RESUMO
This paper reports synthesis of a bioreducible hyperbranched (HB) polymer by A2+B3 approach from commercially available dithiothreitol (DTT) (A2) and an easily accessible trifunctional monomer (B3) containing three reactive pyridyl-disulfide groups. Highly efficient thiol-activated disulfide exchange reaction leads to the formation of the HB polymer (Mw = 21000; D = 2.3) with bioreducible disulfide linkages in the backbone and two different functional groups, namely, hydroxyl and pyridyl-disulfide in the core and periphery, respectively, of the HB-polymer. Postpolymerization functionalization of the hydroxyl-groups with camptothecin (CPT), a topoisomerase inhibitor and known anticancer drug, followed by replacing the terminal pyridyl-disulfide groups with oligo-oxyethylene-thiol resulted in easy access to an amphiphilic HB polydisulfide-CPT conjugate (P1) with a very high drug loading content of â¼40%. P1 aggregated in water (above â¼10 µg/mL) producing drug-loaded nanoparticles (Dh â¼ 135 nm), which showed highly efficient glutathione (GSH)-triggered release of the active CPT. Mass spectrometry analysis of the GSH-treated P1 showed the presence of the active CPT drug as well as a cyclic monothiocarbonate product, which underpins the cascade-degradation mechanism involving GSH-triggered cleavage of the labile disulfide linkage, followed by intramolecular nucleophilic attack by the in situ generated thiol to the neighboring carbonate linkage, resulting in release of the active CPT drug. The P1 nanoparticle showed excellent cellular uptake as tested by confocal fluorescence microscopy in HeLa cells by predominantly endocytosis mechanism, resulting in highly efficient cell killing (IC50 â¼ 0.6 µg/mL) as evident from the results of the MTT assay, as well as the apoptosis assay. Comparative studies with an analogous linear polymer-CPT conjugate showed much superior intracellular drug delivery potency of the hyperbranched polymer.
Assuntos
Nanopartículas , Polímeros , Humanos , Polímeros/química , Células HeLa , Portadores de Fármacos/química , Nanopartículas/química , Dissulfetos/química , Compostos de Sulfidrila , Camptotecina/farmacologia , Liberação Controlada de FármacosRESUMO
Mucins are the key component of the defensive mucus barrier. They are extended fibers of very high molecular weight with diverse biological functions depending strongly on their specific structural parameters. Here, we present a mucin-inspired nanostructure, produced via a synthetic methodology to prepare methacrylate-based dendronized polysulfates (MIP-1) on a multi gram-scale with high molecular weight (MW=450â kDa) and thiol end-functionalized mucin-inspired polymer (MIP) via RAFT polymerization. Cryo-electron tomography (Cryo-ET) analysis of MIP-1 confirmed a mucin-mimetic wormlike single-chain fiber structure (length=144±59â nm) in aqueous solution. This biocompatible fiber showed promising activity against SARS-CoV-2 and its mutant strain, with a remarkable low half maximal (IC50 ) inhibitory concentration (IC50 =10.0â nM). Additionally, we investigate the impact of fiber length on SARS-CoV-2 inhibition by testing other functional polymers (MIPs) of varying fiber lengths.
Assuntos
COVID-19 , Impressão Molecular , Humanos , Mucinas , SARS-CoV-2 , Polímeros/farmacologia , Polímeros/química , Impressão Molecular/métodosRESUMO
Mucus hydrogels at biointerfaces are crucial for protecting against foreign pathogens and for the biological functions of the underlying cells. Since mucus can bind to and host both viruses and bacteria, establishing a synthetic model system that can emulate the properties and functions of native mucus and can be synthesized at large scale would revolutionize the mucus-related research that is essential for understanding the pathways of many infectious diseases. The synthesis of such biofunctional hydrogels in the laboratory is highly challenging, owing to their complex chemical compositions and the specific chemical interactions that occur throughout the gel network. In this perspective, we discuss the basic chemical structures and diverse physicochemical interactions responsible for the unique properties and functions of mucus hydrogels. We scrutinize the different approaches for preparing mucus-inspired hydrogels, with specific examples. We also discuss recent research and what it reveals about the challenges that must be addressed and the opportunities to be considered to achieve desirable de novo synthetic mucus hydrogels.
