ABSTRACT
BACKGROUND: Heterogeneous tumor cells are thought to be a significant factor in the failure of endocrine therapy in estrogen receptor-positive (ER+) cancers. Culturing patient-derived breast cancer cells (PDBCCs) provides an invaluable tool in pre-clinical and translational research for the heterogeneity of cancer cells. This study aimed to investigate the effects of different media components and culture methods on the BCSC-associated immunophenotypes and gene expression in ER + PDBCCs. METHODS: Ten patients with ER + breast cancer were employed in this study, six of whom had neoadjuvant chemotherapy and four of whom did not. PDBCCs were isolated by enzymatic methods using collagen I and hyaluronidase. PDBCCs were grown as monolayers in mediums with different compositions and as multicellular spheroid in a suspended condition. Collagen I-coated plate and ultralow attachment plate coated with polymer-X were used for monolayer and spheroid culture. Flow cytometry, immunofluorescent staining, RT-PCR, and RNA-sequencing were employed to examine the immunophenotype and genetic profile of PDBCCs. RESULTS: More than 95% of PDBCCs sustain EpCAM high/+/fibroblast marker- phenotypes in monolayer conditions by subculturing 3-4 times. A83-01 removal induced senescent cells with high ß-galactosidase activity. PDBCCs grown as monolayers were characterized by the majority of cells having an EpCAM+/CD49f + phenotype. Compared to full media in monolayer culture, EGF removal increased EpCAM+/CD49f - phenotype (13.8-fold, p = 0.028), whereas R-spondin removal reduced it (0.8-fold, p = 0.02). A83-01 removal increased EpCAM+/CD24 + phenotype (1.82-fold, p = 0.023) and decreased EpCAM low/-/CD44+/CD24- phenotype (0.45-fold, p = 0.026). Compared to monolayer, spheroid resulted in a significant increase in the population with EpCAM-/CD49+ (14.6-fold, p = 0.006) and EpCAM low/-/CD44+/CD24- phenotypes (4.16-fold, p = 0.022) and ALDH high activity (9.66-fold, p = 0.037). ALDH1A and EMT-related genes were upregulated. In RNA-sequencing analysis between spheroids and monolayers, a total of 561 differentially expressed genes (2-fold change, p < 0.05) were enriched in 27 KEGG pathways including signaling pathways regulating pluripotency of stem cells. In a recurrence-free survival analysis based on the Kaplan-Meier Plotter database of the up-and down-regulated genes identified in spheroids, 15 up-, and 14 down-regulated genes were associated with poor prognosis of breast cancer patients. CONCLUSION: The media composition and spheroid culture method change in the BCSCs and EMT markers of PDBCCs, implying the importance of defining the media composition and culture method for studying PDBCCs in vitro.
Subject(s)
Collagen Type I , Neoplasms , Epithelial Cell Adhesion Molecule , Integrin alpha6 , RNAABSTRACT
Common medications for treating inflammatory bowel disease (IBD) have limited therapeutic efficacy and severe adverse effects. This underscores the urgent need for novel therapeutic approaches that can effectively target inflamed sites in the gastrointestinal tract upon oral administration, exerting potent therapeutic efficacy while minimizing systemic effects. Here, we report the construction and in vivo therapeutic evaluation of a library of anti-inflammatory glycocalyx-mimicking nanoparticles (designated GlyNPs) in a mouse model of IBD. The anti-inflammatory GlyNP library was created by attaching bilirubin (BR) to a library of glycopolymers composed of random combinations of the five most naturally abundant sugars. Direct in vivo screening of 31â BR-attached anti-inflammatory GlyNPs via oral administration into mice with acute colitis led to identification of a candidate GlyNP capable of targeting macrophages in the inflamed colon and effectively alleviating colitis symptoms. These findings suggest that the BR-attached GlyNP library can be used as a platform to identify anti-inflammatory nanomedicines for various inflammatory diseases.
