Optimizing the performance of silica nanoparticles functionalized with a near-infrared fluorescent dye for bioimaging applications.

Modified fluorescent nanoparticles continue to emerge as promising candidates for drug delivery, bioimaging, and labeling tools for various biomedical applications. The ability of nanomaterials to fluorescently label cells allow for the enhanced detection and understanding of diseases. Silica nanoparticles have a variety of unique properties that can be harnessed for many different applications, causing their increased popularity. In combination with an organic dye, fluorescent nanoparticles demonstrate a vast range of advantageous properties including long photostability, surface modification, and signal amplification, thus allowing ease of manipulation to best suit bioimaging purposes. In this study, the Stöber method with tetraethyl orthosilicate (TEOS) and a fluorescent dye sulfo-Cy5-amine was used to synthesize fluorescent silica nanoparticles. The fluorescence spectra, zeta potential, quantum yield, cytotoxicity, and photostability were evaluated. The increased intracellular uptake and photostability of the dye-silica nanoparticles show their potential for bioimaging.

Fatty Acid Synthase Inhibitors Enhance Microtubule-Stabilizing and Microtubule-Destabilizing Drugs in Taxane-Resistant Prostate Cancer Cells.

Prostate cancer is the third leading cause of cancer-related death in men in the United States. Taxane chemotherapy is a staple therapy for men with metastatic prostate cancer, yet the median survival is less than 2 years in this setting. New strategies are needed to overcome taxane resistance to improve patient survival. Fatty acid synthase (FASN) is overexpressed in many types of cancer, and several inhibitors have been designed in the past 30 years. Previously, we showed that the FASN inhibitor orlistat was able to synergize with taxanes in two established taxane-resistant (TxR) cell lines. In the current study, we investigated five FASN inhibitors-cerulenin, orlistat, triclosan, thiophenopyrimidine fasnall, and pyrazole derivative TVB-3166 for their potential to synergize with docetaxel (a microtubule stabilizer) and vinblastine (a microtubule destabilizer) in TxR cell lines. Orlistat, TVB-3166, and fasnall synergistically inhibited cell viability when combined with docetaxel and vinblastine in PC3-TxR and DU145-TxR cells. Confocal microscopy and immunoblot with an antidetyrosinated tubulin antibody demonstrated that enhanced microtubule stability was induced by the combined treatment of FASN inhibitors and docetaxel compared with docetaxel alone, while combinations of FASN inhibitors with vinblastine diminished microtubule stability compared to vinblastine alone.

Highly Selective Targeting of Pancreatic Cancer in the Liver with a Near-Infrared Anti-MUC5AC Probe in a PDOX Mouse Model: A Proof-of-Concept Study.

Accurately identifying metastatic disease is critical to directing the appropriate treatment in pancreatic cancer. Mucin 5AC is overexpressed in pancreatic cancer but absent in normal pancreas tissue. The present proof-of-concept study demonstrates the efficacy of an anti-mucin 5AC antibody conjugated to an IR800 dye (MUC5AC-IR800) to preferentially label a liver metastasis of pancreatic cancer (Panc Met) in a unique patient-derived orthotopic xenograft (PDOX) model. In orthotopic models, the mean tumor to background ratio was 1.787 (SD ± 0.336), and immunohistochemistry confirmed the expression of MUC5AC within tumor cells. MUC5AC-IR800 provides distinct visualization of pancreatic cancer liver metastasis in a PDOX mouse model, demonstrating its potential utility in staging laparoscopy and fluorescence-guided surgery.

Mucins as contrast agent targets for fluorescence-guided surgery of pancreatic cancer.

Pancreatic cancer is difficult to resect due to its unique challenges, often leading to incomplete tumor resections. Fluorescence-guided surgery (FGS), also known as intraoperative molecular imaging and optical surgical navigation, is an intraoperative tool that can aid surgeons in complete tumor resection through an increased ability to detect the tumor. To target the tumor, FGS contrast agents rely on biomarkers aberrantly expressed in malignant tissue compared to normal tissue. These biomarkers allow clinicians to identify the tumor and its stage before surgical resection and provide a contrast agent target for intraoperative imaging. Mucins, a family of glycoproteins, are upregulated in malignant tissue compared to normal tissue. Therefore, these proteins may serve as biomarkers for surgical resection. Intraoperative imaging of mucin expression in pancreatic cancer can potentially increase the number of complete resections. While some mucins have been studied for FGS, the potential ability to function as a biomarker target extends to the entire mucin family. Therefore, mucins are attractive proteins to investigate more broadly as FGS biomarkers. This review summarizes the biomarker traits of mucins and their potential use in FGS for pancreatic cancer.

