Disulfide-containing Cross-linker Research Articles

PH-induced fluorescent active sodium alginate-based ionically conjugated and REDOX responsive multi-functional microgels for the anticancer drug delivery

A sodium alginate (Alg) based REDOX (reduction and oxidation)-responsive and fluorescent active microgel was prepared via water in oil (w/o) mini-emulsion polymerization technique. Here, we initially synthesized sodium alginate-based disulfide cross linked microgels and after that those microgels were tagged with rhodamine amine derivative (RhB-NH2) by ionic interaction to get the pH-responsive fluorescent property. Functionalized microgels were characterized using 1H NMR, FTIR, DLS, HRTEM, FESEM, UV–vis, and fluorescence spectroscopy analyses. Presence of the REDOX-responsive disulfide-containing crosslinkers in the microgels enhances the release of doxorubicin (DOX), an anti-cancer drug in the reducing environment of the cancer-cells (simulated). Existence of the rhodamine-amine derivative in the microgels triggers the pH-dependent fluorescence property by showing fluorescence emission at 560–580 nm at pH 5.5 (cancer cell pH). The cytotoxicity of the biopolymer based microgel was assessed over both cancerous HeLa (IC50 100 µg/mL) and non-cancerous MDCK (IC50 200 µg/mL) cells by MTT assay which showed the synthesized microgel is non-toxic whereas DOX-loaded microgels showed significant toxicity. FACS and cell uptake (in vitro) analyses were conducted to understand the cell apoptosis cycle and behavior of the cancer cells in presence of the DOX-loaded microgels. This pH-responsive fluorescent active alginate-based biomaterial could be a promising material for the anti-cancer drug delivery and other medical fields.

Dual-Modified Hyaluronic Acid for Tunable Double Cross-Linked Hydrogel Adhesives.

Conventional techniques for the closure of wounds, such as sutures and staples, have significant drawbacks that can negatively impact wound healing. Tissue adhesives have emerged as promising alternatives, but poor adhesion, low mechanical properties, and toxicity have hindered their widespread clinical adoption. In this work, a dual modified, aldehyde and methacrylate hyaluronic acid (HA) biopolymer (HA-MA-CHO) has been synthesized through a simplified route for use as a double cross-linked network (DCN) hydrogel (HA-MA-CHO-DCN) adhesive for the effective closure and sealing of wounds. HA-MA-CHO-DCN cross-links in two stages: initial cross-linking of the aldehyde functionality (CHO) of HA-MA-CHO using a disulfide-containing cross-linker, 3,3'-dithiobis (propionic hydrazide) (DTPH), leading to the formation of a self-healing injectable gel, followed by further cross-linking via ultraviolet (UV) initiated polymerization of the methacrylate (MA) functionality. This hydrogel adhesive shows a stable swelling behavior and remarkable versatility as the storage modulus (G') has shown to be highly tunable (103-105 Pa) for application to many different wound environments. The new HA-MA-CHO-DCN hydrogel showed excellent adhesive properties by surpassing the burst pressure and lap-shear strength for the widely used bovine serum albumin-glutaraldehyde (BSAG) glue while maintaining excellent cell viability.

Redox-Responsive Core-Cross-Linked Micelles of Miktoarm Poly(ethylene oxide)-b-poly(furfuryl methacrylate) for Anticancer Drug Delivery.

The physiological instability of nanocarriers, premature drug leakage during blood circulation, and associated severe side effects cause compromised therapeutic efficacy, which have significantly hampered the progress of nanomedicines. The cross-linking of nanocarriers while keeping the effectiveness of their degradation at the targeted site to release the drug has emerged as a potent strategy to overcome these flaws. Herein, we have designed novel (poly(ethylene oxide))2-b-poly(furfuryl methacrylate) ((PEO2K)2-b-PFMAnk) miktoarm amphiphilic block copolymers by coupling alkyne-functionalized PEO (PEO2K-C≡H) and diazide-functionalized poly(furfuryl methacrylate) ((N3)2-PFMAnk) via click chemistry. (PEO2K)2-b-PFMAnk self-assembled to form nanosized micelles (mikUCL) with hydrodynamic radii in the range of 25∼33 nm. The hydrophobic core of mikUCL was cross-linked by a disulfide-containing cross-linker using the Diels-Alder reaction to avoid unwanted leakage and burst release of a payload. As expected, the resulting core-cross-linked (PEO2K)2-b-PFMAnk micelles (mikCCL) exhibited superior stability under a normal physiological environment and were de-cross-linked to rapidly release doxorubicin (DOX) upon exposure to a reduction environment. The micelles were compatible with HEK-293 normal cells, while DOX-loaded micelles (mikUCL/DOX and mikCCL/DOX) induced high antitumor activity in HeLa and HT-29 cells. mikCCL/DOX preferentially accumulated at the tumor site and was more efficacious than free DOX and mikUCL/DOX for tumor inhibition in HT-29 tumor-bearing nude mice.

