity, cellular uptake and hemolysis were examined on 'cold' mCa and nCa to reveal any impacts. Furthermore, we analyzed in vivo biodistribution of 'cold' mCa and 'cold' nCa and their estimated therapeutic efficiency after drug loading. The main characteristics of mCa and nCa were collected and compared with each other. It was shown that mCa and nCa can be effectively accumulated within the tumors in vivo without any release into healthy organs. Notably, the 'cold' labeling procedure did not have an impact on the therapeutic effect of drug-loaded mCa and nCa. Due to this, the developed pharmaceutical forms of mCa and nCa have remarkable antitumor effects against melanoma showing tumor inhibition of ∼76-84% without any significant side effects. This investigation can contribute to the development of calcium carbonate particles as a universal multifunctional delivery platform for effective tumor therapy and simultaneously accelerates clinical translation for various types of treatments.

Ionizable lipids, widely employed as pharmaceutical excipients for gene delivery, exhibit acidity-responsive membrane-destabilizing activity. This property prompted us to hypothesize that such membrane-destabilizing activity could be harnessed and selectively confined to the acidic tumor microenvironment (aTME) to achieve tumor-selective oncolysis. Herein, we report the rational design of tumor acidity-activatable oncolytic lipid nanoparticles (aoLNPs) through programmed structural modulation of ionizable lipids. By designing and screening an ionizable lipid library with systematic variations in alkyl tail length and number, we found a clear correlation between the total tail carbon number and the pH-dependent cytotoxicity of the corresponding lipid nanoparticles. Subsequent library expansion led to the identification of aoLNPE14A6-2 as the optimal candidate, which exhibited selective cytotoxicity toward cancer cells at pH 6.8 and demonstrated potent antitumor efficacy in vivo. Mechanistic investigations revealed that aoLNPE14A6-2 selectively induces lysosomal vacuolation and endoplasmic reticulum stress under tumor acidic conditions, ultimately leading to plasma membrane rupture. Notably, aoLNPE14A6-2 did not cause obvious chronic toxicity or structural damage to normal tissues after intravenous injection. This study expands the application of ionizable lipids for selective oncolytic therapy.

The differences in the phospholipid profile between bacterial and mammalian membranes present a promising direction for designing antimicrobial peptides (AMPs). A key challenge, however, is engineering AMPs with a high affinity for bacterial anionic phospholipid (phosphatidylglycerol, PG) while minimizing off-target interactions with mammalian phosphatidylserine (PS). Herein, we report a multiarmed radially amphiphilic AMP (maRAP) design that enhances PG recognition, exhibiting potent antibacterial activity and exceptional antibacterial selectivity. Compared to the single-arm RAP (1aRAP), maRAPs exhibited superior affinity for PG, yet they exhibited negligible binding to PS. They also demonstrated significantly enhanced bactericidal efficacy and selectivity over 1aRAP in complex physiological environments. In the murine bladder-infection model, maRAPs more effectively reduced the bacterial load and attenuated infection-induced tissue damage, as demonstrated by an intact urothelial lining and minimal lamina propria edema at the infection site. This work presents the multiarm strategy as an effective approach for advanced AMP development.

A rational design for facilitating innovative cell death modes can substantially aid advances in antitumour therapy1,2. Here we design and implement a unique mode of immunogenic membranolytic cell death (mLCD) in tumour cells, characterized by time-lagged rupture from the lysosomal to plasma membranes; this approach was found to robustly potentiate immune checkpoint blockade therapy. This mode of mLCD was induced by the synthetic-acid-responsive membranolytic peptide (aMP) aMPC16-CA50, which exhibits hierarchical responsiveness to the decreasing pH associated with the tumour extracellular environment and lysosomes. aMPC16-CA50 activated an inflammatory transcriptional program in tumour cells, potentiating their ability to induce antigen presentation on class I major histocompatibility complex molecules on dendritic cells and the subsequent activation of T cells. The pH-responsive kinetics and membranolytic activity of the membranolytic peptides had a critical role in enhancing the immunogenicity of lytic tumour cells through the spatiotemporal regulation of the membrane-rupture processes. Furthermore, aMPC16-CA50 exhibited a considerable advantage in enhancing the antitumour efficacy of immune checkpoint blockade therapy through the promotion of antitumour immune response. Moreover, its systemic administration was well tolerated in mice. Overall, we successfully programmed a unique mode of immunogenic mLCD in tumour cells through the spatiotemporal regulation of membrane-rupture processes using a synthetic pH-responsive membranolytic peptide amenable to manipulation.