Preclinical Evaluation of a Humanized, Near-Infrared Fluorescent Antibody for Fluorescence-Guided Surgery of MUC16-Expressing Pancreatic Cancer.

Surgery remains the only potentially curative treatment option for pancreatic cancer, but resections are made more difficult by infiltrative disease, proximity of critical vasculature, peritumoral inflammation, and dense stroma. Surgeons are limited to tactile and visual cues to differentiate cancerous tissue from normal tissue. Furthermore, translating preoperative images to the intraoperative setting poses additional challenges for tumor detection, and can result in undetected and unresected lesions. Thus, pancreatic ductal adenocarcinoma (PDAC) has high rates of incomplete resections, and subsequently, disease recurrence. Fluorescence-guided surgery (FGS) has emerged as a method to improve intraoperative detection of cancer and ultimately improve surgical outcomes. Initial clinical trials have demonstrated feasibility of FGS for PDAC, but there are limited targeted probes under investigation for this disease, highlighting the need for development of additional novel biomarkers to reflect the PDAC heterogeneity. MUCIN16 (MUC16) is a glycoprotein that is overexpressed in 60-80% of PDAC. In our previous work, we developed a MUC16-targeted murine antibody near-infrared conjugate, termed AR9.6-IRDye800, that showed efficacy in detecting pancreatic cancer. To build on the translational potential of this imaging probe, a humanized variant of the AR9.6 fluorescent conjugate was developed and investigated herein. This conjugate, termed huAR9.6-IRDye800, showed equivalent binding properties to its murine counterpart. Using an optimized dye:protein ratio of 1:1, in vivo studies demonstrated high tumor to background ratios in MUC16-expressing tumor models, and delineation of tumors in a patient-derived xenograft model. Safety, biodistribution, and toxicity studies were conducted. These studies demonstrated that huAR9.6-IRDye800 was safe, did not yield evidence of histological toxicity, and was well tolerated in vivo. The results from this work suggest that AR9.6-IRDye800 is an efficacious and safe imaging agent for identifying pancreatic cancer intraoperatively through fluorescence-guided surgery.

Simpler Procedure and Improved Performance for Pathogenic Bacteria Analysis with a Paper-Based Ratiometric Fluorescent Sensor Array.

Bacterial infections are the leading cause of morbidity and mortality in the world, particularly due to a delay in treatment and misidentification of the bacterial species causing the infection. Therefore, rapid and accurate identification of these pathogens has been of prime importance. The conventional diagnostic techniques include microbiological, biochemical, and genetic analyses, which are time-consuming, require large sample volumes, expensive equipment, reagents, and trained personnel. In response, we have now developed a paper-based ratiometric fluorescent sensor array. Environment-sensitive fluorescent dyes (3-hydroxyflavone derivatives) pre-adsorbed on paper microzone plates fabricated using photolithography, upon interaction with bacterial cell envelopes, generate unique fluorescence response patterns. The stability and reproducibility of the sensor array response were thoroughly investigated, and the analysis procedure was refined for optimal performance. Using neural networks for response pattern analysis, the sensor was able to identify 16 bacterial species and recognize their Gram status with an accuracy rate greater than 90%. The paper-based sensor was stable for up to 6 months after fabrication and required 30 times lower dye and sample volumes as compared to the analogous solution-based sensor. Therefore, this approach opens avenues to a state-of-the-art diagnostic tool that can be potentially translated into clinical applications in low-resource environments.

Dynamic hyaluronic acid hydrogel with covalent linked gelatin as an anti-oxidative bioink for cartilage tissue engineering.