Macrophage Membrane-Camouflaged Responsive Polymer Nanogels Enable Magnetic Resonance Imaging-Guided Chemotherapy/Chemodynamic Therapy of Orthotopic Glioma.

Development of innovative nanomedicine formulations to traverse the blood-brain barrier (BBB) for effective theranostics of glioma remains a great challenge. Herein, we report the creation of macrophage membrane-camouflaged multifunctional polymer nanogels coloaded with manganese dioxide (MnO2) and cisplatin for magnetic resonance (MR) imaging-guided chemotherapy/chemodynamic therapy (CDT) of orthotopic glioma. Redox-responsive poly(N-vinylcaprolactam) (PVCL) nanogels (NGs) formed via precipitation polymerization were in situ loaded with MnO2 and physically encapsulated with cisplatin to have a mean size of 106.3 nm and coated with macrophage membranes to have a good colloidal stability. The generated hybrid NGs display dual pH- and redox-responsive cisplatin and Mn(II) release profiles and can deplete glutathione (GSH) rich in tumor microenvironment through reaction with disulfide-containing cross-linkers within the NGs and MnO2. The thus created Mn(II) enables enhanced CDT through a Fenton-like reaction and T1-weighted MR imaging, while the loaded cisplatin not only exerts its chemotherapy effect but also promotes the reactive oxygen species generation to enhance the CDT efficacy. Importantly, the macrophage membrane coating rendered the hybrid NGs with prolonged blood circulation time and ability to traverse BBB for specific targeted chemotherapy/CDT of orthotopic glioma. Our study demonstrates a promising self-adaptive and cooperative NG-based nanomedicine platform for highly efficient theranostics of glioma, which may be extended to tackle other difficult cancer types.

Multifunctional poly(quaternary ammonium)/Fe3O4 composite nanogels for integration of antibacterial and degradable magnetic redox-responsive properties

Multifunctional cationic poly(2-(dimethylamino)-ethyl methacrylate)/Fe3O4 composite nanogels (PQDMAEMA/Fe3O4 CNGs) with degradable, magnetic, redox-responsive, and antibacterial properties were tailored through activators generated by electron transfer atom transfer radical polymerization in inverse miniemulsion systems. The as-prepared PQDMAEMA/Fe3O4 CNGs not only inherited good superparamagnetism of Fe3O4 nanoparticles but also displayed a marked redox-responsiveness (using a disulfide-containing cross-linker) and a reversible pH-dependent loading and release behavior of a model protein (bovine serum albumin). Most importantly, the PQDMAEMA/Fe3O4 CNGs exhibited potent broad-spectrum antimicrobial activity towards Escherichia coli, Staphylococcus aureus, and Canidia albicans. The results reveal that the multifunctional PQDMAEMA/Fe3O4 CNGs have essential promise in tackling bacterial infection and stimuli-responsive applications such as protein adsorption and separation.

Inverse Electron-Demand Diels-Alder Methylcellulose Hydrogels Enable the Co-delivery of Chondroitinase ABC and Neural Progenitor Cells.

