Molecular Pharmaceutics最新文献

The programmed cell death protein-1/programmed cell death ligand-1 (PD-1/PD-L1) axis plays a central role in tumor immune regulation, with PD-L1 expression serving as a critical biomarker for patient stratification and response prediction. Accurate, noninvasive assessment of PD-L1 expression is, therefore, essential for guiding clinical decision-making. KN035 is an ∼79.6 kDa fusion protein comprising a humanized single-domain antibody linked to an Fc fragment, offering a smaller molecular size than conventional monoclonal antibodies. In this study, KN035 was conjugated with p-SCN-Bn-NOTA and radiolabeled with ⁶⁴Cu to generate [⁶⁴Cu]Cu-NOTA-KN035 for PET imaging of PD-L1. The tracer showed high radiochemical purity (>95%) and strong binding specificity in vitro. In vivo PET imaging and biodistribution studies were performed in H1975 (high PD-L1 expression) and A549 (low PD-L1 expression) nonsmall cell lung cancer (NSCLC) xenograft models. Clear tumor visualization was achieved at 4 h postinjection (5.62 ± 0.55%ID/g in H1975; 4.16 ± 0.18%ID/g in A549), with peak uptake at 48 h (12.32 ± 0.66 and 5.72 ± 0.21%ID/g, respectively). Tumor uptake decreased significantly after blocking with excess KN035, confirming the specificity. These results demonstrate the high PD-L1-targeting specificity of [⁶⁴Cu]Cu-NOTA-KN035, suggesting its great potential as a noninvasive diagnostic tool for immunotherapy-based treatments in the future.

Pretargeting based on the inverse electron-demand Diels–Alder reaction between a tetrazine and a trans-cyclooctene-modified (TCO) antibody has emerged as a powerful approach to the imaging and therapy of cancer. However, poorly constructed immunoconjugates with TCOs in ill-defined places often leads to suboptimal image contrast in pretargeted immune-PET and SPECT. To address these limitations, site-specific and site-selective approaches to antibody bioconjugation may be used for pretargeted imaging. Herein, we present a comparative study between stochastically, site-specifically, and site-selectively modified immunoconjugates for pretargeted SPECT. First, a trio of immunoconjugates─TCO–CC49, TCO-^Gly(Az)CC49, TCO-^PFP(Az)CC49─were produced with active TCO moieties sufficient for tetrazine ligation. All immunoconjugates retained high affinity for their target antigen, TAG-72, in enzyme-linked immunosorbent assay experiments and in antigen-expressing tumor tissue. A DOTA-modified tetrazine was labeled with indium-111 in high radiochemical yield and used for in vivo experiments. Then, mice bearing subcutaneous LS 174T xenografts received either TCO–CC49, TCO-^Gly(Az)CC49, TCO-^PFP(Az)CC49 followed 72 h later by [¹¹¹In]In-DOTA-Tz (16–20 MBq). SPECT scans were acquired at 1 and 24 h postinjection of radiotracer, and all three constructs produced clear tumor delineation. Ex vivo biodistribution data at 24 h showed [¹¹¹In]In-DOTA-Tz–TCO–CC49 with the highest tumoral uptake (10.4 ± 1.2% ID/g) probably due to the highest TCO loading, but [¹¹¹In]In-DOTA-Tz–TCO-^Gly(Az)CC49 and [¹¹¹In]In-DOTA-Tz–TCO-^PFP(Az)CC49 increased tumor-to-blood and tumor-to-lung ratios. Ultimately, this comparative study serves as a critical step toward identifying the optimal bioconjugation strategy that may be used in pretargeted systems.

