High-Throughput Fluorometric Assay For Quantifying Polysorbate In Biopharmaceutical Products Using Micelle Activated Fluorescence Probe N-Phenyl-1-Naphthylamine.

PURPOSE: Polysorbates are among the most used surfactants in biopharmaceutical products containing proteins. Our work aims to develop a high-throughput fluorometric assay to further diversify the analytical toolbox for quantification of PSs. METHOD: The assay leverages the micelle activated fluorescence signal from N-Phenyl-1-Naphthylamine (NPN). The development and optimization of assay parameters were guided by the pre-defined analytical target profile. Furthermore, NMR was used to probe the interaction between protein, PS80 and NPN in the measurement system and understand protein interference. RESULTS: All assay parameters including excitation and emission wavelengths, standard curve, NPN concentration, and incubation time have been optimized and adapted to a microplate format, making it compatible with automated solutions that will be pursued in the near future to drive consistency and efficiency in our workflows. The specificity, accuracy, and precision of the assay have been demonstrated through a case study. Furthermore, NMR results provided additional insight into the change of the interaction dynamics between PS80 and NPN as the protein concentration increases. The results indicate minimal interaction between the protein and PS80 at lower concentration. However, when the concentration exceeds 75 mg/mL, there is a significant interaction between the protein and PS-80 micelle and monomer. CONCLUSION: A high-throughput fluorometric assay has been developed for quantification of polysorbates in biopharmaceutical samples including in-process samples, drug substance and drug product. The assay reported herein could serve as a powerful analytical tool for polysorbate quantification and control, complementing the widely used liquid chromatography with charged aerosol detection method.

Metabolic Response to Small Molecule Therapy in Colorectal Cancer Tracked with Raman Spectroscopy and Metabolomics.

Despite numerous screening tools for colorectal cancer (CRC), 25% of patients are diagnosed with advanced disease.  Novel diagnostic technologies that are early, accurate, and rapid are imperative to assess the therapeutic efficacy of clinical drugs and identify new biomarkers of treatment response. Here Raman spectroscopy (RS) was used to track metabolic reprogramming in KRAS-mutant HCT116 and SW837 cells, and KRAS wild-type CC cells. RS combined with multivariate analysis methods distinguished nonresponsive, partially responsive, and responsive cells treated with cetuximab, a monoclonal antibody for EGFR inhibition, sotorasib, a clinically approved KRAS inhibitor, and various doses of trametinib, an inhibitor of the MAPK pathway. Cells treated with a combination of subtoxic doses of trametinib and BKM120, an inhibitor of the PI3K pathway, showed a synergistic response between the two pathways. Using a supervised machine learning regression model, we established a scoring methodology trained to a priori predict therapeutic response to new treatment combinations. RS metabolites were verified with mass spectrometry, and enrichment pathways were identified, including amino acid, purine, and nicotinate and nicotinamide metabolism that differentiated monotherapy from combination therapy. Our approach may ultimately be applicable to patient-derived primary cells and cultures of patient tumors to predict effective drugs for individualized care.

Mammary tissue-derived extracellular matrix hydrogels reveal the role of irradiation in driving a pro-tumor and immunosuppressive microenvironment.

Radiation therapy (RT) is essential for triple negative breast cancer (TNBC) treatment. However, patients with TNBC continue to experience recurrence after RT. The role of the extracellular matrix (ECM) of irradiated breast tissue in tumor recurrence is still unknown. In this study, we evaluated the structure, molecular composition, and mechanical properties of irradiated murine mammary fat pads (MFPs) and developed ECM hydrogels from decellularized tissues (dECM) to assess the effects of RT-induced ECM changes on breast cancer cell behavior. Irradiated MFPs were characterized by increased ECM deposition and fiber density compared to unirradiated controls, which may provide a platform for cell invasion and proliferation. ECM component changes in collagens I, IV, and VI, and fibronectin were observed following irradiation in both MFPs and dECM hydrogels. Encapsulated TNBC cell proliferation and invasive capacity was enhanced in irradiated dECM hydrogels. In addition, TNBC cells co-cultured with macrophages in irradiated dECM hydrogels induced M2 macrophage polarization and exhibited further increases in proliferation. Our study establishes that the ECM in radiation-damaged sites promotes TNBC invasion and proliferation as well as an immunosuppressive microenvironment. This work represents an important step toward elucidating how changes in the ECM after RT contribute to breast cancer recurrence.

Monitoring Metabolic Changes in Response to Chemotherapies in Cancer with Raman Spectroscopy and Metabolomics.