A hydrogel that can deliver both proteins and cells enables the local microenvironment of transplanted cells to be manipulated with a single injection. Toward this goal, we designed a hydrogel suitable for the co-delivery of neural stem cells and chondroitinase ABC (ChABC), a potent enzyme that degrades the glial scar that forms after central nervous system (CNS) injury. We leveraged the inverse electron-demand Diels-Alder reaction between norbornene and methylphenyltetrazine to form rapidly gelling (<15 min) crosslinked methylcellulose (MC) hydrogels at physiological temperature and pH, with Young's modulus similar to that of brain tissue (1-3 kPa), and degradable, disulfide-containing crosslinkers. To achieve tunable, affinity-controlled release of a ChABC-Src homology 3 (SH3) fusion protein, we immobilized norbornene-functionalized SH3-binding peptides onto MC-methylphenyltetrazine and observed release of bioactive ChABC-SH3 over 4 days. We confirmed cytocompatibility by evaluating neural progenitor cell survival and proliferation. The combined encapsulation of neural stem cells and chondroitinase ABC from one hydrogel lays the framework for future in vivo studies to treat CNS injuries.

Reduction-responsive molecularly imprinted nanogels for drug delivery applications.

Degradable molecularly imprinted polymers (MIPs) with affinity for S-propranolol were prepared by the copolymerization of methacrylic acid as functional monomer and a disulfide-containing cross-linker, bis(2-methacryloyloxyethyl)disulfide (DSDMA), using bulk polymerization or high dilution polymerization for nanogels synthesis. The specificity and the selectivity of DSDMA-based molecularly imprinted polymers toward S-propranolol were studied in batch binding experiments, and their binding properties were compared to a traditional ethylene glycol dimethacrylate (EDMA)-based MIP. Nanosized MIPs prepared with DSDMA as crosslinker could be degraded into lower molecular weight linear polymers by cleaving the disulfide bonds and thus reversing cross-linking using different reducing agents (NaBH4, DTT, GSH). Turbidity, viscosity, polymer size and IR-spectra were measured to study the polymer degradation. The loss of specific recognition and binding capacity of S-propranolol was also observed after MIP degradation. This phenomenon was applied to modulate the release properties of the MIP. In presence of GSH at its intracellular concentration, the S-propranolol release was higher, showing that these materials could potentially be applied as intracellular controlled drug delivery system.

Preparation of degradable magnetic temperature- and redox-responsive polymeric/Fe3O4 nanocomposite nanogels in inverse miniemulsions for loading and release of 5-fluorouracil

This work reports the synthesis of novel degradable magnetic temperature- and redox-responsive polymer/Fe3O4 nanocomposite nanogels (NCGs) through inverse miniemulsion polymerization and their potential application as nanocarriers for controlled drug delivery. Surface of Fe3O4 magnetic nanoparticles (MNPs) were vinyl-modified with 2-isocyanatoethyl methacrylate. Poly(N-vinylcaprolactam) (PNVCL)/Fe3O4 NCGs were prepared via inverse miniemulsion polymerization by using a disulfide-containing cross-linker. The Fe3O4 MNPs were chemically attached to the polymer matrix of NCGs, offering a good structural integrity of NCGs in aqueous systems. The PNVCL/Fe3O4 NCGs not only inherited the good superparamagnetism of Fe3O4 MNPs, but also displayed a reversible temperature-responsiveness as well as marked redox-responsiveness. The release behavior of a model anticancer drug 5-fluorouracil from the PNVCL/Fe3O4 NCGs could be conveniently tuned by the external temperature, redox condition of media, or their combination. The prepared PNVCL/Fe3O4 NCGs displayed a low cytotoxicity, holding a great potential in functioning as magnetic guiding nanocarriers for controlled drug delivery.

Fluorinated Glycopolymers as Reduction-responsive 19F MRI Agents for Targeted Imaging of Cancer.

Imaging agents that can be targeted to specific diseases and respond to the microenvironment of the diseased tissue are of considerable interest due to their potential in diagnosing and managing diseases. Here we report a new class of branched fluorinated glycopolymers as 19F MRI contrast agents that respond to a reductive environment, for targeted imaging of cancer. The fluorinated glycopolymers can be readily prepared by a one-pot RAFT polymerization of glucose- and fluorine-containing monomers in the presence of a disulfide-containing cross-linking monomer. The incorporation of glucose units along the polymer chain enables these fluorinated glycopolymers to effectively target cancer cells due to interactions with the overexpressed sugar transporters present on the cell surface. In addition, the polymers exhibit an enhanced 19F MRI signal in response to a reductive environment, one of the unique hallmarks of many cancer cells, demonstrating their potential as promising candidates for targeted imaging of cancer.

