ABSTRACT
Slow-healing and chronic wounds represent a major global economic and medical burden, and there is significant unmet need for novel therapies which act to both accelerate wound closure and enhance biomechanical recovery of the skin. Here, we report a new approach in which bioactives that augment early stages of wound healing can kickstart and engender effective wound closure in healthy and diabetic, obese animals, and set the stage for subsequent tissue repair processes. We demonstrate that a nanomaterial dressing made of silk fibroin and gold nanorods (GNR) stimulates a pro-neutrophilic, innate immune, and controlled inflammatory wound transcriptomic response. Further, Silk-GNR, lasered into the wound bed, in combination with exogeneous histamine, accelerates early-stage processes in tissue repair leading to effective wound closure. Silk-GNR and histamine enhanced biomechanical recovery of skin, increased transient neoangiogenesis, myofibroblast activation, epithelial-to-mesenchymal transition (EMT) of keratinocytes and a pro-resolving neutrophilic immune response, which are hitherto unknown activities for these bioactives. Predictive and temporally coordinated delivery of growth factor nanoparticles that modulate later stages of tissue repair further accelerated wound closure in healthy and diabetic, obese animals. Our approach of kickstarting healing by delivering the "right bioactive at the right time" stimulates a multifactorial, pro-reparative response by augmenting endogenous healing and immunoregulatory mechanisms and highlights new targets to promote tissue repair.
Subject(s)
Diabetes Mellitus , Nanostructures , Animals , Wound Healing , Histamine , Silk , ObesityABSTRACT
BACKGROUND AND OBJECTIVE: Chrysin and its derivatives proved to possess potential anti-tumour activity. MATERIALS AND METHODS: A new series of chrysin analogs containing 1,2,3-triazoles with different substituent groups (5a-5l) was designed, synthesized, and evaluated as potential anticancer agents. The synthesized compounds were characterized using FT-IR, 1H NMR 13C NMR spectroscopy and mass spectrometry. RESULTS: The anticancer activities of the synthesized compounds were studied in four cancer cell lines viz. PC3, PC3-PSMA, MCF-7 and UM-UC-3 using doxorubicin as standard. Among all the tested compounds, 5c was found as most active with IC50 value of 10.8 ± 0.04 µM in PC3 cells and 20.53 ± 0.21 µMin MCF-7 cells, respectively. Flow cytometry analyses indicated that synthesized compounds 5a, 5c, and 5h arrested MCF-7 cells at the G2/M phase in a dose-dependent manner. CONCLUSION: Chyrsin derivatives could be novel anticancer agents.
Subject(s)
Antineoplastic Agents/pharmacology , Flavonoids/pharmacology , Triazoles/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Flavonoids/chemistry , Humans , Molecular Structure , Structure-Activity Relationship , Triazoles/chemical synthesis , Triazoles/chemistryABSTRACT
Simultaneous delivery of small molecules and nucleic acids using a single vehicle can lead to novel combination treatments and multifunctional carriers for a variety of diseases. In this study, we report a novel library of aminoglycoside-derived lipopolymers nanoparticles (LPNs) for the simultaneous delivery of different molecular cargoes including nucleic acids and small-molecules. The LPN library was screened for transgene expression efficacy following delivery of plasmid DNA, and lead LPNs that showed high transgene expression efficacies were characterized using hydrodynamic size, zeta potential, 1H NMR and FT-IR spectroscopy, and transmission electron microscopy. LPNs demonstrated significantly higher efficacies for transgene expression than 25 kDa polyethyleneamine (PEI) and lipofectamine, including in presence of serum. Self-assembly of these cationic lipopolymers into nanoparticles also facilitated the delivery of small molecule drugs (e.g. doxorubicin) to cancer cells. LPNs were also employed for the simultaneous delivery of the small-molecule histone deacetylase (HDAC) inhibitor AR-42 together with plasmid DNA to cancer cells as a combination treatment approach for enhancing transgene expression. Taken together, our results indicate that aminoglycoside-derived LPNs are attractive vehicles for simultaneous delivery of imaging agents or chemotherapeutic drugs together with nucleic acids for different applications in medicine and biotechnology.
