Herb Extracellular Vesicle-Chitosan-PEGylated Graphene Oxide Conjugate Delivers Estrogen Receptor α Targeting siRNA to Breast Cancer Cells.

Herb-based extracellular vesicles (EV), inherently replete with bioactive proteins, RNA, lipids, and other medicinal compounds, are noncytotoxic and uniquely capable of cellular delivery to meet the ever-stringent challenges of ongoing clinical applications. EVs are abundant in nature, affordable, and scalable, but they are also incredibly fragile and stuffed with many biomolecules. To address the low drug binding abilities and poor stability of EVs, we demonstrated herb-based EVs (isolated from neem, mint, and curry leaves) conjugated with chitosan (CS) and PEGylated graphene oxide (GP) that led to their transformation into robust and efficient vectors. The designed conjugates successfully delivered estrogen receptor α (ERα1)-targeting siRNA to breast cancer MCF7 cells. Our data revealed that neem-based EV-CS-GP conjugates were most efficient in cellular siRNA delivery, which could be attributed to hyaluronic acid-mediated recognition of neem EVs by MCF7 cells via CD44 receptors. Our approach shows a futuristic direction in designing clinically viable, sustainable, nontoxic EV-based vehicles that can deliver a variety of functional siRNA cargos.

Mechanochemistry and oleochemistry: a green combination for the production of high-value small chemicals.

Mechanochemistry and oleochemistry and their combination have been known for centuries. Nevertheless, bioeconomy and circular economy concepts is much more recent and has motivated a regain of interest of dedicated research to improve alternative technologies for the valorization of biomass feedstocks. Accordingly, this review paper aims essentially at outlining recent breakthroughs obtained in the field of mechanochemistry and oleochemicals such as triglycerides, fatty acids, and glycerol derivatives. The review discusses advances obtained in the production of small chemicals derived from oils with a brief overview of vegetable oils, mechanochemistry and the use of mechanochemistry for the synthesis of biodiesel, lipidyl-cyclodextrine, dimeric and labelled fatty acids, calcium diglyceroxide, acylglycerols, benzoxazine and solketal. The paper also briefly overviews advances and limits for an industrial application.

Benzoxazine-grafted-chitosan biopolymer films with inherent disulfide linkage: Antimicrobial properties.

Synthesis and fabrication of naturally sourced biopolymers, especially chitosan, grafted with renewable small molecules have recently attracted attention as efficient antimicrobial agents and are highly desired for sustainable material development. Advantageous inherent functionalities in biobased benzoxazine extend the possibility of crosslinking with chitosan which holds immense potential. Herein, a low-temperature, greener facile methodology is adopted for the covalent confinement of benzoxazine monomers bearing aldehyde and disulfide linkages within chitosan to form benzoxazine-grafted-chitosan copolymer films. The association of benzoxazine as Schiff base, hydrogen bonding, and ring-opened structures enabled the exfoliation of chitosan galleries, and such host-guest mediated interactions demonstrated outstanding properties like hydrophobicity, good thermal, and solution stability due to the synergistic effects. Furthermore, the structures empowered excellent bactericidal properties against both E. coli and S. aureus as investigated by GSH loss, live/dead fluorescence microscopy, and morphological alteration on the cell surface by SEM. The work provides the benefits of disulfide-linked benzoxazines on chitosan, offering a promising avenue for general and eco-friendly usage in wound-healing and packaging material.

Combinatorial delivery of CPI444 and vatalanib loaded on PEGylated graphene oxide as an effective nanoformulation to target glioblastoma multiforme: In vitro evaluation.