Assuntos
Hidrogéis , Muco , Hidrogéis/química , Muco/química , Bactérias/metabolismoRESUMO
The intracellular transport of molecules, macromolecules or materials is a key step in probing cellular structure and function, as well as regulating a plethora of physical and chemical events for treating disease. This communication reveals direct cellular uptake of pyridyl-disulfide (Py-Ds)-conjugated nonionic and biocompatible macromolecules with the aid of rapid exchange of the highly reactive Py-Ds groups with exofacial cell-surface thiols. Confocal microscopy and flow cytometry analysis confirmed highly efficient cellular uptake of Py-Ds-appended polymers (>50 % in 15â min) by avoiding lysosome as a consequence of thiol-disulfide exchange in the cell surface. In contrast, a control polymer lacking the Py-Ds group followed caveolae-mediated endocytosis. Other control polymers containing either the pyridine group (but not disulfide) or the disulfide group (but not pyridine) revealed significantly low cellular uptake, and thus essential role of the highly reactive Py-Ds group was established beyond doubt.
Assuntos
Dissulfetos/metabolismo , Polímeros/metabolismo , Piridinas/metabolismo , Compostos de Sulfidrila/metabolismo , Transporte Biológico , Dissulfetos/química , Células HeLa , Humanos , Estrutura Molecular , Polímeros/química , Piridinas/química , Compostos de Sulfidrila/químicaRESUMO
This article reports the synthesis, spontaneous self-assembly, highly efficient drug encapsulation, and glutathione (GSH)-triggered intracellular sustained drug delivery of an ABA-type amphiphilic triblock copolymer, namely, polyglycerol-b-poly(disulfide)-b-polyglycerol (PG-b-PDS-b-PG). The bioreducible PDS block with reactive pyridyldisulfide groups present at the chain terminals was attached to thiol-terminated heterotelechelic PG by a thiol-disulfide exchange reaction producing the amphiphilic PG-b-PDS-b-PG. It formed a stable polymersome in aqueous medium with a critical aggregation concentration of 0.02 mg/mL and average hydrodynamic diameter (Dh) of 230 nm and showed highly efficient and stable encapsulation of doxorubicin (Dox) with a remarkably high drug loading efficiency (DLE) and drug loading content (DLC) of 54% and 16%, respectively. Fluorescence spectroscopy studies revealed GSH-triggered drug release and strong dependence of the release kinetics on the GSH concentration due to degradation of the amphiphilic block copolymer and disassembly of the polymersome. MTT assay indicated excellent biocompatibility of the block copolymer as >90% cells (HeLa or MDA-MB-231) were found to be alive after 96 h of incubation with a polymer concentration of up to 1.0 mg/mL, which was further validated by the hemolysis assay. Cytotoxicity assay of the Dox-loaded polymersome exhibited time and dose-dependent sustained killing of HeLa as well as MDA-MB-231 cells wherein after 48 h of incubation >50% cell killing was noticed with a Dox concentration of â¼4.0 and â¼8.7 µg/mL, respectively, while the free Dox showed faster cell killing. Flow cytometry and live cell fluorescence microscopy studies revealed time-dependent cellular uptake of the drug-loaded polymersome followed by diffusion of the drug to the nucleus. Cells with artificially enhanced GSH were killed at a much faster rate indicating that intracellular GSH-triggered disassembly is the key drug release mechanism.