Subject(s)
Antineoplastic Agents/pharmacology , DNA/pharmacology , Drug Carriers/chemistry , Histone Deacetylase Inhibitors/pharmacology , Nanoparticles/chemistry , Polymers/chemistry , Aminoglycosides/chemistry , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , DNA/genetics , Doxorubicin/chemistry , Doxorubicin/pharmacology , Drug Liberation , Gene Transfer Techniques , Glycolipids/chemistry , Green Fluorescent Proteins/genetics , Histone Deacetylase Inhibitors/chemistry , Humans , Mice , Phenylbutyrates/pharmacology , Plasmids/genetics , Plasmids/pharmacology , TNF-Related Apoptosis-Inducing Ligand/geneticsABSTRACT
Copper ions play an important role in several physiological processes, including angiogenesis, growth factor induction and extracellular matrix remodeling, that modulate wound healing and tissue repair. In this work, copper-loaded alginate fibers were generated and used as surgical sutures for repair of incisional wounds in live mice. Approximately 95% of initially loaded copper ions were released from the sutures within the first 24 h following an initial burst release. This localized delivery of copper at the incision site resulted in significantly higher recovery in tissue biomechanical strengths compared to conventional nylon and calcium alginate sutures at early times following surgery. Irradiation of copper alginate sutures with near-infrared light resulted in a robust photothermal response and led to efficacies similar to those seen with nonirradiated sutures. Histopathology and immunohistological analyses indicated significantly reduced epithelial gap and higher number of CD31+ cells, which is indicative of increased angiogenesis around the incision site. Delivery of copper ions did not result in toxicity under the conditions employed. Our findings demonstrate that delivery of ionic copper from sutures resulted in efficacious approximation and healing of incisional wounds, and copper-eluting fibers may have translational potential for accelerating repair in surgical and trauma wounds.
Subject(s)
Copper/chemistry , Copper/pharmacology , Alginates/chemistry , Animals , Cells, Cultured , Male , Mice , Neovascularization, Physiologic/drug effects , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Skin/cytology , Sutures , Wound Healing/drug effectsABSTRACT
Anticancer drugs exert their effects on cancer cells by deregulating many pathways linked to cell cycle, apoptosis, etc. but cancer cells gradually become resistive against anticancer drugs, thereby necessitating the development of newer generation anticancer molecules. N-end rule pathway has been shown to be involved in the degradation of many cell cycle and apoptosis-related proteins. However, the involvements of this pathway in cancer are not well established. Recently, we developed a non-peptide-based N-end rule pathway inhibitor, RF-C11 for type 1 and 2 recognition domains of E3 ubiquitin ligases. The inhibitor significantly increased the half-life of potential N-degrons leading to significant physiological changes in vivo. We hypothesized RF-C11 may be used to decipher the N-end rule pathway's role in cancer towards the development of anticancer therapeutics. In this study, we showed that RF-C11, barring noncancer cells, significantly sensitizes cancer cells towards different anticancer agents tested. We further find that the profound cellular sensitization to anticancer drugs was affected by (a) downregulation of X-linked inhibitor of apoptosis protein, an antiapoptotic protein and (b) by stabilization of RAD21, and thereby inhibiting metaphase to anaphase promotion. The study shows that RF-C11 or its analogs may be used as a novel additive in combination therapy against cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic , Neoplasms/drug therapy , Ubiquitin-Protein Ligases/antagonists & inhibitors , X-Linked Inhibitor of Apoptosis Protein/metabolism , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Cycle Proteins/genetics , Cell Proliferation , DNA-Binding Proteins/genetics , Drug Resistance, Neoplasm , Drug Synergism , Humans , Neoplasms/metabolism , Neoplasms/pathology , Tumor Cells, Cultured , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , X-Linked Inhibitor of Apoptosis Protein/geneticsABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand that can preferentially induce apoptosis in cancer cells over normal cells. The transmembrane form of TRAIL has been shown to elicit much stronger activity than its soluble counterpart but delivery is a potential challenge. Here, we investigated the potential of aminoglycoside-derived polymers to enhance delivery of a plasmid (pEF-TRAIL) that expresses the transmembrane form of TRAIL in order to determine the effect on cell death in vitro and tumor growth in vivo. Transgene delivery efficacy and toxicity of aminoglycoside-derived polymers was first evaluated using a GFP-expressing plasmid (pEF-GFP) at different plasmid amounts and plasmid : polymer ratios in UMUC3 bladder cancer and HeLa cervical cancer cells. Delivery of the TRAIL plasmid using aminoglycoside-derived polymers resulted in up to 60% cell death in UMUC3 and HeLa cells; TRAIL protein expression was confirmed using Western blots. TRAIL plasmid delivery resulted in a decrease in cellular procaspase-8 and an increase in TRAIL receptor DR5 levels, suggesting a role for the death receptor and caspase cascade in TRAIL-mediated apoptosis. The TRAIL plasmid did not cause cell death in normal human or mouse fibroblasts. The in vivo delivery of the TRAIL plasmid using a paromomycin-derived polymer resulted in significant reduction in tumor burden and increased survival in tumor-bearing live mice.