Glioblastoma multiforme (GBM) is known as the primary malignant and most devastating form of tumor found in the central nervous system of the adult population. The active pharmaceutical component in current chemotherapy regimens is mostly hydrophobic and poorly water-soluble, which hampers clinical implications. Nanodrug formulations using nanocarriers loaded with such drugs assisted in water dispersibility, improved cellular permeability, and drug efficacy at a low dose, thus adding to the overall practical value. Here, we successfully developed a water-dispersible and biocompatible nanocargo (GO-PEG) based on covalently modified graphene oxide (GO) with a 6-armed poly(ethylene glycol) amine dendrimer for effective loading of the two hydrophobic anticancer drug molecules, CPI444 and vatalanib. These drug molecules target adenosine receptor (A2AR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and type III stem cell receptor tyrosine kinase (c-KIT), which plays a crucial role in cancers. The effective cellular delivery of the drugs when loaded on GO-PEG is attributed to the increased permeability of the drug-nanoconjugate formulation. We observed that this combinatorial drug treatment with nanocargo resulted in a significant reduction in the overall cell survival as supported by reduced calcium levels and stem cell markers such as Oct4 and Nanog, which are two of the prime factors for GBM stem cell proliferation. Furthermore, reduced expression of CD24 upon treatment with nanoformulation impeded cellular migration. Cellular assays confirmed inhibition of cell proliferation, migration, and angiogenic potential of GBM treated with GO-PEG-Drug conjugates. Ultimately, GBM U87 cells assumed programmed cell death at a very low concentration due to nanocarrier-mediated drug delivery along with the chosen combination of drugs. Together, this study demonstrated the advantage of GO-PEG mediated combined delivery of CPI444 and vatalanib drugs with increased permeability, a three-pronged combinatorial strategy toward effective GBM treatment.

Dendrimer-Functionalized Nanodiamonds as Safe and Efficient Drug Carriers for Cancer Therapy: Nucleus Penetrating Nanoparticles.

Nanodiamonds (NDs) are increasingly being assessed as potential candidates for drug delivery in cancer cells and they hold great promise in overcoming the side effects of traditional chemotherapeutics. In the current work, carboxylic acid functionalized nanodiamonds (ND-COOH) were covalently modified with poly(amidoamine) dendrimer (PAMAM) to form amine-terminated nanodiamonds (NP). Unlike ND-COOH, the chemically modified nanodiamond platform NP revealed a pH-independent aqueous dispersion stability, enhancing its potential as an effective carrier. Physical encapsulation of poorly water soluble cabazitaxel (CTX) drug on NP formed ND-PAMAM-CTX (NPC) nanoconjugates and substantially reduced the size of CTX from micrometer to nanometer. CTX was localized within the pores of nanoparticle aggregates and the cavities of the PAMAM dendrimer, thus facilitating the loaded drug's controlled and sustained release. NPC's cumulative CTX release efficiency was determined to be ∼95% at pH 4 after 96 h. A high cellular uptake of NPC both within the cytoplasm and nucleus of U87 cells is confirmed, accounting for a reduced IC50 value (1 nM). Both the cell cycle and Western blot analyses confirmed enhanced cell death and suppressed tubulin protein expression in NPC-treated cells. A significantly high inhibition to cell division with early apoptosis and reduced metastasis demonstrates the effective loading of CTX dosages on the nanocarrier. The present work highlights the potential of a newly designed nanocarrier NP as an efficient nanocargo for cellular delivery applications and may provide future insights to treat one of the most aggressive tumors in neuro-oncological research, glioblastoma multiforme (GBM).

Self-Polymerization Promoting Monomers: In Situ Transformation of Disulfide-Linked Benzoxazines into the Thiazolidine Structure.

Polybenzoxazines obtained especially from green synthons are facing challenges of the requirement of high ring-opening polymerization (ROP) temperature of the monomer, thus affecting their exploration at the industrial front. This demands effective structural changes in the monomer itself, to mediate catalyst-free polymerization at a low energy via one-step synthesis protocol. In this regard, monomers based on disulfide-linked bisbenzoxazine were successfully synthesized using cystamine (biobased) and cardanol (agro-waste)/phenol. Reduction of the disulfide bridge in the monomer using dithiothreitol under mild conditions in situ transformed the oxazine ring in the monomer, via neighboring group participation of the -SH group in a transient intermediate monomer, into a thiazolidine structure, which is otherwise difficult to synthesize. Structural transformation of ring-opening followed by the ring-closing intramolecular reaction led to an interconversion of O-CH2-N containing a six-membered oxazine ring to S-CH2-N containing a five-membered thiazolidine ring and a phenolic-OH. The structure of the monomer with the oxazine ring and its congener with the thiazolidine ring was characterized by spectroscopic methods and X-ray analysis. Kinetics of structural transformation at a molecular level is studied in detail, and it was found that the reaction proceeded via a transient 2-aminoethanethiol-linked benzoxazine intermediate, as supported by nuclear magnetic resonance spectroscopy and density functional theory studies. The thiazolidine-ring-containing monomer promotes ROP at a substantially low temperature than the reported mono-/bisoxazine monomers due to the dual mode of facilitation of the ROP reaction, both by phenolic-OH and by ring strain. Surprisingly, both the monomer structures led to the formation of a similar polymer structure, as supported by thermogravimetric analysis and Fourier transform infrared study. The current work highlights the benefits of inherent functionalities in naturally sourced feedstocks as biosynthons for the new latest generation of benzoxazine monomers.