The theranostic approach, which employs diagnostic radiopharmaceuticals to select patients who would benefit from targeted radiotherapy agents, has become an invaluable strategy for effective medical care. Scandium radionuclides offer the advantage of forming elementally matched and chemically identical diagnostic and therapeutic compounds, making them ideal candidates for this strategy. PSMA-617 is an established prostate-specific membrane antigen targeting agent and can be used as a proof of concept to investigate ⁴³Sc, the diagnostic nuclide, and ⁴⁷Sc, the therapeutic nuclide, as a theranostic pair. Methods: Cellular uptake, competitive binding assays, and internalization studies were carried out using LNCaP or PC-3 cell lines. [⁴³Sc]Sc-PSMA-617 was used in PET imaging studies in LNCaP or PC-3 tumor models, with time points ranging from 1–9 h. LNCaP tumor-bearing mice injected with [⁴⁷Sc]Sc-PSMA-617 were imaged using SPECT up to 48 h. A longitudinal study was carried out using LNCaP tumor-bearing mice imaged with [⁴³Sc]Sc-PSMA-617 prior to receiving a therapeutic dose of [⁴⁷Sc]Sc-PSMA-617. Results: ⁴³Sc and ⁴⁷Sc were incorporated into PSMA-617 at radiochemical yields of >99%. Cellular uptake studies demonstrated high uptake and specificity to PSMA receptors for [⁴⁷Sc]Sc-PSMA-617. In vivo PET studies showed specificity of [⁴³Sc]Sc-PSMA-617 while SPECT studies demonstrated tumor retention of [⁴⁷Sc]Sc-PSMA-617 up to 48 h. [⁴⁷Sc]Sc-PSMA-617 demonstrated therapeutic efficacy by delaying tumor growth and increasing survival rates from a single administered dose in xenograft models. More importantly, the PET results from [⁴³Sc]Sc-PSMA-617 PET were highly correlated with the therapeutic response from [⁴⁷Sc]Sc-PSMA-617, showing that ⁴³Sc PET data can predict therapeutic outcomes in individual animals from ⁴⁷Sc agents, even in animals sharing a genetic background and implanted with tumors from the same cell line. Conclusions: Two chemically identical, PSMA-targeting radioscandium pharmaceuticals demonstrated in vivo stability, specificity and retention in PSMA+ tumor models. A theranostic study showed that a higher ⁴³Sc PET SUV_mean was strongly correlated to therapeutic response from the ⁴⁷Sc agent, demonstrating that ⁴³Sc and ⁴⁷Sc can be used as an elementally matched theranostic pair.

Cell-penetrating peptides (CPPs) and supercharged proteins (SPs) enable efficient intracellular delivery of macromolecules, with expanding applications in basic research and in therapeutic development. Despite their potential, reproducible workflows for isolation, biochemical characterization, and quantitative uptake analysis remain limited. Here, we present a comprehensive and replicable protocol for the isolation, characterization, and cellular uptake analysis of CPP-fusion proteins (CPP-FPs) and SPs using methyl-CpG-binding protein 2 (MeCP2) constructs as a proof-of-principle model. This workflow combines native protein purification with dynamic light scattering (DLS)-based buffer optimization. Cellular uptake is then assessed and quantified under live-cell conditions using high-content imaging and imaging flow cytometry, with additional assays to probe endocytic trafficking routes, identify CPP-like motifs in SPs, and validate transducing CPP-FP/SP functionality. The protein isolation and DLS-guided buffer screen yield samples with long-term stability. Live-cell fluorescence microscopy and imaging flow cytometry enable discrimination between membrane-bound and internalized signal, providing higher accuracy compared to plate-based readouts. MeCP2 sequence probing has revealed the presence of a CPP-like motif that is critical to its internalization. Finally, validation assays clearly demonstrated CPP-FP/SP activity. This protocol integrates advances in protein biochemistry, structural analysis, and live-cell imaging into a reproducible pipeline adaptable to a wide range of CPP- and SP-based protein constructs and provides a practical framework for downstream mechanistic and therapeutic interventions.