Assuntos
Portadores de Fármacos , Glutationa , Sobrevivência Celular , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Glicerol , Humanos , Micelas , Polietilenoglicóis , PolímerosRESUMO
The dynamic nature of the disulfide bond has enhanced the potential for disulfide based amphiphiles in the emerging biomedical field. Disulfide containing amphiphiles have extensively been used for constructing wide ranging soft nanostructures as potential candidates for delivery of drugs, proteins and genes owing to their degradable nature in the presence of intracellular glutathione (present in a many fold excess compared to the extracellular milieu). This degradable nature of amphiphiles is not only useful to deliver therapeutics but it also eliminates the toxicity issues associated with the carrier after delivery of such therapeutics. Therefore, these bioreducible and biocompatible nano-aggregates inspired researchers to use them as vehicles for therapeutic delivery and as a result the literature of disulfide containing amphiphiles has been intensified. This review article highlights the structural diversity in disulfide containing amphiphilic small molecule and polymeric systems, structural effects on their aqueous aggregation, redox-responsive disassembly and biological applications. Furthermore, the use of disulfide chemistry towards the design of cell penetrating polymers has also been discussed. Finally a brief perspective on some future opportunities of these systems is provided.
Assuntos
Dissulfetos/química , Ciclodextrinas/química , Portadores de Fármacos/química , Glutationa/química , Humanos , Micelas , Nanoestruturas/química , Polímeros/químicaRESUMO
A bioreducible poly(disulfide)-derived amphiphilic block copolymer-drug conjugate (loading content 31%) was synthesized by post-polymerization modification. It shows redox-responsive polymersome assembly in water with aggregation induced emission property arising from the appended Camptothecin (CPT) drug. Glutathione (GSH), a tripeptide overexpressed in cancer cells, triggers a cascade reaction resulting in simultaneous degradation of the polymer backbone (consisting of disulfide linkage) and the release of the pendant drug. The cascade reaction involves GSH trigger cleavage of the backbone disulfide bond producing free thiol followed by its intrachain nucleophilic attack to the adjacent carbonate group that links the appended drug molecule. The polymeric pro-drug exhibits killing efficiency to a cancer cell with remarkably low IC50 value of 3.1 µg/mL (based on the CPT concentration) while it shows negligible toxicity to a normal cell up to polymer concentration 300 µg/mL.
Assuntos
Dissulfetos/química , Glutationa/metabolismo , Polímeros/química , Antineoplásicos Fitogênicos/administração & dosagem , Camptotecina/administração & dosagem , Portadores de Fármacos , Liberação Controlada de Fármacos , Células HeLa , Humanos , Concentração Inibidora 50 , Pró-Fármacos/administração & dosagem , Pró-Fármacos/químicaRESUMO
Mucus lines the epithelial cells at the biological interface and is the first line of defense against multiple viral infections. Mucins, the gel-forming components of mucus, are high molecular weight glycoproteins and crucial for preventing infections by binding pathogens. Consequently, mimicking mucins is a promising strategy for new synthetic virus inhibitors. In this work, synthetic mucin-inspired polymers (MIPs) as potential inhibitors of herpes simplex virus 1 (HSV-1) are investigated. By using a telechelic reversible addition-fragmentation chain-transfer (RAFT) polymerization technique, a new dendronized polysulfate p(G1AAm-OSO3)PDS with an amide-backbone similar to the native mucin glycoproteins is synthesized. p(G1AAm-OSO3)PDS shows mucin-like elongated fiber structure, as revealed in cryo-electron microscopy (cryo-EM) imaging, and its HSV-1 inhibition activity together with its previously reported methacrylate analogue p(G1MA-OSO3)PDS is tested. Both of the sulfated MIPs show strong HSV-1 inhibition in plaque reduction assays with IC50 values in lower nanomolar range (<3 × 10-9 m) and demonstrate a high cell compatibility (CC50 > 1.0 mg mL-1) with lower anticoagulant activity than heparin. In addition, the prophylactic and therapeutic activity of both MIPs is assessed in pre- and post-infection inhibition assays and clearly visualize their high potential for application using fluorescent microscopy imaging of infected cells.