Endosomal escape is a limiting factor for the in vivo application of nucleic acid therapeutics and remains a major challenge in the development of drug delivery systems. In this study, we developed a pH-ultrasensitive membranolytic polymer (P(C6-BnX))-assisted delivery system (NPBnX) to facilitate siRNA endosomal escape and enhance the gene silencing efficiency. The incorporation of P(C6-BnX) imparts the siRNA delivery system with enhanced cellular uptake, effective membranolytic activity under endosomal pH conditions, and improved endosomal escape efficiency of siRNA. The significantly enhanced siRNA-mediated gene silencing efficacy was also confirmed in multiple tumor cell lines. Further investigations demonstrated that the delivery of siRNA targeting CD47 via this system effectively suppressed the expression of CD47 at both the cellular level and in vivo, thereby enhancing macrophage-mediated phagocytosis of tumor cells and promoting anti-tumor immune responses. This study provides a promising and viable strategy for the design and development of siRNA delivery systems.

Cancer vaccines have gained considerable attention for tumor prevention and therapy; however, their clinical efficacy remains limited by insufficient activation of antigen-specific cytotoxic T lymphocytes (CTLs). This limitation is primarily due to the inability of conventional vaccine components to effectively promote dendritic-cell-mediated cross-presentation and activation. While adjuvants are often used to enhance this interaction, many conventional adjuvants suffer from poor biocompatibility and systemic toxicity, limiting their clinical utility. Here, we report a vaccine strategy that leverages the bioorthogonal click chemistry reagent dibenzocyclooctyne (DBCO) as an adjuvant-like immune enhancer to promote cross-presentation and a CTL response. DBCO modification to antigen significantly increased antigen uptake by dendritic cells in vitro and promoted MHC class I-mediated cross-presentation via activation of the NF-κB/iNOS signaling pathway. In murine tumor models, systemic administration of DBCO-conjugated protein vaccines elicited enhanced CD8+ T-cell activation, leading to improved tumor control and long-term immune memory. Furthermore, DBCO-modified protein vaccines significantly suppressed tumor metastasis when combined with anti-PD-1 treatment. Collectively, our findings extend the immunotherapeutic potential of click chemistry reagents and establish a promising platform for enhancing CTL-mediated cancer vaccine efficacy in clinical settings.

pH-ultra-sensitive oncolytic polymers hold great promise for cancer therapy due to their selective oncolytic activity in the tumor microenvironment. However, achieving well-defined structures and finely tunable pH responsiveness for these polymers remains a challenge. Herein, we developed a straightforward blending strategy using two well-defined alternating polyesters with distinct pH sensitivities to create pH-ultra-sensitive hybrid polyester nanoparticles (Hps) with precisely engineered responsiveness. By varying the mass ratio of the two components, the pKa of the Hps could be continuously tuned from 6.37 to 7.04. The resulting Hps demonstrated a sharp pH-dependent membranolytic activity switch within a 0.3 pH unit window, with minimal cytotoxicity observed at pH values above the transition pH. A lead Hp formulation identified through screening exhibited potent cytotoxicity at pH 6.8, along with a favorable safety profile and significant antitumor efficacy in vivo. This blending strategy establishes a flexible and simple platform for customizing pH responsiveness of oncolytic polymers, advancing oncolytic therapy.