Subject(s)
Colitis , Inflammatory Bowel Diseases , Nanoparticles , Animals , Mice , Glycocalyx , Colitis/drug therapy , Inflammatory Bowel Diseases/drug therapy , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic useABSTRACT
Affinity chromatography utilizing specific interactions between therapeutic proteins and bead-immobilized capturing agents is a standard method for protein purification, but its scalability is limited by long purification times, activity loss by the capturing molecules and/or purified protein, and high costs. Here, we report a platform for purifying therapeutic antibodies via affinity precipitation using the endogenous calcium ion-binding protein, calsequestrin (CSQ), which undergoes a calcium ion-dependent phase transition. In this method, ZZ-CSQ fusion proteins with CSQ and an affinity protein (Z domain of protein A) capture antibodies and undergo multimerization and subsequent aggregation in response to calcium ions, enabling the antibody to be collected by affinity precipitation. After robustly validating and optimizing the performance of the platform, the ZZ-CSQ platform can rapidly purify therapeutic antibodies from industrial harvest feedstock with high purity (>97%) and recovery yield (95% ± 3%). In addition, the ZZ-CSQ platform outperforms protein A-based affinity chromatography (PAC) in removing impurities, yielding â¼20-fold less DNA and â¼4.8-fold less host cell protein (HCP) contamination. Taken together, this platform is rapid, recyclable, scalable, and cost-effective, and it shows antibody-purification performance superior or comparable to that of the standard affinity chromatography method.
Subject(s)
Calcium , Calsequestrin , Antibodies/metabolism , Calcium/metabolism , Calcium-Binding Proteins , Calsequestrin/chemistry , Calsequestrin/genetics , Calsequestrin/metabolism , Chromatography, Affinity/methods , Staphylococcal Protein A/metabolismABSTRACT
Most therapeutic peptides available on the market today are naturally occurring hormones or protein fragments that were serendipitously discovered to possess therapeutic effects. However, the limited repertoire of available natural resources presents difficulties for the development of new peptide drug candidates. Traditional peptides possess several shortcomings that must be addressed for biomedical applications, including relatively low affinity or specificity toward biological targets compared to antibody- and protein scaffold-based affinity molecules, poor in vivo stability owing to rapid enzymatic degradation, and rapid clearance from circulation owing to their small size. Going forward, it will be increasingly important for scientists to develop novel classes of high-affinity and -specificity peptides against desired targets that mitigate these limitations while remaining compatible with pharmaceutical manufacturing processes. Recently, several highly constrained, artificial cyclic peptides have emerged as platforms capable of generating high-affinity peptide binders against various disease-associated protein targets by combining with phage or mRNA display method, some of which have entered clinical trials. In contrast, although linear peptides are relatively easy to synthesize cost-effectively and modify site-specifically at either N- or C-termini compared to cyclic peptides, there have been few linear peptide-based platforms that can provide high-affinity and -specificity peptide binders.In this Account, we describe the creation and development of a novel class of high-affinity peptides, termed "aptide"-from the Latin word "aptus" meaning "to fit" and "peptide"-and summarize their biomedical applications. In the first part, we consider the design and creation of aptides, with a focus on their unique structural features and binding mode, and address screening and identification of target protein-specific aptides. We also discuss advantages of the aptide platform over ordinary linear peptides lacking preorganized structures in terms of the affinity and specificity of identified peptide binders against target molecules. In the second part, we describe the potential biomedical applications of various target-specific aptides, ranging from imaging and therapy to theranostics, according to the types of aptides and diseases. We show that certain aptides can not only bind to a target protein but also inhibit its biological function, thereby showing potential as therapeutics per se. Further, aptides specific for cancer-associated protein antigens can be used as escort molecules or targeting ligands for delivery of chemotherapeutics, cytokine proteins, and nanomedicines, such as liposomes and magnetic particles, to tumors, thereby substantially improving therapeutic effects. Finally, we present a strategy capable of overcoming the critical issue of short blood circulation time associated with most peptides by constructing a hybrid system between an aptide and a hapten cotinine-specific antibody.