RESUMO
Mucus is a dynamic biological hydrogel, composed primarily of the glycoprotein mucin, exhibits unique biophysical properties and forms a barrier protecting cells against a broad-spectrum of viruses. Here, this work develops a polyglycerol sulfate-based dendronized mucin-inspired copolymer (MICP-1) with ≈10% repeating units of activated disulfide as cross-linking sites. Cryo-electron microscopy (Cryo-EM) analysis of MICP-1 reveals an elongated single-chain fiber morphology. MICP-1 shows potential inhibitory activity against many viruses such as herpes simplex virus 1 (HSV-1) and SARS-CoV-2 (including variants such as Delta and Omicron). MICP-1 produces hydrogels with viscoelastic properties similar to healthy human sputum and with tuneable microstructures using linear and branched polyethylene glycol-thiol (PEG-thiol) as cross-linkers. Single particle tracking microrheology, electron paramagnetic resonance (EPR) and cryo-scanning electron microscopy (Cryo-SEM) are used to characterize the network structures. The synthesized hydrogels exhibit self-healing properties, along with viscoelastic properties that are tuneable through reduction. A transwell assay is used to investigate the hydrogel's protective properties against viral infection against HSV-1. Live-cell microscopy confirms that these hydrogels can protect underlying cells from infection by trapping the virus, due to both network morphology and anionic multivalent effects. Overall, this novel mucin-inspired copolymer generates mucus-mimetic hydrogels on a multi-gram scale. These hydrogels can be used as models for disulfide-rich airway mucus research, and as biomaterials.
Assuntos
Herpesvirus Humano 1 , Hidrogéis , Muco , SARS-CoV-2 , Hidrogéis/química , Hidrogéis/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Muco/metabolismo , SARS-CoV-2/efeitos dos fármacos , Mucinas/química , Mucinas/metabolismo , Antivirais/farmacologia , Antivirais/química , Polímeros/química , Polímeros/farmacologia , Animais , Dissulfetos/química , Polietilenoglicóis/química , Microscopia Crioeletrônica , COVID-19/virologia , GlicerolRESUMO
This paper describes the synthesis of a polymer-prodrug conjugate, its aqueous self-assembly, noncovalent encapsulation of a second drug, and stimuli-responsive intracellular dual drug delivery. Condensation polymerization between a functionalized diol and a commercially available diisocyanate in the presence of poly(ethylene glycol) hydroxide (PEG-OH) as the chain stopper produces an ABA-type amphiphilic block copolymer (PU-1) in one pot, with the middle hydrophobic block being a polyurethane containing a pendant tert-butyloxycarbonyl (Boc)-protected amine in every repeating unit. Deprotection of the Boc group, followed by covalent attachment of the Pt(IV) prodrug using the pendant amine groups, produces the polymer-prodrug conjugate PU-Pt-1, which aggregates to nanocapsule-like structures in water with a hydrophilic interior. In the presence of sodium ascorbate, the Pt(IV) prodrug can be detached from the polymer backbone, producing the active Pt(II) drug. Cell culture studies show appreciable cell viability by the parent polymer. However, the polymer-prodrug conjugate nanocapsules exhibit cellular uptake and intracellular release of the active drug under a reducing environment. The capsule-like aggregates of the polymer-prodrug conjugate were used for noncovalent encapsulation of a second drug, doxorubicin (Dox), and Dox-loaded PU-Pt-1 aggregate showed a significantly superior cell killing efficiency compared to either of the individual drugs, highlighting the promising application of such a dual-drug-delivery approach.
Assuntos
Neoplasias , Pró-Fármacos , Cisplatino , Pró-Fármacos/farmacologia , Poliuretanos , Sistemas de Liberação de Medicamentos , Polietilenoglicóis/química , Polímeros/química , Doxorrubicina/farmacologia , Doxorrubicina/química , Micelas , Aminas , Portadores de Fármacos/químicaRESUMO
A strategy for a redox-activatable heavy-atom-free photosensitizer (PS) based on thiolated naphthalimide has been demonstrated. The PS exhibits excellent reactive oxygen species (ROS) generation in the monomeric state. However, when encapsulated in a disulfide containing bioreducible amphiphilic triblock copolymer aggregate (polymersome), the PS exhibits aggregation in the confined hydrophobic environment, which results in a smaller exciton exchange rate between the singlet and triplet excited states (TDDFT studies), and consequently, the ROS generation ability of the PS was almost fully diminished. Such a PS (in the dormant state)-loaded redox-responsive polymersome showed excellent cellular uptake and intracellular release of the PS in its active form, which enabled cell killing upon light irradiation due to ROS generation. In a control experiment involving aggregates of a similar block copolymer, but lacking the bioreducible disulfide linkage, no intracellular reactivation of the PS was noticed, highlighting the importance of stimuli-responsive polymer assemblies in the area of targeted photodynamic therapy.