Imaging-guided radiotherapy can improve the precision and therapeutic efficacy of prostate cancer (PCa). However, advanced PCa often exhibits diffuse intraprostatic infiltration, extracapsular extension, and intrinsic radioresistance, complicating accurate tumor identification and adaptive radiation delivery without increasing damage to surrounding healthy tissues. Herein, we report the design of prostate-specific membrane antigen (PSMA) targeted platinum(IV) prodrug nanoprobes (GPNPs) with high MR imaging sensitivity and PSMA specificity for enhanced MR-guided chemoradiotherapy of locally advanced PCa. The GPNPs are constructed through the co-assembly of a Pt(IV)-PSMA-1 targeting prodrug and a Pt(IV)-Gd(III) imaging prodrug, yielding stable nanoprobes with r₁ relaxivity as high as 18.59 mM-1 s-1. Through selective targeting of PSMA-positive tumor cells, GPNPs achieved enhanced cellular uptake and tumor accumulation, enabling more accurate MR imaging and tumor lesion identification. The GPNPs underwent activation at reductive tumor microenvironment and upon X-ray irradiation, they can overcome radioresistance by improved reactive oxygen species generation and DNA damage amplification. The GPNPs induced robust tumor growth inhibition in vivo with minimal systemic toxicity, while concurrently suppressing lung metastasis through enhanced CD8+ T-cell infiltration and increased effector cytokine production. These results highlight GPNPs as a potent and versatile nanoplatform for precision imaging of locally advanced PCa and enhanced MR-guided chemoradiotherapy.

Bacterial biofilm infections, a key contributor to antibiotic resistance, pose a critical global health challenge. Although antimicrobial peptides are promising candidates, their cationic amphipathic structures often lead to nonspecific sequestration by polyanionic biofilm matrix components. Here, we report a class of primary amine-functionalized radially amphiphilic antimicrobial polypeptides (paRAPs) that achieve potent antibiofilm activity by selectively targeting bacterial phosphatidylglycerol (PG) in polyanionic biofilm matrices. Simulation studies support a mechanism of PG-responsive structural rearrangement in paRAPs. In contrast to the compact form of quaternary amine analogs, paRAPs adopt an extended conformation, with outward-facing cationic amine termini that shield the hydrophobic core and thereby reduce nonspecific protein binding. Upon encountering bacterial membranes, strong PG recognition triggers a side-chain rearrangement, reorienting the cationic groups toward the membrane surface and exposing hydrophobic motifs for progressive bilayer insertion and disruption. Supportingly, lengthening the exposed terminal hydrophobic group increased interactions with proteins and mammalian lipids, reduced PG selectivity, and compromised antibiofilm efficacy, underscoring the importance of hidden hydrophobic domains for biofilm bacteria targeting. paRAP showed potent antibiofilm efficacy in vitro and in murine models of both periodontitis and urinary tract infections. Our study provides a PG-targeting strategy for designing matrix-resistant antibiofilm polypeptides.

Ionizable lipids, widely employed as pharmaceutical excipients for gene delivery, exhibit acidity-responsive membrane-destabilizing activity. This property prompted us to hypothesize that such membrane-destabilizing activity could be harnessed and selectively confined to the acidic tumor microenvironment (aTME) to achieve tumor-selective oncolysis. Herein, we report the rational design of tumor acidity-activatable oncolytic lipid nanoparticles (aoLNPs) through programmed structural modulation of ionizable lipids. By designing and screening an ionizable lipid library with systematic variations in alkyl tail length and number, we found a clear correlation between the total tail carbon number and the pH-dependent cytotoxicity of the corresponding lipid nanoparticles. Subsequent library expansion led to the identification of aoLNPE14A6-2 as the optimal candidate, which exhibited selective cytotoxicity toward cancer cells at pH 6.8 and demonstrated potent antitumor efficacy in vivo. Mechanistic investigations revealed that aoLNPE14A6-2 selectively induces lysosomal vacuolation and endoplasmic reticulum stress under tumor acidic conditions, ultimately leading to plasma membrane rupture. Notably, aoLNPE14A6-2 did not cause obvious chronic toxicity or structural damage to normal tissues after intravenous injection. This study expands the application of ionizable lipids for selective oncolytic therapy.