Subject(s)
Nanomedicine , Peptides/metabolism , Animals , Antibodies/chemistry , Antibodies/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Diabetic Retinopathy/drug therapy , Humans , Kinetics , Magnetics , Mice , Nanoparticles/chemistry , Neoplasms/drug therapy , Peptides/chemistry , Peptides/therapeutic use , Protein Structure, Tertiary , STAT3 Transcription Factor/chemistry , Transplantation, Heterologous , Vascular Endothelial Growth Factor A/chemistryABSTRACT
RATIONALE: Central nervous system has low vascular permeability by organizing tight junction (TJ) and limiting endothelial transcytosis. While TJ has long been considered to be responsible for vascular barrier in central nervous system, suppressed transcytosis in endothelial cells is now emerging as a complementary mechanism. Whether transcytosis regulation is independent of TJ and its dysregulation dominantly causes diseases associated with edema remain elusive. Dll4 signaling is important for various vascular contexts, but its role in the maintenance of vascular barrier in central nervous system remains unknown. OBJECTIVE: To find a TJ-independent regulatory mechanism selective for transcytosis and identify its dysregulation as a cause of pathological leakage. METHODS AND RESULTS: We studied transcytosis in the adult mouse retina with low vascular permeability and employed a hypertension-induced retinal edema model for its pathological implication. Both antibody-based and genetic inactivation of Dll4 or Notch1 induce hyperpermeability by increasing transcytosis without junctional destabilization in arterial endothelial cells, leading to nonhemorrhagic leakage predominantly in the superficial retinal layer. Endothelial Sox17 deletion represses Dll4 in retinal arteries, phenocopying Dll4 blocking-driven vascular leakage. Ang II (angiotensin II)-induced hypertension represses arterial Sox17 and Dll4, followed by transcytosis-driven retinal edema, which is rescued by a gain of Notch activity. Transcriptomic profiling of retinal endothelial cells suggests that Dll4 blocking activates SREBP1 (sterol regulatory element-binding protein 1)-mediated lipogenic transcription and enriches gene sets favorable for caveolae formation. Profiling also predicts the activation of VEGF (vascular endothelial growth factor) signaling by Dll4 blockade. Inhibition of SREBP1 or VEGF-VEGFR2 (VEGF receptor 2) signaling attenuates both Dll4 blockade-driven and hypertension-induced retinal leakage. CONCLUSIONS: In the retina, Sox17-Dll4-SREBP1 signaling axis controls transcytosis independently of TJ in superficial arteries among heterogeneous regulations for the whole vessels. Uncontrolled transcytosis via dysregulated Dll4 underlies pathological leakage in hypertensive retina and could be a therapeutic target for treating hypertension-associated retinal edema.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Blood-Retinal Barrier/metabolism , Calcium-Binding Proteins/metabolism , Hypertensive Retinopathy/metabolism , Transcytosis , Adaptor Proteins, Signal Transducing/genetics , Animals , Arteries/metabolism , Calcium-Binding Proteins/genetics , Caveolae/metabolism , Endothelial Cells/metabolism , HMGB Proteins/metabolism , Homeostasis , Mice , Mice, Inbred C57BL , Receptor, Notch1/genetics , Receptor, Notch1/metabolism , SOXF Transcription Factors/metabolism , Signal Transduction , Sterol Regulatory Element Binding Protein 1/metabolism , Tight Junctions/metabolism , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
While conventional approaches for inflammatory bowel diseases mainly focus on suppressing hyperactive immune responses, it remains unclear how to address disrupted intestinal barriers, dysbiosis of the gut commensal microbiota and dysregulated mucosal immune responses in inflammatory bowel diseases. Moreover, immunosuppressive agents can cause off-target systemic side effects and complications. Here, we report the development of hyaluronic acid-bilirubin nanomedicine (HABN) that accumulates in inflamed colonic epithelium and restores the epithelium barriers in a murine model of acute colitis. Surprisingly, HABN also modulates the gut microbiota, increasing the overall richness and diversity and markedly augmenting the abundance of Akkermansia muciniphila and Clostridium XIVα, which are microorganisms with crucial roles in gut homeostasis. Importantly, HABN associated with pro-inflammatory macrophages, regulated innate immune responses and exerted potent therapeutic efficacy against colitis. Our work sheds light on the impact of nanotherapeutics on gut homeostasis, microbiome and innate immune responses for the treatment of inflammatory diseases.