Amplified Activity of Artesunate Mediated by Iron Oxide Nanoparticles Loaded on a Graphene Oxide Carrier for Cancer Therapeutics.

Development of drugs to tackle the ever-increasing cases of cancer and many other diseases including any pandemic is itself challenging. Repurposing existing drugs is an upcoming drug development strategy established for the reuse of existing licensed drugs to ensure accessible, sustainable, and affordable care against cancer. Herein, we presented a nanochemotherapeutic approach based on PEGylated graphene oxide (GO-PEG) loaded with superparamagnetic iron oxide nanoparticles (NPs) and a sustainable natural origin drug, artesunate (ART) to kill cancerous cells. GO-PEG provided a larger surface area to load the dual cargo, iron oxide NPs (∼40%) and ART (∼13%), at a high loading efficiency and simultaneously affected nanotization and crystallinity of the iron oxide NPs. The morphology and internalization of NPs were determined qualitatively and quantitatively by atomic force microscopy (AFM)-Raman imaging and atomic absorption spectroscopy (AAS) analysis, respectively. Furthermore, the loading and unloading of iron reserves were characterized by high-resolution transmission electron microscopy (TEM) images. The loaded iron oxide NPs underwent a pH-triggered release of iron ions, which is higher in acidic pH than in neutral pH. A ∼sevenfold reduction in the IC50 value of ART upon treatment with the designed nanoconjugate is observed. ART is repositioned as a therapeutic drug against cancer cells, and its efficacy is amplified by the Fenton reaction due to iron oxide NPs, as confirmed by a high oxidative stress generated within the cells. The current work suggests that ART and iron oxide NPs loaded on GO-PEG, a biocompatible carrier, are a promising drug-nanoparticle conjugate for cancer treatment.

Pre-clinical study of iron oxide nanoparticles fortified artesunate for efficient targeting of malarial parasite.

BACKGROUND: Artesunate the most potent antimalarial is widely used for the treatment of multidrug-resistant malaria. The antimalarial cytotoxicity of artesunate has been mainly attributed to its selective, irreversible and iron- radical-mediated damage of parasite biomolecules. In the present research, iron oxide nanoparticle fortified artesunate was tested in P. falciparum and in an experimental malaria mouse model for enhancement in the selectivity and toxicity of artesunate towards parasite. Artesunate was fortified with nontoxic biocompatible surface modified iron oxide nanoparticle which is specially designed and synthesized for the sustained pH-dependent release of Fe2+ within the parasitic food vacuole for enhanced ROS spurt. METHODS: Antimalarial efficacy of Iron oxide nanoparticle fortified artesunate was evaluated in wild type and artemisinin-resistant Plasmodium falciparum (R539T) grown in O + ve human blood and in Plasmodium berghei ANKA infected swiss albino mice. Internalization of nanoparticles, the pH-dependent release of Fe2+, production of reactive oxygen species and parasite biomolecule damage by iron oxide nanoparticle fortified artesunate was studied using various biochemical, biophysical, ultra-structural and fluorescence microscopy. For determining the efficacy of ATA-IONP+ART on resistant parasite ring survival assay was performed. RESULTS: The nanoparticle fortified artesunate was highly efficient in the 1/8th concentration of artesunate IC50 and led to retarded growth of P. falciparum with significant damage to macromolecules mediated via enhanced ROS production. Similarly, preclinical In vivo studies also signified a radical reduction in parasitemia with ~8-10-fold reduced dosage of artesunate when fortified with iron oxide nanoparticles. Importantly, the ATA-IONP combination was efficacious against artemisinin-resistant parasites. INTERPRETATION: Surface coated iron-oxide nanoparticle fortified artesunate can be developed into a potent therapeutic agent towards multidrug-resistant and artemisinin-resistant malaria in humans. FUND: This study is supported by the Centre for Study of Complex Malaria in India funded by the National Institute of Health, USA.