Hybridization of graphene oxide into nanogels to acquire higher photothermal effects for therapeutic delivery

Although the special architecture of two-dimensional (2D) nanomaterials endows them with unique properties, their poor colloidal stability remains a main bottleneck to fully exploit their applications in the biomedical field. Herein, this study aims to develop a simple and effective approach to in situ incorporate 2D graphene oxide (GO) nanoplatelets into a thermosensitive matrix to acquire hybrid nanogels with good stability and photothermal effect. In order to improve its stability, GO firstly underwent silanization to its surface with double bonds, followed by intercalation with N-isopropylacrylamide (NIPAM) in the presence of a disulfide-containing crosslinker via an emulsion method. Radical polymerization was then initiated to accelerate direct GO exfoliation in PNIPAM nanogels by forming covalent bonds between them. The well-dispersed GO nanopletlets in the nanogels not only displayed an enhanced photothermal effect, but also improved the encapsulation efficiency of an anticancer drug. The hybrid nanogels accelerate drug release under conditions mimicking the acidic/reducible solid tumor and intracellular microenvironments, most importantly, it can be further enhanced via remote photothermal treatment. The multifunctional nanogels potentiate their synergistic anticancer bioactivity as an ideal nanoplatform for cancer treatment.

Photothermally Enhanced Chemotherapy Delivered by Graphene Oxide-Based Multiresponsive Nanogels.

To develop multifunctional anticancer nanomedicines, photothermal nanogels with multistimulative properties are fabricated by hybridizing graphene oxide (GO) with poly(N-isopropylacrylamide, PNIPAM) matrix. This technique allows for easy monomer-intercalation between GO sheets, followed by in situ polymerization to promote GO exfoliation as nanoplatelets inside emulsified PNIPAM nanodrops, followed by fixation using a disulfide-containing cross-linker. The resulting nanogels own significantly improved colloidal stability and biocompatibility as compared to native GO. They can effectively encapsulate an anticancer drug and accelerate its release under conditions mimicking acidic/reducible solid tumor and intracellular microenvironments. Drug delivery can be further enhanced via remote photothermal treatment. The local photothermal effect and drug release smartness make the nanogels as an ideal nanoplatform for synergistic anticancer therapy upon their arrival at tumor tissue or inside cancer cells.

Abstract 3565: T cell receptor signaling-responsive single chain IL-12 and IL-15 superagonist nanogel “backpacks” to enhance adoptive cell therapy in solid tumors

Abstract Adoptive cell therapy (ACT) using patient-derived tumor-specific T cells is a promising approach for cancer treatment especially for the treatment of hematological malignancies. However, therapeutic approaches using ACT in patients with solid tumors, such as hepatocellular carcinoma (HCC), has not shown substantially improved clinical outcomes in particular due to immunosuppressive tumor microenvironments. Therefore, strategies to enhance ACT T cell functionality in vivo and to overcome the immunosuppressive microenvironments in solid tumors are strongly needed. We developed a strategy combining nanomedicine with ACT, based on the chemical conjugation of cytokine-loaded nanoparticles (NPs) as synthetic “backpacks” to the surfaces of live lymphocytes for adoptive therapy. Novel protein nanoparticles were synthesized by crosslinking of single chain IL-12 (scIL-12)- or IL-15 superagonist through disulfide-containing crosslinkers, forming nanogels that when bound to the surface of T cells, release the supporting cytokines in response to antigen receptor signaling-mediated changes in cell surface redox state. This TCR-mediated drug release allowed drug delivery to the T cells to be focused in tumors and tumor-draining lymph nodes. Coupling of cytokine nanogels to T cell membranes induced significant cell activation in terms of interferon-γ production or proliferation in a concentration-dependent manner. Antigen-specific CD8+ T cells “backpacked” with scIL-12 or IL-15SA nanogels were capable of substantial tumor cell killing in a B16F10 melanoma cancer model in vivo, while avoiding lethal systemic toxicities triggered by systemically-administered immunomodulatory cytokines. Ongoing studies are seeking to apply this approach in HCC, and this approach bears the potential for the development of successful immunotherapeutic strategies against this and other solid tumors where effective treatment options are still lacking. Citation Format: Michael Fichter. T cell receptor signaling-responsive single chain IL-12 and IL-15 superagonist nanogel “backpacks” to enhance adoptive cell therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3565.