Subject(s)
Bilirubin/pharmacology , Colitis/immunology , Colitis/therapy , Hyaluronic Acid/pharmacology , Akkermansia , Animals , Dysbiosis/immunology , Female , Gastrointestinal Microbiome/immunology , HT29 Cells , Homeostasis , Humans , Immune System , Immunosuppressive Agents/therapeutic use , Inflammation , Intestinal Mucosa/pathology , Intestines/pathology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Microbiota , Nanomedicine , Nanoparticles/chemistry , Permeability , Reactive Oxygen Species/metabolism , VerrucomicrobiaABSTRACT
Catalytic precipitation and subsequent electrochemical oxidation or reduction of a redox-active precipitate has been widely used in electrochemical biosensors. However, such biosensors often do not allow for low detection limits due to a low rate of precipitation, nonspecific precipitation, loose binding of the precipitate to the electrode surface, and insulating behavior of the precipitate within a normal potential window. Here, we report an ultrasensitive electrochemical immunosensor for parathyroid hormone (PTH) detection based on DT-diaphorase (DT-D)-catalyzed formation of an organic precipitate and electrochemical oxidation of the precipitate. In the present study we found that DT-D can be used as a catalytic label in precipitation-based affinity biosensors because DT-D catalyzes fast reduction of 3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to MTT-formazan precipitate; the MTT reduction does not occur in the absence of DT-D; and a high electrochemical signal is obtained at low potentials during electrodissolution of MTT-formazan precipitate. The immunosensor is fabricated using a silane copolymer-modified ITO electrode surface that is suitable for both efficient and strong adsorption of MTT-formazan precipitate. When the enzymatic MTT-formazan precipitation and subsequent MTT-formazan electrodissolution is applied to a sandwich-type immunosensor, PTH can be detected over a wide range of concentrations with a very low detection limit (â¼1 pg/mL) in artificial serum. The measured concentrations of PTH in clinical serum samples showed high similarity with those obtained using a commercial instrument.
Subject(s)
Biosensing Techniques/methods , Formazans/chemistry , NAD(P)H Dehydrogenase (Quinone)/chemistry , Parathyroid Hormone/analysis , Tetrazolium Salts/chemistry , Catalysis , Electrochemical Techniques , Electrodes , Humans , Oxidation-Reduction , Parathyroid Hormone/bloodABSTRACT
We describe a small lipid nanoparticle (SLNP)-based nanovaccine platform and a new combination treatment regimen. Tumor antigen-displaying, CpG adjuvant-embedded SLNPs (OVAPEP -SLNP@CpG) were prepared from biocompatible phospholipids and a cationic cholesterol derivative. The resulting nanovaccine showed highly potent antitumor efficacy in both prophylactic and therapeutic E.G7 tumor models. However, this vaccine induced T cell exhaustion by elevating PD-L1 expression, leading to tumor recurrence. Thus, the nanovaccine was combined with simultaneous anti-PD-1 antibody treatment, but the therapeutic efficacy of this regimen was comparable to that of the nanovaccine alone. Finally, mice that showed a good therapeutic response after the first cycle of immunization with the nanovaccine underwent a second cycle together with anti-PD-1 therapy, resulting in suppression of tumor relapse. This suggests that the antitumor efficacy of combinations of nanovaccines with immune checkpoint blockade therapy is dependent on treatment sequence and the timing of each modality.
Subject(s)
Cancer Vaccines/administration & dosage , Cell Proliferation , Immune Checkpoint Inhibitors/administration & dosage , Nanotechnology , Neoplasms/therapy , Animals , Enzyme-Linked Immunosorbent Assay , Humans , Mice , Neoplasms/pathologyABSTRACT
A number of cancer-targeting peptide-drug conjugates (PDCs) have been explored as alternatives to antibody-drug conjugates (ADCs) for targeted cancer therapy. However, the much shorter circulation half-life of PDCs compared with ADCs in vivo has limited their therapeutic value and thus their translation into the clinic, highlighting the need to develop new approaches for extending the half-life of PDCs. Here, we report a new strategy for targeted cancer therapy of a PDC based on a molecular hybrid between an antihapten antibody and a hapten-labeled PDC. An anticotinine antibody (Abcot) was used as a model antihapten antibody. The anticancer drug SN38 was linked to a cotinine-labeled aptide specific to extra domain B of fibronectin (cot-APTEDB), yielding the model PDC, cot-APTEDB-SN38. The cotinine-labeled PDC showed specific binding to and cytotoxicity toward an EDB-overexpressing human glioblastoma cell line (U87MG) and also formed a hybrid complex (HC) with Abcot in situ, designated HC[cot-APTEDB-SN38/Abcot]. In glioblastoma-bearing mice, in situ HC[cot-APTEDB-SN38/Abcot] significantly extended the circulation half-life of cot-APTEDB-SN38 in blood, and it enhanced accumulation and penetration within the tumor and, ultimately, inhibition of tumor growth. These findings suggest that the present platform holds promise as a new, targeted delivery strategy for PDCs in anticancer therapy.