Resistance to clinical therapies remains a major barrier in cancer management. There is a critical need for rapid and highly sensitive diagnostic tools that enable early prediction of treatment response to allow accurate clinical decisions. Here, Raman spectroscopy was employed to monitor changes in key metabolites as early predictors of response in KRAS-mutant colorectal cancer (CRC) cells, HCT116, treated with chemotherapies. We show at the single cell level that HCT116 is resistant to cetuximab (CTX), the first-line treatment in CRC, but this resistance can be overcome with pre-sensitization of cells with oxaliplatin (OX). In combination treatment of CTX + OX, sequential delivery of OX followed by CTX rather than simultaneous administration of drugs was observed to be critical for effective therapy. Our results demonstrated that metabolic changes are well aligned to cellular mechanical changes where Young's modulus decreased after effective treatment, indicating that both changes in mechanical properties and metabolism in cells are likely responsible for cancer proliferation. Raman findings were verified with mass spectrometry (MS) metabolomics, and both platforms showed changes in lipids, nucleic acids, and amino acids as predictors of resistance/response. Finally, key metabolic pathways enriched were identified when cells are resistant to CTX but downregulated with effective treatment. This study highlights that drug-induced metabolic changes both at the single cell level (Raman) and ensemble level (MS) have the potential to identify mechanisms of response to clinical cancer therapies.

The Emerging Role of Raman Spectroscopy as an Omics Approach for Metabolic Profiling and Biomarker Detection toward Precision Medicine.

Omics technologies have rapidly evolved with the unprecedented potential to shape precision medicine. Novel omics approaches are imperative toallow rapid and accurate data collection and integration with clinical information and enable a new era of healthcare. In this comprehensive review, we highlight the utility of Raman spectroscopy (RS) as an emerging omics technology for clinically relevant applications using clinically significant samples and models. We discuss the use of RS both as a label-free approach for probing the intrinsic metabolites of biological materials, and as a labeled approach where signal from Raman reporters conjugated to nanoparticles (NPs) serve as an indirect measure for tracking protein biomarkers in vivo and for high throughout proteomics. We summarize the use of machine learning algorithms for processing RS data to allow accurate detection and evaluation of treatment response specifically focusing on cancer, cardiac, gastrointestinal, and neurodegenerative diseases. We also highlight the integration of RS with established omics approaches for holistic diagnostic information. Further, we elaborate on metal-free NPs that leverage the biological Raman-silent region overcoming the challenges of traditional metal NPs. We conclude the review with an outlook on future directions that will ultimately allow the adaptation of RS as a clinical approach and revolutionize precision medicine.

STING-activating nanoparticles normalize the vascular-immune interface to potentiate cancer immunotherapy.

The tumor-associated vasculature imposes major structural and biochemical barriers to the infiltration of effector T cells and effective tumor control. Correlations between stimulator of interferon genes (STING) pathway activation and spontaneous T cell infiltration in human cancers led us to evaluate the effect of STING-activating nanoparticles (STANs), which are a polymersome-based platform for the delivery of a cyclic dinucleotide STING agonist, on the tumor vasculature and attendant effects on T cell infiltration and antitumor function. In multiple mouse tumor models, intravenous administration of STANs promoted vascular normalization, evidenced by improved vascular integrity, reduced tumor hypoxia, and increased endothelial cell expression of T cell adhesion molecules. STAN-mediated vascular reprogramming enhanced the infiltration, proliferation, and function of antitumor T cells and potentiated the response to immune checkpoint inhibitors and adoptive T cell therapy. We present STANs as a multimodal platform that activates and normalizes the tumor microenvironment to enhance T cell infiltration and function and augments responses to immunotherapy.

The tumor phototherapeutic application of nanoparticles constructed by the relationship between PTT/PDT efficiency and 2,6- and 3,5-substituted BODIPY derivatives.

BODIPY dyes have recently been used for photothermal and photodynamic therapy of tumors. However, complex multi-material systems, multiple excitation wavelengths and the unclear relationship between BODIPY structures and their PTT/PDT efficiency are still major issues. In our study, nine novel BODIPY near-infrared dyes were designed and successfully synthesized and then, the relationships between BODIPY structures and their PTT/PDT efficiency were investigated in detail. The results showed that modifications at position 3,5 of the BODIPY core with conjugated structures have better effects on photothermal and photodynamic efficiency than the modifications at position 2,6 with halogen atoms. Density functional theory (DFT) calculations showed that this is mainly due to the extension of the conjugated chain and the photoinduced electron transfer (PET) effect. By encapsulating BDPX-M with amphiphilic DSPE-PEG2000-RGD and lecithin, the obtained NPs not only show good water solubility and biological stability, but also could act as superior agents for photothermal and photodynamic synergistic therapy of tumors. Finally, we obtained BODIPY NPs that exhibited excellent photothermal and photodynamic effects at the same time under single irradiation with an 808 nm laser (photothermal conversion efficiency: 42.76%, A/A0: ∼0.05). In conclusion, this work provides a direction to design and construct phototherapeutic nanoparticles based on BODIPY dyes for tumor treatment.

Multi-functional Nanodrug Based on a Three-dimensional Framework for Targeted Photo-chemo Synergetic Cancer Therapy.