Subject(s)
Aminoglycosides/chemistry , DNA/genetics , Polymers/chemistry , TNF-Related Apoptosis-Inducing Ligand/metabolism , Animals , Carcinoma/therapy , Cell Line, Tumor , Cell Survival , Female , Genetic Therapy , Humans , Mice , Mice, Nude , Molecular Structure , NIH 3T3 Cells , Neoplasms, Experimental , Plasmids , TNF-Related Apoptosis-Inducing Ligand/genetics , Urinary Bladder Neoplasms/therapyABSTRACT
Under the heading "Methods-Synthesis of the Bioreducible Modified-PAE (mPAE)", on page 3, line 14-17, there is an error. The quantity unit of PAE and 2-iminothiolane hydrochloride needs to be corrected to mg instead of g.
ABSTRACT
PURPOSE: Lung cancer is one of the leading causes of deaths in the United States, but currently available therapies for lung cancer are associated with reduced efficacy and adverse side effects. Small interfering RNA (siRNA) can knock down the expression of specific genes and result in therapeutic efficacy in lung cancer. Recently, mTOR siRNA has been shown to induce apoptosis in NSCLC cell lines but its use is limited due to poor stability in biological conditions. METHODS: In this study, we modified an aminoglyocisde-derived cationic poly (amino-ether) by introducing a thiol group using Traut's reagent to generate a bio-reducible modified-poly (amino-ether) (mPAE). The mPAE polymer was used to encapsulate mTOR siRNA by nanoprecipitation method, resulting in the formation of stable and bio-reducible nanoparticles (NPs) which possessed an average diameter of 114 nm and a surface charge of approximately +27 mV. RESULTS: The mTOR siRNA showed increased release from the mTS-mPAE NPs in the presence of 10 mM glutathione (GSH). The polymeric mTS-mPAE-NPs were also capable of efficient gene knockdown (60 and 64%) in A549 and H460 lung cancer cells, respectively without significant cytotoxicity at 30 µg/ml concentrations. The NPs also showed time-dependent cellular uptake for up to 24 h as determined using flow cytometry. Delivery of the siRNA using these NPs also resulted in significant inhibition of A549 and H460 cell proliferation in vitro, respectively. CONCLUSIONS: The results demonstrate that the mPAE polymer based NPs show strong potential for siRNA delivery to lung cancer cells. It is anticipated that future modification can help improve the efficacy of nucleic acid delivery, leading to higher inhibition of lung cancer growth in vitro and in vivo.
Subject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Ethers/chemistry , Lung Neoplasms/therapy , Polymers/chemical synthesis , RNA, Small Interfering/administration & dosage , Antineoplastic Agents/metabolism , Biological Transport , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Compounding , Humans , Lung Neoplasms/metabolism , Oxidation-Reduction , Rhodamines/metabolismABSTRACT
Effective transgene expression in mammalian cells relies on successful delivery, cytoplasmic trafficking, and nuclear translocation of the delivered vector, but delivery is impeded by several formidable physicochemical barriers on the surface of and within the target cell. Although methods to overcome cellular exclusion and endosomal entrapment have been studied extensively, strategies to overcome inefficient nuclear entry and subsequent intranuclear barriers to effective transient gene expression have only been sparsely explored. In particular, the role of nuclear packaging of DNA with histone proteins, which governs endogenous gene expression, has not been extensively elucidated in the case of exogenously delivered plasmids. In this work, a parallel screen of small molecule inhibitors of chromatin-modifying enzymes resulted in the identification of class I/II HDACs, sirtuins, LSD1, HATs, and the methyltransferases EZH2 and MLL as targets whose inhibition led to the enhancement of transgene expression following polymer-mediated delivery of plasmid DNA. Quantitative PCR studies revealed that HDAC inhibition enhances the amount of plasmid DNA delivered to the nucleus in UMUC3 human bladder cancer cells. Native chromatin immunoprecipitation (N-ChIP)-qPCR experiments in CHO-K1 cells indicated that plasmids indeed interact with intracellular core Histone H3, and inhibitors of HDAC and LSD1 proteins are able to modulate this interaction. Pair-wise treatments of effective inhibitors led to synergistic enhancement of transgene expression to varying extents in both cell types. Our results demonstrate that the ability to modulate enzymes that play a role in epigenetic processes can enhance the efficacy of non-viral gene delivery, resulting in significant implications for gene therapy and industrial biotechnology.