Subject(s)
Antibodies/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Immunoconjugates/therapeutic use , Peptides/chemistry , Animals , Cell Line, Tumor , Drug Delivery Systems , Female , Glioma/drug therapy , Humans , Immunoconjugates/chemistry , In Situ Nick-End Labeling , Irinotecan/chemistry , Irinotecan/therapeutic use , Mice , Mice, Inbred BALB C , Mice, NudeABSTRACT
Despite the appreciable success of synthetic nanomaterials for targeted cancer therapy in preclinical studies, technical challenges involving their large-scale, cost-effective production and intrinsic toxicity associated with the materials, as well as their inability to penetrate tumor tissues deeply, limit their clinical translation. Here, we describe biologically derived nanocarriers developed from a bioengineered yeast strain that may overcome such impediments. The budding yeast Saccharomyces cerevisiae was genetically engineered to produce nanosized vacuoles displaying human epidermal growth factor receptor 2 (HER2)-specific affibody for active targeting. These nanosized vacuoles efficiently loaded the anticancer drug doxorubicin (Dox) and were effectively endocytosed by cultured cancer cells. Their cancer-targeting ability, along with their unique endomembrane compositions, significantly enhanced drug penetration in multicellular cultures and improved drug distribution in a tumor xenograft. Furthermore, Dox-loaded vacuoles successfully prevented tumor growth without eliciting any prolonged immune responses. The current study provides a platform technology for generating cancer-specific, tissue-penetrating, safe, and scalable biological nanoparticles for targeted cancer therapy.
Subject(s)
Bioengineering , Molecular Targeted Therapy , Organ Specificity , Saccharomyces cerevisiae/metabolism , Vacuoles/metabolism , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Death/drug effects , Doxorubicin/blood , Doxorubicin/pharmacokinetics , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Drug Delivery Systems , Drug Liberation , Kinetics , Mice , Mice, Inbred C57BL , Neoplasms/blood , Neoplasms/drug therapy , RAW 264.7 Cells , Receptor, ErbB-2/metabolism , Tissue Distribution/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Peptide-based therapeutics have suffered from a short plasma half-life. On the other hand, antibodies suffer from poor penetration into solid tumors owing to their large size. Herein, we present a new molecular form, namely a hybrid complex between a hapten-labeled bispecific peptide and an anti-hapten antibody ("HyPEP-body"), that may be able to overcome the aforementioned limitation. The bispecific peptide containing a cotinine tag was synthesized by linking a peptide specific to fibronectin extra domainâ B (EDB) and a peptide able to bind and inhibit vascular endothelial growth factor (VEGF), yielding cot-biPEPEDB-VEGF . Simple mixing of cot-biPEPEDB-VEGF and anti-cotinine antibody (Abcot ) yielded the hybrid complex, HyPEPEDB-VEGF . HyPEPEDB-VEGF retained the characteristics of the included peptides, and showed improved pharmacokinetic behavior. Moreover, HyPEPEDB-VEGF showed tumor growth inhibition with excellent tumor accumulation and penetration. These findings suggest that the hybrid platform described here offers a solution for most peptide therapeutics that suffer from a short circulation half-life in blood.
Subject(s)
Antibodies, Bispecific/pharmacology , Antineoplastic Agents/pharmacology , Peptides/pharmacology , Animals , Antibodies, Bispecific/chemistry , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Humans , Mice , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , PC-3 Cells , Peptides/chemistry , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Direct electron transfer between a redox label and an electrode requires a short working distance (<1-2 nm), and in general an affinity biosensor based on direct electron transfer requires a finely smoothed Au electrode to support efficient target binding. Here we report that direct electron transfer over a longer working distance is possible between (i) an anionic π-conjugated polyelectrolyte (CPE) label having many redox-active sites and (ii) a readily prepared, thin polymeric monolayer-modified indium-tin oxide electrode. In addition, the long CPE label (â¼18 nm for 10 kDa) can approach the electrode within the working distance after sandwich-type target-specific binding, and fast CPE-mediated oxidation of ammonia borane along the entire CPE backbone affords high signal amplification.