Poly(2-methacryloyloxyethyl phosphorylcholine)-based biodegradable nanogels for controlled drug release

We developed reduction degradable PMPC nanogels for controlled drug releaseviaprecipitation polymerization using a disulfide-containing crosslinker.

Novel fluorescent pH/reduction dual stimuli-responsive polymeric nanoparticles for intracellular triggered anticancer drug release

Fluorescent poly(methacrylic acid-co-poly(ethylene glycol) methyl ether methacrylate-co-N′-rhodamine B-acrylhydrazine) (P(MAA-co-PEGMA-co-RhBAh), PMPR) nanoparticles were designed as pH/reduction dual stimuli-responsive drug delivery system (DDS) for anti-cancer drug via facile distillation-precipitation copolymerization with bio-reducible disulfide-containing crosslinker. As a carrier for doxorubicin (DOX), high drug-loading capacity (DLC) and drug-loading efficiency (DLE) of 53.1% and 97.3% were achieved respectively, via the strong electrostatic interaction between the carboxyl groups of PMAA segments and the amino group of DOX. Due to the disulfide crosslinking structure and their drug-loading mode, the DOX-loaded PMPR nanoparticles were relatively stable under extracellular conditions with low drug leakage, while a rapid intracellular drug release took place in acidic and reductive environment of tumor cells. The fluorescence microscope analysis showed that the DOX-loaded PMPR nanoparticles could be successfully endocytosed by HepG2 cells and the released DOX was mainly accumulated in cell nuclei. These features make them potential DDS for tumor-environment-responsive controlled release. Furthermore, the PMPR nanoparticles showed strong fluorescence only at low pH media, which might be used for the real-time fluorescent imaging in cancer diagnosis as a potential theranostic nanoplatform for imaging-guided therapy of cancer.

A redox-labile poly(oligo(ethylene glycol)methacrylate)-based nanogel with tunable thermosensitivity for drug delivery

We developed a redox degradable P(MEO2MA-s-s-OEGMA) nanogel with tunable volume phase transition temperature for drug delivery via precipitation polymerization using a disulfide-containing crosslinker.

One-Pot Synthesis of Redox-Labile Polymer Capsules via Emulsion Droplet-Mediated Precipitation Polymerization

Monodisperse poly(vinylcaprolactam) (PVCL)-based capsules are prepared by precipitation polymerization of vinylcaprolactam (VCL) onto dimethyldiethoxysilane (DMDES) emulsion droplets and removal of the DMDES templates by ethanol. Polymer chains in the shells can be cross-linked during the polymerization by disulfide-containing cross-linker N,N′-bis(acryloyl) cystamine, which endows the capsules with an excellent redox-labile property. Versatility of this technique to prepare capsules with diverse components is demonstrated by the copolymerization of methacrylic acid (MAA) and VCL in the shell to prepare poly(vinylcaprolactam-co-methacrylic acid) (P(VCL-co-MAA)) capsules. The disulfide-bonded capsules can degrade efficiently into low molecular weight species (ca. 1200 Da) when the capsules are incubated with 10 mM glutathione (GSH) as the reducing agent. Delivery of the anticancer drug (doxorubicin, DOX) was also investigated in the P(VCL-co-MAA) capsules. The cumulative in vitro release of DOX-loaded capsules allows a relatively low DOX release at pH 7.4. However, a burst release (ca. 90% in 6 h) of DOX was observed in the presence of 10 mM GSH. Cell viability assays show that the P(VCL-co-MAA) capsules have negligible cytotoxicity to HeLa cancer cells. In comparison, DOX-loaded P(VCL-co-MAA) capsules cause significant cell death following internalization. The reported capsules represent a novel and versatile class of stimuli-responsive carriers for controlled drug delivery.