Tumor hypoxia is a major contributor to therapeutic resistance, including radio-, chemo-, and immunotherapy, and is closely linked to unfavorable clinical outcomes. PET imaging has shown promise in guiding the clinical treatment of hypoxic tumors. Here, we developed an NTR-responsive PET strategy for in vivo tumor hypoxia imaging using radiolabeled nitrogen mustard analogues (¹⁸F-NTRP and ¹⁸F-NCRP). Cell uptake studies with A549 cells showed that ¹⁸F-NTRP and ¹⁸F-NCRP exhibited approximately two fold higher uptake and good retention under hypoxic conditions. MicroPET imaging of A549 tumor-bearing mice revealed pronounced tumor accumulation of both ¹⁸F-NTRP and ¹⁸F-NCRP, with uptake values of 1.95 ± 0.37 and 2.86 ± 0.49 %ID/g, respectively, and a rapid clearance rate in normal organs. At 45 min postinjection, the tumor/muscle (T/M) ratios of ¹⁸F-NTRP and ¹⁸F-NCRP were 2.62 and 3.44, respectively, both substantially higher than that of ¹⁸F-FMISO (1.40, p < 0.05). Following dicoumarin-mediated inhibition of tumor NTR, the uptake and T/M ratio of ¹⁸F-NCRP were decreased to 1.25 ± 0.32 %ID/g and 1.26 (p < 0.001), respectively. Moreover, T/M ratios correlated well with HIF-1α (r² = 0.71) and NTR (r² = 0.66) levels in the A549 tumor. Collectively, PET imaging demonstrated that ¹⁸F-NCRP specifically targets tumor NTR and can potentially discriminate between varying degrees of hypoxia.

Nanomaterials have shown great potential in promoting wound healing; however, most studies focus only on describing their apparent functions, lacking an in-depth exploration of their underlying molecular mechanisms. This has significantly hindered their clinical translation and application. Herein, this study developed a nanomaterial, carbon quantum dots (Ct-CQDs), using natural catechin as a precursor, with a focus on systematically elucidating the precise molecular mechanism by which Ct-CQDs promote wound healing. Through comprehensive in vitro cell experiments, we confirmed that Ct-CQDs exhibit biosafety and the ability to promote cell migration. Meanwhile, this study is the first to reveal that Ct-CQDs promote wound healing by specifically activating phosphorylation of ERK and p38 in the MAPK signaling pathway. Innovatively, we used specific inhibitors (PD98059 and SB203580) to verify the mechanism both in vitro and in animal models, confirming that once the ERK/p38 pathway is blocked, the wound-healing-promoting effect of Ct-CQDs is significantly inhibited. In conclusion, this study provides a theoretical basis for the development of novel nano wound dressings based on natural products and offers solid theoretical and experimental support for their application as a nanodrug therapeutic strategy with clear mechanisms, high efficiency, and safety.

Silica nanoparticles are widely studied nanomaterials for biomedical applications owing to their tunable physicochemical properties, such as size, porosity, geometry, and surface modification. Despite their promising potential, concerns regarding their safety continue to limit clinical translation. In this study, we systematically investigated how key physicochemical parameters and surface attachment of poly(ethylene glycol) (PEG) affect the cytotoxicity and immune activation profiles of silica nanoparticles in macrophages. A structurally diverse set of silica nanoparticles (rod, spherical, porous, nonporous, and surface-modified) was synthesized and characterized. RAW 264.7 macrophages were used as a model cell line to evaluate nanoparticle internalization, membrane integrity, apoptosis, cell cycle progression, and macrophage activation. While PEGylation and physicochemical variations significantly influenced both cellular uptake and maximum nontoxic dose, none of the tested nanoparticles impaired macrophage viability or baseline functionality at their respective saturation points. Notably, PEGylated silica nanoparticles approximately 100 nm in diameter and rod-shaped nanoparticles elicited pronounced immune activation, highlighting their distinct immunomodulatory potential despite the preserved cellular integrity.