Subject(s)
DNA/analysis , Polyelectrolytes/chemistry , Electron TransportABSTRACT
The development of novel fluorescent probes for monitoring the concentration of various biomolecules in living systems has great potential for eventual early diagnosis and disease intervention. Selective detection of competitive species in biological systems is a great challenge for the design and development of fluorescent probes. To improve on the design of fluorescent coumarin-based biothiol sensing technologies, we have developed herein an enhanced dual emission doubly activated system (DACP-1 and the closely related DACP-2) for the selective detection of glutathione (GSH) through the use of one optical channel and the detection of cysteine (Cys) by another channel. A phenylselenium group present at the 4-position completely quenches the fluorescence of the probe via photoinduced electron transfer to give a nonfluorescent species. Probes are selective for glutathione (GSH) in the red region and for cysteine/homocysteine (Cys/Hcy) in the green region. When they were treated with GSH, DACP-1 and DACP-2 showed strong fluorescence enhancement in comparison to that for closely related species such as amino acids, including Cys/Hcy. Fluorescence quantum yields (ΦF) increased for the red channel (<0.001 to 0.52 (DACP-1) and 0.48 (DACP-2)) and green channel (Cys) (<0.001 to 0.030 (DACP-1) and 0.026 (DACP-2)), respectively. Competing fluorescent enhancements upon addition of closely related species were negligible. Fast responses, improved water solubility, and good cell membrane permeability were all properly established with the use of DACP-1 and DACP-2. Live human lung cancer cells and fibroblasts imaged by confocal microscopy, as well as live mice tumor model imaging, confirmed selective detection.
Subject(s)
Cysteine/analysis , Fibroblasts/chemistry , Fluorescent Dyes/chemistry , Glutathione/analysis , Lung Neoplasms/chemistry , Optical Imaging , Animals , Cell Survival/drug effects , Density Functional Theory , Female , Fluorescent Dyes/administration & dosage , Fluorescent Dyes/pharmacology , Humans , Injections, Intravenous , Lung Neoplasms/diagnostic imaging , Mice , Mice, Inbred BALB C , Mice, Nude , Microscopy, Confocal , Molecular Structure , Neoplasms, Experimental/chemistry , Neoplasms, Experimental/diagnostic imaging , Tumor Cells, CulturedABSTRACT
Cysteine plays a crucial role in cellular functions and in human pathologies. However, the development of cysteine probes with extremely accurate detection is still a key challenge for the field. Herein, we have fully characterized and developed a novel selective fluorescent probe: red emission, aqueous detection and large Stokes' shift for cysteine (Reals-C). Key in the probe synthesis is a Michael addition onto an acroylate group and subsequent intramolecular cyclization. The probe exhibits analyte detection via an intricate role set up by the leaving groups so to discriminate and form the red-emissive analyte sensing platform (λex =471â nm, λem =637â nm) through a chemical cascade pathway. Furthermore, the sensing ability of the probe was demonstrated by both in vitro and in vivo assays. This probe enables for successfully endogenous cysteine sensing in HaCaT human keratinocytes through comparison with a commercial thiol-sensitive probe; Reals-C shows excellent in vivo cysteine detection in a drug-induced animal liver injury model.
Subject(s)
Cysteine/analysis , Fluorescent Dyes/chemistry , Animals , Chemical and Drug Induced Liver Injury , Cyclization , Cysteine/chemistry , Cysteine/metabolism , Disease Models, Animal , Fluorescent Dyes/chemical synthesis , Humans , Keratinocytes/drug effects , Spectrometry, Fluorescence/methods , Sulfhydryl Compounds/chemistryABSTRACT
Despite growing interest in targeted cancer therapy with small molecule drug conjugates (SMDCs), the short half-life of these conjugates in blood associated with their small size has limited their efficacy in cancer therapy. In this report, we propose a new approach for improving the antitumor efficacy of SMDCs based on nanoparticle-assisted delivery. Ideally, a nanoparticle-based delivery vehicle would prolong the half-life of an SMDC in blood and then release it in response to stimuli in the tumor microenvironment (TME). In this study, PEGylated bilirubin-based nanoparticles (BRNPs) were chosen as an appropriate delivery carrier because of their ability to release drugs in response to TME-associated reactive oxygen species (ROS) through rapid particle disruption. As a model SMDC, ACUPA-SN38 was synthesized by linking the prostate-specific membrane antigen (PSMA)-targeting ligand, ACUPA, to the chemotherapeutic agent, SN38. ACUPA-SN38 was loaded into BRNPs using a film-formation and rehydration method. The resulting ACUPA-SN38@BRNPs exhibited ROS-mediated particle disruption and rapid release of the SMDC, resulting in greater cytotoxicity toward PSMA-overexpressing prostate cancer cells (LNCaP) than toward ROS-unresponsive ACUPA-SN38@Liposomes. In a pharmacokinetic study, the circulation time of ACUPA-SN38@BRNPs in blood was prolonged by approximately 2-fold compared with that of the SMDC-based micellar nanoparticles. Finally, ACUPA-SN38@BRNPs showed greater antitumor efficacy in a PSMA-overexpressing human prostate xenograft tumor model than SN38@BRNPs or the SMDC alone. Collectively, these findings suggest that BRNPs are a viable delivery carrier option for various cancer-targeting SMDCs that suffer from short circulation half-life and limited therapeutic efficacy.