Redox‐Triggered Self‐Rolling Robust Hydrogel Tubes for Cell Encapsulation

In this study, mechanically strong hydrogels are synthesized by photopolymerization of 2-vinyl-4,6-diamino-1,3,5-triazine, poly(ethylene glycol) methacrylate, and disulfide-containing cross-linker, N'N-bis(acryloyl)cystamine. The bilayer hydrogel with distinct cross-linking density is shown to self-roll into a 3D tube, which could still be well reinforced by hydrogen bondings, upon exposing reductants such as 1,4-dithio-DL-threitol (DTT) or L-glutathione (GSH), because the redox-induced cleavage of disulfide bonds results in the imbalanced internal shrinking stress between two layers. At an intracellular level of GSH, model L929 cells-seeded bilayer gel sheet could curl up into a 3D tubular scaffold where the cells maintained good viability.

Polymeric redox-responsive delivery systems bearing ammonium salts cross-linked via disulfides.

A redox-responsive polycationic system was synthesized via copolymerization of N,N-diethylacrylamide (DEAAm) and 2-(dimethylamino)ethyl methacrylate (DMAEMA). N,N’-bis(4-chlorobutanoyl)cystamine was used as disulfide-containing cross-linker to form networks by the quaternization of tertiary amine groups. The insoluble cationic hydrogels become soluble by reduction of disulfide to mercaptanes by use of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) or cysteamine, respectively. The soluble polymeric system can be cross-linked again by using oxygen or hydrogen peroxide under basic conditions. The redox-responsive polymer networks can be used for molecular inclusion and controlled release. As an example, phenolphthalein, methylene blue and reactive orange 16 were included into the network. After treatment with DTT a release of the dye could be recognized. Physical properties of the cross-linked materials, e.g., glass transition temperature (Tg), swelling behavior and cloud points (Tc) were investigated. Redox-responsive behavior was further analyzed by rheological measurements.

Novel shell-cross-linked micelles with detachable PEG corona for glutathione-mediated intracellular drug delivery

A series of novel disulfide-containing triblock copolymers, poly(ethylene glycol)-b-poly(L-lysine)-b-poly(rac-leucine) (PEG-SS-PLys-PLeu), were prepared. In an aqueous solution, the copolymers could self-assemble to form core–shell-corona micelles with a disulfide-linked detachable PEG corona, since the PLys middle shell with primary amine groups was linked by a disulfide-containing cross-linker. The morphology and stability of self-assembled micelles were characterized by TEM, DLS and SEM. In the intracellular environment, the micelles underwent destruction of the cross-linked shell with detachment of the PEG corona due to the cleavage of disulfide bonds, followed by the collapse of micelles. The in vitro drug release in response to GSH was further studied. Interestingly, it was found that the micelles not only exhibited reduced drug loss in extracellular environments, but also drastically accelerated drug release at the cytoplasmic GSH level, leading to enhanced growth inhibition of HeLa cells. The glutathione-responsive micelles might have great potential in intracellular drug delivery.

Negative ion fragmentations of disulfide-containing cross-linking reagents are competitive with aspartic acid side-chain-induced cleavages

It has been shown that the disulfide moiety in the chemical cross-linking reagent dithiobis(succinimidyl)propionate (DSP), which is similar in structure to the natural cystine disulfide, cleaves preferentially to the peptide backbone in the negative ion mode. However, the tandem mass (MS/MS) spectra of peptides in the negative ion mode are often dominated by products arising from low-energy, side-chain-induced processes, which may compete with any facile cross-linker fragmentations and complicate identification of chemical cross-links in a complex mixture. Two disulfide-containing crosslinking reagents similar to DSP, but with varying spacer arm lengths, were synthesized and the MS/MS spectra of several model peptides cross-linked with these reagents were investigated. Theoretical calculations were used to describe the energetics of the cross-linker fragmentations as well as several low-energy side-chain-induced fragmentations which compete with disulfide cleavages. Altering the spacer arm length of the cross-linker, such that there is one methylene group less than in DSP, results in a more facile cleavage process, whilst the opposite is true when a methylene group is added. Of the low-energy side-chain-induced fragmentations studied, only those from aspartic acid compete significantly with those of the cross-linker disulfide. Low-energy cleavage processes from aspartic acid that compete with cross-linker fragmentations occur in the negative ion MS/MS spectra of the cross-linked peptides, irrespective of the spacer arm length. Other fragmentation pathways do not significantly interfere with low-energy disulfide cleavage, making the presence of additional product ions in the MS/MS spectrum diagnostic for the presence of aspartic acid.