Subject(s)
DNA/genetics , Gene Expression/drug effects , Gene Transfer Techniques , Histone Deacetylase Inhibitors/pharmacology , Histone Demethylases/antagonists & inhibitors , Plasmids/genetics , Transgenes , Animals , CHO Cells , Cell Line, Tumor , Cricetulus , Genetic Therapy , Histones/metabolism , Humans , Neoplasms/therapy , Transgenes/drug effectsABSTRACT
Scaffolds generated from naturally occurring and synthetic polymers have been investigated in several applications because of their biocompatibility and tunable chemo-mechanical properties. Existing methods for generation of 3D polymeric scaffolds typically cannot be parallelized, suffer from low throughputs, and do not allow for quick and easy removal of the fragile structures that are formed. Current molds used in hydrogel and scaffold fabrication using solvent casting and porogen leaching are often single-use and do not facilitate 3D scaffold formation in parallel. Here, we describe a simple device and related approaches for the parallel fabrication of macroporous scaffolds. This approach was employed for the generation of macroporous and non-macroporous materials in parallel, in higher throughput and allowed for easy retrieval of these 3D scaffolds once formed. In addition, macroporous scaffolds with interconnected as well as non-interconnected pores were generated, and the versatility of this approach was employed for the generation of 3D scaffolds from diverse materials including an aminoglycoside-derived cationic hydrogel ("Amikagel"), poly(lactic-co-glycolic acid) or PLGA, and collagen. Macroporous scaffolds generated using the device were investigated for plasmid DNA binding and cell loading, indicating the use of this approach for developing materials for different applications in biotechnology. Our results demonstrate that the device-based approach is a simple technology for generating scaffolds in parallel, which can enhance the toolbox of current fabrication techniques.
Subject(s)
Biocompatible Materials , Biotechnology/methods , Cell Culture Techniques/methods , Tissue Scaffolds , Cells, Cultured , Plasmids/isolation & purification , PorosityABSTRACT
The development of effective drug carriers can lead to improved outcomes in a variety of disease conditions. Aminoglycosides have been used as antibacterial therapeutics, and are attractive as monomers for the development of polymeric materials in various applications. Here, we describe the development of novel aminoglycoside-derived amphiphilic nanoparticles for drug delivery, with an eye towards ablation of cancer cells. The aminoglycoside paromomycin was first cross-linked with resorcinol diglycidyl ether leading to the formation of a poly (amino ether), PAE. PAE molecules were further derivatized with methoxy-terminated poly(ethylene glycol) or mPEG resulting in the formation of mPEG-PAE polymer, which self-assembled to form nanoparticles. Formation of the mPEG-PAE amphiphile was characterized using (1)H NMR, (13)C NMR, gel permeation chromatography (GPC) and FTIR spectroscopy. Self-assembly of the polymer into nanoparticles was characterized using dynamic light scattering, zeta potential analyses, atomic force microscopy (AFM) and the pyrene fluorescence assay. mPEG-PAE nanoparticles were able to carry significant amounts of doxorubicin (DOX), presumably by means of hydrophobic interactions between the drug and the core. Cell-based studies indicated that mPEG-PAE nanoparticles, loaded with doxorubicin, were able to induce significant loss in viabilities of PC3 human prostate cancer, MDA-MB-231 human breast cancer, and MB49 murine bladder cancer cells; empty nanoparticles resulted in negligible losses of cell viability under the conditions investigated. Taken together, our results indicate that the mPEG-PAE nanoparticle platform is attractive for drug delivery in different applications, including cancer.
Subject(s)
Aminoglycosides/chemistry , Doxorubicin/pharmacology , Drug Delivery Systems , Nanoparticles/administration & dosage , Neoplasms/drug therapy , Antibiotics, Antineoplastic/administration & dosage , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacology , Cell Survival/drug effects , Doxorubicin/administration & dosage , Doxorubicin/chemistry , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Micelles , Molecular Structure , Nanoparticles/chemistry , Neoplasms/pathology , Polymers/chemistry , Tumor Cells, CulturedABSTRACT
Targeted delivery of anticancer therapeutics can potentially overcome the limitations associated with current chemotherapeutic regimens. Folate receptors are overexpressed in several cancers, including ovarian, triple-negative breast and bladder cancers, making them attractive for targeted delivery of nucleic acid therapeutics to these tumors. This work describes the synthesis, characterization and evaluation of folic acid-conjugated, aminoglycoside-derived polymers for targeted delivery of transgenes to breast and bladder cancer cell lines. Transgene expression was significantly higher with FA-conjugated aminoglycoside-derived polymers than with Lipofectamine, and these polymers demonstrated minimal cytotoxicty. Competitive inhibition using free folic acid significantly reduced transgene expression efficacy of folate-targeted polymers, suggesting a role for folate receptor-mediated uptake. High efficacy FA-targeted polymers were employed to deliver a plasmid expressing the TRAIL protein, which induced death in cancer cells. These results indicate that FA-conjugated aminoglycoside-derived polymers are promising for targeted delivery of nucleic acids to cancer cells that overexpress folate receptors.