Assuntos
Fotoquimioterapia , Fármacos Fotossensibilizantes , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio , Polímeros/química , Dissulfetos/química , OxirreduçãoRESUMO
Photodynamic therapy (PDT) is emerging as an efficient strategy to combat multidrug-resistant (MDR) cancer. However, the short half-life and limited diffusion of reactive oxygen species (ROS) undermine the therapeutic outcomes of this therapy. To address this issue, a tumor-targeting nanoplatform was developed to precisely deliver mitochondria- and endoplasmic reticulum (ER)-targeting PDT agents to desired sites for dual organelle-targeted PDT. The nanoplatform is constructed by functionalizing molybdenum disulfide (MoS2) nanoflakes with glucose-modified hyperbranched polyglycerol (hPG), and then loading the organelle-targeting PDT agents. The resultant nanoplatform Cy7.5-TG@GPM is demonstrated to mediate both greatly enhanced internalization within MDR cells and precise subcellular localization of PDT agents, facilitating in situ near-infrared (NIR)-triggered ROS generation for augmented PDT and reversal of MDR, causing impressive tumor shrinkage in a HeLa multidrug-resistant tumor mouse model. As revealed by mechanistic studies of the synergistic mitochondria- and ER-targeted PDT, ROS-induced ER stress not only activates the cytosine-cytosine-adenosine-adenosine thymidine/enhancer-binding protein homologous protein (CHOP) pro-apoptotic signaling pathway, but also cooperates with ROS-induced mitochondrial dysfunction to trigger cytochrome C release from the mitochondria and induce subsequent cell death. Furthermore, the mitochondrial dysfunction reduces ATP production and thereby contributes to the reversal of MDR. This nanoplatform, with its NIR-responsive properties and ability to target tumors and subcellular organelles, offers a promising strategy for effective MDR cancer therapy.
Assuntos
Nanopartículas , Fotoquimioterapia , Camundongos , Animais , Espécies Reativas de Oxigênio/metabolismo , Molibdênio , Glucose , Linhagem Celular Tumoral , Adenosina , Citosina , Fármacos Fotossensibilizantes/uso terapêutico , Fármacos Fotossensibilizantes/farmacologia , Nanopartículas/químicaRESUMO
In two ABA type amphiphilic block copolymers (P1, P2), the hydrophobic B block consists of a bioreducible segmented poly(disulfide) (PDS), while poly-N-isopropylacrylamide (PNIPAM) or poly(triethyleneglycol)methylether-methacrylate (PTEGMA) serve as the hydrophilic A blocks in P1 and P2, respectively, leading to the formation of polymersome and micelle, owing to the difference in the packing parameters. Both exhibit comparable doxorubicin (Dox) encapsulation efficiency, but glutathione (GSH) triggered release appears much faster from the polymersome than micelle owing to the complete degradation of the PDS segment in polymersome morphology unlike in micelle. Dox-loaded polymers (P1-Dox and P2-Dox) exhibit minimum toxicity to normal cells like C2C12. By contrast, P1-Dox shows excellent killing efficiency to the HeLa cells (cancer cell) (in which the GSH concentration is significantly higher). However, P2-Dox reveals a rather poor activity even to HeLa cells. Fluorescence microscopy studies show comparable cellular uptake of P1-Dox and P2-Dox. But the polymersome entrapped dye escapes fast from the cargo and reach the nucleus, while the drug-loaded micelle remains trapped in the perinuclear zone explaining the significant difference in the drug delivery performance of polymersome and micelle.