Targeted synergistic therapy has broad prospects in tumor treatments. Here, a multi-functional nanodrug GDYO-CDDP/DOX@DSPE-PEG-MTX (GCDM) based on three traditional anticancer drugs (doxorubicin (DOX), cisplatin (CDDP) and methotrexate (MTX)) modified graphdiyne oxide (GDYO) is described, for diagnosis and targeted cancer photo-chemo synergetic therapy. In this system, for the first time, these three traditional anti-cancer drugs have played new roles and can reduce multidrug resistance through synergistic anti-tumor effects. Cisplatin can be hybridized with GDYO to form a multifunctional and well-dispersed three-dimensional framework, which can not only be used as nano-drug carriers to achieve high drug loading rates (40.3%), but also exhibit excellent photothermal conversion efficiency (47%) and good photodynamic effects under NIR irradiation. Doxorubicin (DOX) is loaded onto GDYO-CDDP through π-π stacking, which is used as an anticancer drug and as a fluorescent probe for nanodrug detection. Methotrexate (MTX) can be applied in tumor targeting and play a role in synergistic chemotherapy with DOX and CDDP. The synthesized multi-functional nanodrug GCDM has good biocompatibility, active targeting, long-term retention, sustained drug release, excellent fluorescence imaging capabilities, and remarkable photo-chemo synergistic therapeutic effects.

Probing metabolic alterations in breast cancer in response to molecular inhibitors with Raman spectroscopy and validated with mass spectrometry.

Rapid and accurate response to targeted therapies is critical to differentiate tumors that are resistant to treatment early in the regimen. In this work, we demonstrate a rapid, noninvasive, and label-free approach to evaluate treatment response to molecular inhibitors in breast cancer (BC) cells with Raman spectroscopy (RS). Metabolic reprogramming in BC was probed with RS and multivariate analysis was applied to classify the cells into responsive or nonresponsive groups as a function of drug dosage, drug type, and cell type. Metabolites identified with RS were then validated with mass spectrometry (MS). We treated triple-negative BC cells with Trametinib, an inhibitor of the extracellular-signal-regulated kinase (ERK) pathway. Changes measured with both RS and MS corresponding to membrane phospholipids, amino acids, lipids and fatty acids indicated that these BC cells were responsive to treatment. Comparatively, minimal metabolic changes were observed post-treatment with Alpelisib, an inhibitor of the mammalian target of rapamycin (mTOR) pathway, indicating treatment resistance. These findings were corroborated with cell viability assay and immunoblotting. We also showed estrogen receptor-positive MCF-7 cells were nonresponsive to Trametinib with minimal metabolic and viability changes. Our findings support that oncometabolites identified with RS will ultimately enable rapid drug screening in patients ensuring patients receive the most effective treatment at the earliest time point.

Cancer Immunoimaging with Smart Nanoparticles.

Dynamic immunoimaging in vivo is crucial in patient-tailored immunotherapies to identify patients who will benefit from immunotherapies, monitor therapeutic efficacy post treatment, and determine alternative strategies for nonresponders. Nanoparticles have played a major role in the immunotherapy landscape. In this review, we summarize recent findings in immunoimaging where smart nanoparticles target, detect, stimulate, and deliver therapeutic dose in vivo. Nanoparticles interfaced with an immunoimaging toolbox enable the use of multiple modalities and achieve depth-resolved whole-body tracking of immunomarkers with high accuracy both before and after treatment. We highlight how functional nanoparticles track T cells, dendritic cells (DCs), tumor-associated macrophages (TAMs), and immune checkpoint receptors (ICRs), and facilitate image-guided interventions.

Multimodal Multiplexed Immunoimaging with Nanostars to Detect Multiple Immunomarkers and Monitor Response to Immunotherapies.

The overexpression of immunomarker programmed cell death protein 1 (PD-1) and engagement of PD-1 to its ligand, PD-L1, are involved in the functional impairment of cluster of differentiation 8+ (CD8+) T cells, contributing to cancer progression. However, heterogeneities in PD-L1 expression and variabilities in biopsy-based assays render current approaches inaccurate in predicting PD-L1 status. Therefore, PD-L1 screening alone is not predictive of patient response to treatment, which motivates us to simultaneously detect multiple immunomarkers engaged in immune modulation. Here, we have developed multimodal probes, immunoactive gold nanostars (IGNs), that accurately detect PD-L1+ tumor cells and CD8+ T cells simultaneously in vivo, surpassing the limitations of current immunoimaging techniques. IGNs integrate the whole-body imaging of positron emission tomography with high sensitivity and multiplexing of Raman spectroscopy, enabling the dynamic tracking of both immunomarkers. IGNs also monitor response to immunotherapies in mice treated with combinatorial PD-L1 and CD137 agonists and distinguish responders from those nonresponsive to treatment. Our results showed a multifunctional nanoscale probe with capabilities that cannot be achieved with either modality alone, allowing multiplexed immunologic tumor profiling critical for predicting early response to immunotherapies.