Antimicrobial peptide (AMP)-based treatments have exhibited significant therapeutic potential with anti-inflammatory, anticancer, and immunomodulatory activities. While most AMPs exert effects by permeabilizing bacterial cell membranes through poration and hydrolysis, recent findings have revealed a distinct membrane interaction mechanism employed by L10-MMBen, a cationic amphiphilic helical peptide with potent antimicrobial efficacy and moderate cytotoxicity. Herein, we elucidate the ability of L10-MMBen to remodel bacterial cell membranes into double-layered structures rather than inducing permeabilization. Quantitative real-time giant unilamellar vesicle assays, atomic force microscopy characterizations, and simulations demonstrate that L10-MMBen selectively adsorbs onto phosphatidylglycerol-containing membranes with shallow insertion at approximately 0.6 nm; once reaching a threshold local concentration (peptide-to-lipid ratio of 1:20), it extracts lipids from the bilayer and facilitates the formation of double-layered peptide-membrane composite structures. Single-molecule tracking analysis indicates that peptide-induced molecular reorganization significantly reduces membrane fluidity, impeding lipid lateral diffusion from 2.8 μm2/s to an immobile state. These findings may contribute to the design of innovative membrane-active agents for biomedical applications.

Over the past 120 years, significant efforts are dedicated to delivering maximum radiation doses to tumor sites while sparing adjacent normal tissues as much as possible. Despite encouraging progress in the development of heavy metal-based nanoscale radiosensitizers, radiotherapy often fails to fully eradicate hypoxic tumors, leading to local recurrence or even progression to distant metastasis. In this study, a versatile biomimetic hybrid radiosensitizer is engineered by integrating the hypoxia-activated prodrug banoxantrone and CeO2 nanozymes into mesoporous silica-coated Bi2O3 nanoparticles (NPs), followed by camouflage coating with cancer-cell-derived membranes. Compared to naked Bi2O3 NPs and free banoxantrone alone, the radiosensitization efficacy of the biomimetic NPs is substantially enhanced toward both normoxic and hypoxic cancer cells. Moreover, lung metastasis is markedly inhibited by reactive oxygen species-mediated remodeling of the extracellular matrix through the activity of CeO2 nanozymes. As confirmed by in vitro and in vivo results, the biomimetic hybrid radiosensitizer enhances radiotherapy against lung metastasis with fewer side effects. This study provides compelling evidence for the development of next-generation radiosensitizers with optimized functionalities using biomimetic hybrid engineering to finely balance the benefits and risks of radiotherapy.

The distinct phospholipid compositions of bacterial and mammalian cell membranes offer a promising target for the development of antimicrobial peptides (AMPs). However, distinguishing between the similarly charged anionic phospholipids-bacterial phosphatidylglycerol (PG) and mammalian phosphatidylserine (PS)-poses a significant challenge. Here we introduce a competitive inhibition strategy that leverages host-guest interactions to enable AMPs to selectively recognize PG without engaging with PS. After analyzing the binding interactions of various radially amphiphilic AMPs (RAPs), host molecules, and phospholipids, we discovered that a RAP, named C6HO, exhibited a higher affinity for cucurbit[7]uril (CB[7]) compared to PS, yet a lower affinity than for PG. Consequently, CB[7] functions as a competitive inhibitor: by forming a complex with C6HO upon simple mixing, it prevents C6HO from interacting with PS. Notably, PG can outcompete CB[7] for binding to C6HO within the complex, leading to the aggregation of PG molecules and the subsequent disruption of membranes rich in PG. Furthermore, the competitive inhibitor CB[7] effectively neutralizes C6HO's cytotoxic effects on mammalian cells while preserving the antimicrobial potency of C6HO. In vivo experiments in a subcutaneous infection model demonstrated that CB[7] reduced both systemic and local toxicity of C6HO without compromising its antimicrobial efficacy. Our study presents a strategy for the specific recognition of bacterial phospholipids and the design of highly selective AMPs.