Subject(s)
Antineoplastic Agents , Bilirubin , Drug Delivery Systems , Nanoparticles , Prostatic Neoplasms/drug therapy , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Bilirubin/chemistry , Bilirubin/pharmacokinetics , Bilirubin/pharmacology , Cell Line, Tumor , Humans , Kallikreins/metabolism , Liposomes , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Nanoparticles/chemistry , Nanoparticles/therapeutic use , Prostate-Specific Antigen/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Reactive Oxygen Species/metabolism , Tumor Microenvironment/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Both high sensitivity and high specificity are crucial for detection of miRNAs that have emerged as important clinical biomarkers. Just Another Zinc finger proteins (JAZ, ZNF346) bind preferably (but nonsequence-specifically) to DNA-RNA hybrids over single-stranded RNAs, single-stranded DNAs, and double-stranded DNAs. We present an ultrasensitive and highly specific electrochemical method for miRNA-21 detection based on the selective binding of JAZ to the DNA-RNA hybrid formed between a DNA capture probe and a target miRNA-21. This enables us to use chemically stable DNA as a capture probe instead of RNA as well as to apply a standard sandwich-type assay format to miRNA detection. High signal amplification is obtained by (i) enzymatic amplification by alkaline phosphatase (ALP) coupled with (ii) electrochemical-chemical-chemical (ECC) redox cycling involving an ALP product (hydroquinone). Low nonspecific adsorption of ALP-conjugated JAZ is obtained using a polymeric self-assembled-monolayer-modified and casein-treated indium-tin oxide electrode. The detection method can discriminate between target miRNA-21 and nontarget nucleic acids (DNA-DNA hybrid, single-stranded DNA, miRNA-125b, miRNA-155, single-base mismatched miRNA, and three-base mismatched miRNA). The detection limits for miRNA-21 in buffer and 10-fold diluted serum are approximately 2 and 30 fM, respectively, indicating that the detection method is ultrasensitive. This detection method can be readily extended to multiplex detection of miRNAs with only one ALP-conjugated JAZ probe due to its nonsequence-specific binding character. We also believe that the method could offer a promising solution for point-of-care testing of miRNAs in body fluids.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Electrochemical Techniques/methods , MicroRNAs/analysis , RNA-Binding Proteins/metabolism , RNA/metabolism , Zinc Fingers , Alkaline Phosphatase/chemistry , Electrochemical Techniques/standards , Electrodes , Humans , Limit of Detection , Nucleic Acid HybridizationABSTRACT
Tumor necrosis factor-α has shown potent antitumor effects in preclinical and clinical studies. However, severe side effects at less than therapeutic doses have limited its systemic delivery, prompting the need for a new strategy for targeted delivery of the protein to tumors. Here, we report a fusion protein of mouse tumor necrosis factor (TNF)-α (mTNFα) and a cancer-targeting, high-affinity aptide and investigate its therapeutic efficacy in tumor-bearing mice. A fusion protein consisting of mTNFα, a linker, and an aptide specific to extra domain B (EDB) of fibronectin (APTEDB), designated mTNFα-APTEDB, was successfully produced by expression in Escherichia coli. mTNFα-APTEDB retained specificity and affinity for its target, EDB. In mice bearing EDB-overexpressing fibrosarcomas, mTNFα-APTEDB showed greater efficacy in inhibiting tumor growth than mTNFα alone or mTNFα linked to a nonrelevant aptide, without causing an appreciable loss in body weight. Moreover, in vivo antitumor efficacy was further significantly increased by combination treatment with the chemotherapeutic drug, melphalan, suggesting a synergistic effect attributable to enhanced drug uptake into the tumor as a result of TNFα-mediated enhanced vascular permeability. These results suggest that a fusion protein of mTNFα with a cancer-targeting peptide could be a new anticancer therapeutic option for ensuring potent antitumor efficacy after systemic delivery.