In the treatment of breast cancer, loading chemotherapy drugs into nanoparticles can accumulate in tumor tissues by virtue of the enhanced permeability and retention (EPR) effect, thereby reducing the systemic toxicity of chemotherapy drugs and improving the therapeutic effect. However, the interior of solid tumors contains a dense extracellular matrix (ECM) composed largely of cancer-associated fibroblasts (CAFs), which severely hinders the deep penetration of drugs and limits their full therapeutic efficacy. Although the antifibrotic effects of α-mangostin (α-M) have been reported, its potential in remodeling the tumor microenvironment (TME) to synergistically enhance nanomedicine penetration and antimetastatic efficacy remains underexplored. For this reason, our study proposes a combined therapeutic strategy that inhibits the activity of CAFs through the antifibrotic drug α-mangostin, in order to promote better penetration of shikonin nanomedicine (NP/SHK) into the tumor tissue and enhance the therapeutic effect of breast cancer. Chitosan thermosensitive hydrogel loaded with α-M (HG@α-M) is designed to transform CAFs from an activated state to a quiescent state and reduce the deposition of ECM. In addition, we designed glutathione (GSH)-responsive nanomedicine (NP/SHK) carrying shikonin (SHK), which solves the problem of poor water solubility of SHK itself. In breast cancer model mice, compared with the single NP/SHK treatment group, the combined treatment down-regulated the expressions of CAF markers α-SMA and FAP-α by 52.70 and 56.77%, respectively, increased the tumor growth inhibition rate by 32.27%, and reduced lung metastatic nodules by 26.06%. This study effectively inhibited the growth and metastasis of tumors, providing a new and efficient combined treatment approach for metastatic breast cancer.

Amorphous solid dispersions (ASDs) are often used to increase the bioavailability of poorly water-soluble drugs. However, there are substantial formulation challenges associated with optimizing their performance. The high polymer content needed to ensure rapid and extensive release creates a pill burden for patients, leading to lower compliance. To combat this problem, formulations with higher drug loading are desirable, but often have poor release. Previous research suggests that specific interactions between the drug and polymer can have a negative effect on ASD release, but the failure mechanisms are not fully understood. In this study, a model system of the low glass transition temperature drug, ibuprofen, and polyvinylpyrrolidone vinyl acetate (PVPVA) was used to investigate the mechanisms underlying poor release from an ASD with drug-polymer hydrogen bonding at high drug loading. Infrared spectroscopy was used to demonstrate the presence of hydrogen bonds between the carboxylic acid of ibuprofen and the pyrrolidone carbonyl group of PVPVA. ASD phase morphology following exposure to water, either via the vapor phase or following immersion in aqueous media, was studied using confocal fluorescence microscopy. Surface area normalized release studies of ASDs were performed at different drug loadings. Phase separation was readily induced following exposure to water vapor and was also observed following immersion in aqueous media. Furthermore, the resultant phase morphology varied with drug loading, changing from discrete drug-rich regions at low drug loading to continuous drug-rich regions at higher drug loading, explaining why release rate decreased dramatically with increasing drug load. Approximately 40% of PVPVA was present in the insoluble drug-rich phase, indicating a high affinity of the polymer for the drug in an aqueous environment, likely due to drug-polymer hydrogen bonding interactions. The presence of the polymer resulted in an increase in the volume of the insoluble drug-rich phase, underpinning the observed change in phase separation morphology at a relatively low drug loading. This study thus provides mechanistic understanding of the role played by drug-polymer hydrogen bonding in lowering the limit of congruency of PVPVA-based ASDs.

Targeted, small-molecule therapeutics have improved patient survival across cancer types, with >20 tyrosine kinase inhibitors (TKIs) receiving FDA approval as cancer therapies. While the initial TKI response is often promising, it is typically transient due to tumor evolution and subsequent therapeutic resistance driven by primary or acquired resistance mechanisms. While resistance mechanisms vary, there are currently no spatially resolved methodologies that provide a quantitative, mechanistic understanding of drug delivery and therapeutic response across therapeutic modalities (e.g., chemotherapy, radiotherapy, TKIs, immunotherapy) to enable personalized cancer therapy. Herein, we utilize our previously reported fluorescence imaging platform, Therapeutic Response Imaging through Proteomic and Optical Drug Distribution (TRIPODD), which is a quantitative protocol capable of interpreting the relationship between drug delivery and therapeutic response within the spatial context of a tumor to evaluate single-cell response and resistance to epidermal growth factor receptor TKI therapy. In this study, we applied TRIPODD to quantify the therapeutic response of EGFR-TKI-sensitive nonsmall cell lung cancer (NSCLC) xenografts to erlotinib, as a proof of concept for the platform. Through these studies, we were able to identify unique signatures of therapeutic response linked to the accumulation and engagement of erlotinib on a single-cell basis, demonstrating the utility of our TRIPODD platform technology in evaluating the treatment response and resistance at the single-cell level in heterogeneous tumors.