Surface immobilization of antimicrobial peptides (AMPs) on implants offers promising efficacy against drug-resistant bacterial infections. The cationically amphipathic structure of AMPs, while crucial for their potent antimicrobial activity, poses substantial challenges for achieving scalable coatings on implants and contributes to their cytotoxicity toward normal tissues/cells. Herein, we developed hydrophobicity-to-cationic amphipathicity transformable AMPs (HAT-AMPs) for scalable coating on medical implant, effectively preventing bacterial infections while exhibiting low cytotoxicity to normal tissues. The HAT-AMPs, composed of ionizable and hydrophobic residues, are hydrophobic and electrically neutral at physiological pH condition, making them highly suitable for physical immobilization on titanium surfaces while exhibiting minimal toxicity to normal tissues. In acidic environments during infections, the protonation of ionizable units induces HAT-AMPs to adopt a cationically amphipathic structure, remarkably enhancing their antimicrobial activity by targeting bacterial phospholipid phosphatidylglycerol. When immobilized on titanium implant surface, the coating exhibited a favorable safety profile, and demonstrated strong antibacterial efficacy and promising wound-healing potential in a subcutaneous infection mouse model. This strategy offers an efficient approach for fabricating infection-responsive implant coatings.

The highly selective ability of cytotoxic T lymphocytes (CTLs) to eliminate tumor cells has inspired the development of artificial nanomedicine for effective cancer therapy. Here, an artificial CTL (aCTL) that selectively recognizes and kills cancer cells is developed. The membrane target-binding moiety, membrane-perforating peptides, and therapeutic prodrugs are integrated into the aCTL through supramolecular interactions. Consequently, the prepared aCTL selectively binds cancer cells and perforates the surrounding membrane. Subsequently, the conjugated prodrug is cleaved and released by the overexpressed membrane transpeptidase, allowing entry into tumor cells through the perforated holes. Finally, the aCTL significantly suppresses the growth of HepG2 hepatocellular carcinoma, B16-F10 melanomas, and patient-derived xenograft (PDX) breast cancer models.

The growing concern over bacterial multidrug resistance has led to a heightened interest in antimicrobial peptides (AMPs) known for their strong efficacy, albeit with high toxicity. In order to retain the potent antibacterial activity of AMPs while minimizing their systemic toxicity, we introduce a conjugate strategy with selective infection-activatable and bacteria-targeting capabilities. A series of colistin prodrugs with well-defined structures were developed by attaching a reactive oxygen species-responsive phenylboronic acid linker to all amines of colistin, followed by covalent linkage to sugars through the reaction of boronic acid and a diol moiety. Among these prodrugs, a lactosyl-functionalized colistin prodrug, LaP-Col, was identified because of its minimal toxicity toward normal tissues and ability to target bacteria. It exhibits a maximum tolerated dose over 20 times greater than colistin and minimal nephrotoxicity. In a mouse model of bacterial pneumonia, intravenous administration of LaP-Col led to its accumulation in the infected lung. It effectively killed bacteria, significantly improving the therapeutic efficacy and mouse survival rates. Overall, this study presents a prodrug strategy that effectively reduces the inherent high toxicity of colistin and enhances its therapeutic targeting efficacy, which has promising implications for modifying other potent but toxic AMPs.