In the past decade, cartilage tissue engineering has arisen as a promising therapeutic option for degenerative joint diseases, such as osteoarthritis, in the hope of restoring the structure and physiological functions. Hydrogels are promising biomaterials for developing engineered scaffolds for cartilage regeneration. However, hydrogel-delivered mesenchymal stem cells or chondrocytes could be exposed to elevated levels of reactive oxygen species (ROS) in the inflammatory microenvironment after being implanted into injured joints, which may affect their phenotype and normal functions and thereby hinder the regeneration efficacy. To attenuate ROS induced side effects, a multifunctional hydrogel with an innate anti-oxidative ability was produced in this study. The hydrogel was rapidly formed through a dynamic covalent bond between phenylboronic acid grafted hyaluronic acid (HA-PBA) and poly(vinyl alcohol) and was further stabilized through a secondary crosslinking between the acrylate moiety on HA-PBA and the free thiol group from thiolated gelatin. The hydrogel is cyto-compatible and injectable and can be used as a bioink for 3D bioprinting. The viscoelastic properties of the hydrogels could be modulated through the hydrogel precursor concentration. The presence of dynamic covalent linkages contributed to its shear-thinning property and thus good printability of the hydrogel, resulting in the fabrication of a porous grid construct and a meniscus like scaffold at high structural fidelity. The bioprinted hydrogel promoted cell adhesion and chondrogenic differentiation of encapsulated rabbit adipose derived mesenchymal stem cells. Meanwhile, the hydrogel supported robust deposition of extracellular matrix components, including glycosaminoglycans and type II collagen, by embedded mouse chondrocytesin vitro. Most importantly, the hydrogel could protect encapsulated chondrocytes from ROS induced downregulation of cartilage-specific anabolic genes (ACAN and COL2) and upregulation of a catabolic gene (MMP13) after incubation with H2O2. Furthermore, intra-articular injection of the hydrogel in mice revealed adequate stability and good biocompatibilityin vivo. These results demonstrate that this hydrogel can be used as a novel bioink for the generation of 3D bioprinted constructs with anti-ROS ability to potentially enhance cartilage tissue regeneration in a chronic inflammatory and elevated ROS microenvironment.

Correction to "Water-Soluble Blue Fluorescent Nonconjugated Polymer Dots from Hyaluronic Acid and Hydrophobic Amino Acids".

[This corrects the article DOI: 10.1021/acsomega.1c01343.].

Colonoscopy-based intramucosal transplantation of cancer cells for mouse modeling of colon cancer and lung metastasis.

The conventional orthotopic/xenograft models or genetically engineered murine models of colon cancer (CRC) are limited in their scope for a true understanding of tumor growth, progression and eventual metastasis in its natural microenvironment. In the currently used murine models of CRC metastasis, the metastasis occurs primarily in the liver, though lung metastasis accounts for a significant proportion of CRC metastasis. There is an urgent need for a murine model of CRC, which not only allows tumor progression in the colonic mucosa but also metastasis of the lung. The authors describe a minimally invasive murine model of colon cancer progression that may be ideal for a wide range of applications, including evaluating gene function, microenvironment, cancer metastasis and therapeutic translational research.

Water-Soluble Blue Fluorescent Nonconjugated Polymer Dots from Hyaluronic Acid and Hydrophobic Amino Acids.

Fluorescent polymers have been increasingly investigated to improve their water solubility and biocompatibility to enhance their performance in drug delivery and theranostic applications. However, the environmentally friendly synthesis and dual functionality of such systems remain a challenge due to the complicated synthesis of conventional fluorescent materials. Herein, we generated a novel blue fluorescent polymer dot through chemical conjugation of hydrophobic amino acids to hyaluronic acid (HA) under one-pot green chemistry conditions. These nonconjugated fluorescent polymer dots (NCPDs) are water soluble, nontoxic to cells, have high fluorescence quantum yield, and can be used for in vitro bioimaging. HA-derived NCPDs exhibit excitation wavelength-dependent fluorescent properties. In addition, the NCPDs also show enhanced doxorubicin loading and delivery in naive and drug-resistant breast cancer cells in 2D and 3D tumor cellular systems. These results demonstrate the potential for successful synthetic scale-up and applications for HA-derived NCPDs.

Nanoformulation of CCL21 greatly increases its effectiveness as an immunotherapy for neuroblastoma.