Subject(s)
Fibronectins/metabolism , Peptides/metabolism , Recombinant Fusion Proteins/therapeutic use , Tumor Necrosis Factor-alpha/metabolism , Animals , Fibronectins/chemistry , Fibrosarcoma/drug therapy , Melphalan/chemistry , Melphalan/metabolism , Mice , Peptides/chemistry , Recombinant Fusion Proteins/metabolism , Tumor Necrosis Factor-alpha/chemistryABSTRACT
A major problem with cancer chemotherapy begins when cells acquire resistance. Drug-resistant cancer cells typically upregulate multi-drug resistance proteins such as P-glycoprotein (P-gp). However, the lack of overexpressed surface biomarkers has limited the targeted therapy of drug-resistant cancers. Here we report a drug-delivery carrier decorated with a targeting ligand for a surface marker protein extra-domain B(EDB) specific to drug-resistant breast cancer cells as a new therapeutic option for the aggressive cancers. We constructed EDB-specific aptide (APTEDB)-conjugated liposome to simultaneously deliver siRNA(siMDR1) and Dox to drug-resistant breast cancer cells. APTEDB-LS(Dox,siMDR1) led to enhanced delivery of payloads into MCF7/ADR cells and showed significantly higher accumulation and retention in the tumors. While either APTEDB-LS(Dox) or APTEDB-LS(siMDR1) did not lead to appreciable tumor retardation in MCF7/ADR orthotropic model, APTEDB-LS(Dox,siMDR1) treatment resulted in significant reduction of the drug-resistant breast tumor. Taken together, this study provides a new strategy of drug delivery for drug-resistant cancer therapy.
Subject(s)
Breast Neoplasms/drug therapy , Drug Delivery Systems , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Fibronectins , Animals , Cell Line, Tumor , Doxorubicin , Humans , Liposomes , Mice , Mice, Inbred BALB C , Tumor Cells, CulturedABSTRACT
Bilirubin (BR), a bile pigment that exerts potent antioxidant and anti-inflammatory effects, is also a major constituent of black pigment gallstones found in bile ducts under certain pathological conditions. Inspired by the intrinsic metal-chelating power of BR found in gallstones, herein we report a cisplatin-chelated BR-based nanoparticle (cisPt@BRNP) for use as a new photonic nanomedicine for combined photoacoustic imaging and photothermal therapy of cancers. The cisPt@BRNPs were prepared by simply mixing cisplatin with BRNPs, yielding ca. 150-nm-size NPs. Upon near-IR laser irradiation at 808â nm, cisPt@BRNPs generated considerable heat and induced clear death of cancer cells inâ vitro. Following intravenous injection into human colon cancer-bearing mice, cisPt@BRNPs allowed effective tumor visualization by photoacoustic imaging and remarkable antitumor efficacy by photothermal therapy, suggesting their potential for use as a new photonic nanomedicine for cancer therapy.
Subject(s)
Antineoplastic Agents/therapeutic use , Bilirubin/therapeutic use , Cisplatin/therapeutic use , Colonic Neoplasms/diagnostic imaging , Colonic Neoplasms/therapy , Nanoparticles/therapeutic use , Theranostic Nanomedicine/methods , Animals , Antineoplastic Agents/chemistry , Bilirubin/chemistry , Chelating Agents/chemistry , Chelating Agents/therapeutic use , Cisplatin/chemistry , HT29 Cells , Humans , Hyperthermia, Induced/methods , Infrared Rays , Mice , Nanoparticles/chemistry , Photoacoustic Techniques/methods , Phototherapy/methods , Platinum/chemistry , Platinum/therapeutic useABSTRACT
Two closely related phenyl selenyl based boron-dipyrromethene (BODIPY) turn-on fluorescent probes for the detection of hypochlorous acid (HOCl) were synthesized for studies in chemical biology; emission intensity is modulated by a photoinduced electron-transfer (PET) process. Probe 2 intrinsically shows a negligible background signal; however, after reaction with HOCl, chemical oxidation of selenium forecloses the PET process, which evokes a significant increase in fluorescence intensity. The fluorescence intensity of probes 1 and 2 with HOCl involves an â¼18 and â¼50-fold enhancement compared with the respective responses from other reactive oxygen/nitrogen species (ROS/RNS) and low detection limits (30.9â nm for 1 and 4.5â nm for 2). Both probes show a very fast response with HOCl; emission intensity reached a maximum within 1â s. These probes show high selectivity for HOCl, as confirmed by confocal microscopy imaging when testing with RAW264.7 and MCF-7 cells.