Integrative natural medicine inspired graphene nanovehicle-benzoxazine derivatives as potent therapy for cancer.

Natural products from medicinal plants have always attracted a lot of attention due to their diverse and interesting therapeutic properties. We have employed the principles of green chemistry involving isomerization, coupling and condensation reaction to synthesize a class of compounds derived from eugenol, a naturally occurring bioactive phytophenol. The compounds were characterized structurally by 1H-, 13C-NMR, FT-IR spectroscopy and mass spectrometry analysis. The purity of compounds was detected by HPLC. The synthesized compounds exhibited anti-cancer activity. A 10-12-fold enhancement in efficiency of drug molecules (~ 1 µM) was observed when delivered with graphene oxide (GO) as a nanovehicle. Our data suggest cell death via apoptosis in a dose-dependent manner due to increase in calcium levels in specific cancer cell lines. Interestingly, the benzoxazine derivatives of eugenol with GO nanoparticle exhibited enhanced therapeutic potential in cancer cells. In addition to anti-cancer effect, we also observed significant role of these derivatives on parasite suggesting its multi-pharmacological capability.

Stable Dispersions of Covalently Tethered Polymer Improved Graphene Oxide Nanoconjugates as an Effective Vector for siRNA Delivery.

Conjugates of poly(amidoamine) (PAMAM) with modified graphene oxide (GO) are attractive nonviral vectors for gene-based cancer therapeutics. GO protects siRNA from enzymatic cleavage and showed reasonable transfection efficiency along with simultaneous benefits of low cost and large scale production. PAMAM is highly effective in siRNA delivery but suffers from high toxicity with poor in vivo efficacy. Co-reaction of GO and PAMAM led to aggregation and more importantly, have detrimental effect on stability of dispersion at physiological pH preventing their exploration at clinical level. In the current work, we have designed, synthesized, characterized and explored a new type of hybrid vector (GPD), using GO synthesized via improved method which was covalently tethered with poly(ethylene glycol) (PEG) and PAMAM. The existence of covalent linkage, relative structural changes and properties of GPD is well supported by Fourier transform infrared (FTIR), UV-visible (UV-vis), Raman, X-ray photoelectron (XPS), elemental analysis, powder X-ray diffraction (XRD), thermogravimetry analysis (TGA), dynamic light scattering (DLS), and zeta potential. Scanning electron microscopy (SEM), and transmission electron microscopy (TEM) of GPD showed longitudinally aligned columnar self-assembled ∼10 nm thick polymeric nanoarchitectures onto the GO surface accounting to an average size reduction to ∼20 nm. GPD revealed an outstanding stability in both phosphate buffer saline (PBS) and serum containing cell medium. The binding efficiency of EPAC1 siRNA to GPD was supported by gel retardation assay, DLS, zeta potential and photoluminescence (PL) studies. A lower cytotoxicity with enhanced cellular uptake and homogeneous intracellular distribution of GPD/siRNA complex is confirmed by imaging studies. GPD exhibited a higher transfection efficiency with remarkable inhibition of cell migration and lower invasion than PAMAM and Lipofectamine 2000 suggesting its role in prevention of breast cancer progression and metastasis. A significant reduction in the expression of the specific protein against which siRNA was delivered is revealed by Western blot assay. Furthermore, a pH-triggered release of siRNA from the GPD/siRNA complex was studied to provide a mechanistic insight toward unloading of siRNA from the vector. Current strategy is a way forward for designing effective therapeutic vectors for gene-based antitumor therapy.