Neuroblastoma is the most commonly diagnosed extracranial solid tumor in children. The patients with aggressive metastatic disease or refractory/relapsed neuroblastoma currently face a dismally low chance of survival. Thus, there is a great need for more effective therapies for this illness. In previous studies, we, as well as others, showed that the immune cell chemoattractant C-C motif chemokine ligand 21 (CCL21) is effective as an intratumoral therapy able to slow the growth of cancers. In this current study, we developed and tested an injectable, slow-release, uniform, and optimally loaded alginate nanoformulation of CCL21 as a means to provide prolonged intratumoral treatment. The alginate-nanoformulated CCL21, when injected intratumorally into mice bearing neuroblastoma lesions, significantly prolonged survival and decreased the tumor growth rate compared to CCL21 alone, empty nanoparticles, or buffer. Notably, we also observed complete tumor clearance and subsequent full protection against tumor rechallenge in 33% of nanoformulated CCL21-treated mice. Greater intratumoral presence of nanoformulated CCL21, compared to free CCL21, at days 1 and 2 after treatment ended was confirmed through fluorescent labeling and tracking. Nanoformulated CCL21-treated tumors exhibited a general pattern of prolonged increases in anti-tumor cytokines and relatively lower levels of pro-tumor cytokines in comparison to tumors treated with CCL21 alone or buffer only. Thus, this novel nanoformulation of CCL21 is an effective treatment for neuroblastoma, and may have potential for the delivery of CCL21 to other types of solid tumors in the future and as a slow-release delivery modality for other immunotherapies.

Sulfation modulates the targeting properties of hyaluronic acid to P-selectin and CD44.

Many targeting strategies can be employed to direct nanoparticles to tumors for imaging and therapy. However, tumors display a dynamic, heterogeneous microenvironment that undergoes spatiotemporal changes, including the expression of targetable cell-surface biomarkers. Here, we develop a nanoparticle system to effectively target two receptors overexpressed in the microenvironment of aggressive tumors. Hyaluronic acid (HA) was regioselectivity modified using a multi-step synthetic approach to alter binding specificities for CD44 and P-selectin to tumor cell interaction. The dual-targeting strategy utilizes sulfate modifications on HA that targets P-selectin, in addition to native targeting of CD44, which exploits spatiotemporal alterations in the expression patterns of these two receptors in cancer sites. Using biophysical characterization and in vitro studies, we demonstrate that modified HA nanoparticles effectively targets both P-selectin+ and CD44+ cells, which lays the groundwork for future in vivo biomedical applications.

Development of a MUC16-Targeted Near-Infrared Fluorescent Antibody Conjugate for Intraoperative Imaging of Pancreatic Cancer.

Surgical resection is currently the only potentially curative option for patients with pancreatic cancer. However, the 5-year survival rate after resection is only 25%, due in part to high rates of R1 resections, in which cells are left behind at the surgical margin, resulting in disease recurrence. Fluorescence-guided surgery (FGS) has emerged as a method to reduce incomplete resections and improve intraoperative assessment of cancer. Mucin-16 (MUC16), a protein biomarker highly overexpressed in pancreatic cancer, is a potential target for FGS. In this study, we developed a fluorescent MUC16-targeted antibody probe, AR9.6-IRDye800, for image-guided resection of pancreatic cancer. We demonstrated the efficacy of this probe to bind human pancreatic cancer cell lines in vitro and in vivo In an orthotopic xenograft model, AR9.6-IRDye800 exhibited superior fluorescence enhancement of tumors and lower signal in critical background organs in comparison to a nonspecific IgG control. The results of this study suggest that AR9.6-IRDye800 has potential for success as a probe for FGS in pancreatic cancer patients, and MUC16 is a feasible target for intraoperative imaging.

Solvent tuning of photochemistry upon excited-state symmetry breaking.

The nature of the electronic excited state of many symmetric multibranched donor-acceptor molecules varies from delocalized/multipolar to localized/dipolar depending on the environment. Solvent-driven localization breaks the symmetry and traps the exciton in one branch. Using a combination of ultrafast spectroscopies, we investigate how such excited-state symmetry breaking affects the photochemical reactivity of quadrupolar and octupolar A-(π-D)2,3 molecules with photoisomerizable A-π-D branches. Excited-state symmetry breaking is identified by monitoring several spectroscopic signatures of the multipolar delocalized exciton, including the S2 ← S1 electronic transition, whose energy reflects interbranch coupling. It occurs in all but nonpolar solvents. In polar media, it is rapidly followed by an alkyne-allene isomerization of the excited branch. In nonpolar solvents, slow and reversible isomerization corresponding to chemically-driven symmetry breaking, is observed. These findings reveal that the photoreactivity of large conjugated molecules can be tuned by controlling the localization of the excitation.