Transistor-like nanoprobes enable highly sensitive detection of dysregulated pH, showing great promise for tumor imaging. Their pH sensitivity arises from protonation of ionizable tertiary amines on the polymer side chains, which can increase the interaction with negatively charged cell membranes, raising risks of cytotoxicity and biosafety. Herein, we present a platform of pH-ultrasensitive polyester nanoprobes (pUPNs) that exhibit minimal toxicity upon activation and feature tunable pH responsiveness. The pUPNs self-assemble from pH-responsive polyesters, whose backbones possess high steric hindrance to minimize interactions with cell membranes. Their side chains are covalently grafted with ionizable tertiary amines and self-quenched fluorescent dyes to finely adjust the sensitivity to subtle pH variations. Interestingly, pUPN with the lowest transition pH exhibits the best tumor imaging contrast. These pUPNs demonstrated excellent biocompatibility after intravenous administration with no detectable hepatic or renal toxicity. This study presents a design platform for pH-ultrasensitive nanoprobes with excellent sensitivity and biocompatibility through increased steric hindrance in the polymer backbone.

The helical conformation of antimicrobial peptides (AMPs) exerts a dual effect: it enhances bactericidal activity while concurrently increasing cytotoxicity by facilitating penetration into mammalian cells, leading to organelle damage. Herein, we report a bacterial phospholipid-inducible, helix-transformable antimicrobial polypeptide (HT-AMP) for enhancing the antimicrobial selectivity. The HT-AMP, C6-10, which has a charge-to-backbone span of 10 σ-bonds, adopts a moderate intrinsic helicity of 38% due to side-chain charge repulsion. This restrained conformation significantly reduces AMP penetration into mammalian cells, thereby minimizing mitochondrial damage. C6-10 shows a high affinity for the bacterial phospholipid phosphatidylglycerol (PG). Upon PG recognition, the side-chain charge repulsion of C6-10 is reduced, and its helicity increases to ∼77%, exhibiting robust antibacterial activity. Further extending the hydrophobicity of the C-terminal group increases the helicity of polypeptides, leading to enhanced mammalian cellular internalization and mitochondrial damage. C6-10 demonstrated low toxicity toward organs following intravenous administration and exhibited significant antibacterial efficacy in both a bladder infection model and a sepsis model. Overall, this PG-triggered helix-transformable strategy provides an effective approach to improving the antibacterial selectivity of AMPs.

Molecular imaging is a non-invasive imaging method that is widely used for visualization and detection of biological events at cellular or molecular levels. Stimuli-responsive linkers that can be selectively cleaved by specific biomarkers at desired sites to release or activate imaging agents are appealing tools to improve the specificity, sensitivity, and efficacy of molecular imaging. This review summarizes the recent advances of stimuli-responsive linkers and their application in molecular imaging, highlighting the potential of these linkers in the design of activatable molecular imaging probes. It is hoped that this review could inspire more research interests in the development of responsive linkers and associated imaging applications.

The fundamental differences in phospholipids between bacterial and mammalian cell membranes present remarkable opportunities for antimicrobial design. However, it is challenging to distinguish bacterial anionic phospholipid phosphatidylglycerol (PG) from mammalian anionic phosphatidylserine (PS) with the same net charge. Here, we report a class of radially amphiphilic α helix antimicrobial peptides (RAPs) that can selectively discriminate PG from PS, relying on the helix structure. The representative RAP, L 10 -MMBen, can direct the rearrangement of PG vesicles into a lamellar structure with its helix axis parallel to the PG membrane surface. The helical structure imparts both the thermodynamic and kinetic advantages of L 10 -MMBen/PG assembly, and the hiding of hydrophobic regions in RAPs is crucial for PG recognition. L 10 -MMBen exhibits high selectivity against bacteria depending on PG recognition, showing low in vivo toxicity and significant treatment efficacy in mice infection models. Our study introduces a helicity-direct bacterial phospholipid recognition paradigm for designing highly selective antimicrobial peptides.