Tuned near infrared fluorescent hyaluronic acid conjugates for delivery to pancreatic cancer for intraoperative imaging.

The prognosis of pancreatic cancer remains poor. Intraoperative fluorescence imaging of tumors could improve staging and surgical resection, thereby improving prognosis. However, imaging pancreatic cancer with macromolecular delivery systems, is often hampered by nonspecific organ accumulation. Methods: We describe the rational development of hyaluronic acid (HA) conjugates that vary in molecular weight and are conjugated to near infrared fluorescent (NIRF) dyes that have differences in hydrophilicity, serum protein binding affinity, and clearance mechanism. We systematically investigated the roles of each of these properties on tumor accumulation, relative biodistribution, and the impact of intraoperative imaging of orthotopic, syngeneic pancreatic cancer. Results: Each HA-NIRF conjugate displayed intrapancreatic tumor enhancement. Regardless of HA molecular weight, Cy7.5 conjugation directed biodistribution to the liver, spleen, and bowels. Conjugation of IRDye800 to 5 and 20 kDa HA resulted in low liver and spleen signal while enhancing the tumor up to 14-fold compared to healthy pancreas, while 100 kDa HA conjugated to IRDye800 resulting in liver and spleen accumulation. Conclusion: These studies demonstrate that by tuning HA molecular weight and the physicochemical properties of the conjugated moiety, in this case a NIRF probe, peritoneal biodistribution can be substantially altered to achieve optimized delivery to tumors intraoperative abdominal imaging.

Image-guided tumor surgery: The emerging role of nanotechnology.

Surgical resection is a mainstay treatment for solid tumors. Yet, methods to distinguish malignant from healthy tissue are primarily limited to tactile and visual cues as well as the surgeon's experience. As a result, there is a possibility that a positive surgical margin (PSM) or the presence of residual tumor left behind after resection may occur. It is well-documented that PSMs can negatively impact treatment outcomes and survival, as well as pose an economic burden. Therefore, surgical tumor imaging techniques have emerged as a promising method to decrease PSM rates. Nanoparticles (NPs) have unique characteristics to serve as optical contrast agents during image-guided surgery (IGS). Recently, there has been tremendous growth in the volume and types of NPs used for IGS, including clinical trials. Herein, we describe the most recent contributions of nanotechnology for surgical tumor identification. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Implantable Materials and Surgical Technologies > Nanoscale Tools and Techniques in Surgery Diagnostic Tools > in vivo Nanodiagnostics and Imaging.

Nanoparticle Formulation of Indocyanine Green Improves Image-Guided Surgery in a Murine Model of Breast Cancer.

PURPOSE: Negative surgical margins (NSMs) have favorable prognostic implications in breast tumor resection surgery. Fluorescence image-guided surgery (FIGS) has the ability to delineate surgical margins in real time, potentially improving the completeness of tumor resection. We have recently developed indocyanine green (ICG)-loaded self-assembled hyaluronic acid (HA) nanoparticles (NanoICG) for solid tumor imaging, which were shown to enhance intraoperative contrast. PROCEDURES: This study sought to assess the efficacy of NanoICG on completeness of breast tumor resection and post-surgical survival. BALB/c mice bearing iRFP+/luciferase+ 4T1 syngeneic breast tumors were administered NanoICG or ICG, underwent FIGS, and were compared to bright light surgery (BLS) and sham controls. RESULTS: NanoICG increased the number of complete resections and improved tumor-free survival. This was a product of improved intraoperative contrast enhancement and the identification of a greater number of small, occult lesions than ICG and BLS. Additionally, NanoICG identified chest wall invasion and predicted recurrence in a model of late-stage breast cancer. CONCLUSIONS: NanoICG is an efficacious intraoperative contrast agent and could potentially improve surgical outcomes in breast cancer.

Rilpivirine-associated aggregation-induced emission enables cell-based nanoparticle tracking.

Antiretroviral therapy (ART) has improved the quality and duration of life for people living with human immunodeficiency virus (HIV) infection. However, limitations in drug efficacy, emergence of viral mutations and the paucity of cell-tissue targeting remain. We posit that to maximize ART potency and therapeutic outcomes newer drug formulations that reach HIV cellular reservoirs need be created. In a step towards achieving this goal we harnessed the aggregation-induced emission (AIE) property of the non-nucleoside reverse transcriptase inhibitor rilpivirine (RPV) and used it as a platform for drug cell and subcellular tracking. RPV nanocrystals were created with endogenous AIE properties enabling the visualization of intracellular particles in cell and tissue-based assays. The intact drug crystals were easily detected in CD4+ T cells and macrophages, the natural viral target cells, by flow cytometry and ultraperformance liquid chromatography tandem mass spectrometry. We conclude that AIE can be harnessed to monitor cell biodistribution of selective antiretroviral drug nanocrystals.

ω-Hydroxy isoprenoid bisphosphonates as linkable GGDPS inhibitors.

The enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential therapeutic target for multiple myeloma. Malignant plasma cells produce and secrete large amounts of monoclonal protein, and inhibition of GGDPS results in disruption of protein geranylgeranylation which in turn impairs intracellular protein trafficking. Our previous work has demonstrated that some isoprenoid triazole bisphosphonates are potent and selective inhibitors of GGDPS. To explore the possibility of selective delivery of such compounds to plasma cells, new analogues with an ω-hydroxy group have been synthesized and examined for their enzymatic and cellular activity. These studies demonstrate that incorporation of the ω-hydroxy group minimally impairs GGDPS inhibitory activity. Furthermore conjugation of one of the novel ω-hydroxy GGDPS inhibitors to hyaluronic acid resulted in enhanced cellular activity. These results will allow future studies to focus on the in vivo biodistribution of HA-conjugated GGDPS inhibitors.

Targeted Drug Delivery and Image-Guided Therapy of Heterogeneous Ovarian Cancer Using HER2-Targeted Theranostic Nanoparticles.

Cancer heterogeneity and drug resistance limit the efficacy of cancer therapy. To address this issue, we have developed an integrated treatment protocol for effective treatment of heterogeneous ovarian cancer. Methods: An amphiphilic polymer coated magnetic iron oxide nanoparticle was conjugated with near infrared dye labeled HER2 affibody and chemotherapy drug cisplatin. The effects of the theranostic nanoparticle on targeted drug delivery, therapeutic efficacy, non-invasive magnetic resonance image (MRI)-guided therapy, and optical imaging detection of therapy resistant tumors were examined in an orthotopic human ovarian cancer xenograft model with highly heterogeneous levels of HER2 expression. Results: We found that systemic delivery of HER2-targeted magnetic iron oxide nanoparticles carrying cisplatin significantly inhibited the growth of primary tumor and peritoneal and lung metastases in the ovarian cancer xenograft model in nude mice. Differential delivery of theranostic nanoparticles into individual tumors with heterogeneous levels of HER2 expression and various responses to therapy were detectable by MRI. We further found a stronger therapeutic response in metastatic tumors compared to primary tumors, likely due to a higher level of HER2 expression and a larger number of proliferating cells in metastatic tumor cells. Relatively long-time retention of iron oxide nanoparticles in tumor tissues allowed interrogating the relationship between nanoparticle drug delivery and the presence of resistant residual tumors by in vivo molecular imaging and histological analysis of the tumor tissues. Following therapy, most of the remaining tumors were small, primary tumors that had low levels of HER2 expression and nanoparticle drug accumulation, thereby explaining their lack of therapeutic response. However, a few residual tumors had HER2-expressing tumor cells and detectable nanoparticle drug delivery but failed to respond, suggesting additional intrinsic resistant mechanisms. Nanoparticle retention in the small residual tumors, nevertheless, produced optical signals for detection by spectroscopic imaging. Conclusion: The inability to completely excise peritoneal metastatic tumors by debulking surgery as well as resistance to chemotherapy are the major clinical challenges for ovarian cancer treatment. This targeted cancer therapy has the potential for the development of effective treatment for metastatic ovarian cancer.