Wireless electrical-molecular quantum signalling for cancer cell apoptosis.

Quantum biological tunnelling for electron transfer is involved in controlling essential functions for life such as cellular respiration and homoeostasis. Understanding and controlling the quantum effects in biology has the potential to modulate biological functions. Here we merge wireless nano-electrochemical tools with cancer cells for control over electron transfer to trigger cancer cell death. Gold bipolar nanoelectrodes functionalized with redox-active cytochrome c and a redox mediator zinc porphyrin are developed as electric-field-stimulating bio-actuators, termed bio-nanoantennae. We show that a remote electrical input regulates electron transport between these redox molecules, which results in quantum biological tunnelling for electron transfer to trigger apoptosis in patient-derived cancer cells in a selective manner. Transcriptomics data show that the electric-field-induced bio-nanoantenna targets the cancer cells in a unique manner, representing electrically induced control of molecular signalling. The work shows the potential of quantum-based medical diagnostics and treatments.

Biocompatible metallosurfactant-based nanocolloid-loaded Rose Bengal with excellent singlet oxygen-induced phototoxicity efficiency against cancer cells.

Photodynamic therapy (PDT) is facing challenges such as poor solubility, precise delivery, self-aggregation, and photobleaching of photosensitizers with cancer cells due to their less tendency to accumulate in tumor tissues. To address these challenges, we have explored a Rose Bengal (RB)-loaded metallocatanionic vesicles (MCVs) nanosystem for the phototoxicity of cancer cells. Different sets of MCVs were prepared by two different cationic single-chain metallosurfactants, i.e., hexadecylpyridinium trichlorocuprate (CuCPC I) and hexadecylpyridinium trichloroferrate (FeCPC I) in combination with anionic double-chain sodium bis(2-ethylhexyl)sulfosuccinate (AOT) surfactant in phosphate buffer saline of pH 7.4. The RB-loaded CuCPC I:AOT and FeCPC I:AOT vesicles enhanced the maximum singlet oxygen (1O2) generation by 1-fold and 3-fold, respectively, compared to pure RB. Upon irradiation with a 532 nm laser for 10 min, these RB-loaded CuCPC I:AOT and FeCPC I:AOT MCVs significantly decreased the metabolic activity of U-251 cells by 70% and 85% at MCVs concentration of 0.75 µM, respectively. Furthermore, RB-loaded MCVs showed the highest intracellular 1O2-mediated membrane damage and cell-killing effect as confirmed by singlet oxygen sensor green and differential nuclear staining assay, which is attributed to the cellular uptake profile of different RB-loaded MCVs fractions. Caspase 3/7 assay confirmed the apoptotic pathway of cell death by activating caspase. Therefore, the photoactivation of RB-loaded MCVs led to a significant reduction in the viability of U-251 cells (maximum 85%), which resulted in cell death. Our study demonstrated the advantage of using these dual-charge and biocompatible metallocatanionic vesicles as a promising delivery system of photodynamic therapy that can enhance 1O2 generation from PS and can be further utilized in photomedicine.

Intrinsic Permeation and Anti-Inflammatory Evaluation of Curcumin, Bisdemethoxycurcumin and Bisdemethylcurcumin by a Validated HPLC-UV Method.

Curcumin shows anti-inflammatory activity, and it has been widely investigated for neurodegenerative diseases, adjuvant treatment in AIDS and antitumor activity against different tumors, among other activities. The goal of this work was to evaluate the capacity of curcumin and its derivatives (bisdemethoxycurcumin and bisdemethylcurcumin) in preventing the irritant effects of topically applied xylol and to assess the intrinsic capacity of curcuminoids in permeating human skin by ex vivo permeation tests. Its secondary goal was to validate an HPLC method to simultaneously determine the curcuminoids in the samples from the ex vivo permeation studies and drug extraction from the skin. Curcuminoid quantification was performed using an RP-C18 column, at isocratic conditions of elution and a detection wavelength of 265 nm. The method was specific with a suitable peak resolution, as well as linear, precise, and accurate in the range of 0.195-3.125 µg/mL for the three curcuminoids. Bisdemethylcurcumin showed the greatest permeation through the human skin, and it was the curcuminoid that was most retained within the human skin. The anti-inflammatory activity of the curcuminoids was evaluated in vivo using a xylol-induced inflammation model in rats. Histological studies were performed to observe any changes in morphology at the microscopic level, and these three curcuminoids were found to be respectful within the skin structure. These results show that these three curcuminoids are suitable for anti-inflammatory formulations for dermal applications, and they can be properly quantified using HPLC-UV.

Gemini Surfactant Mediated Catansomes for Enhanced Singlet Oxygen Generation of Rose Bengal and Their Phototoxicity against Cancer Cells.

Photodynamic therapy (PDT) is an innovative technique for cancer treatment with minimal side effects, based on the use of a photosensitizer, oxygen, and light. Photosensitizers (PSs) have several limitations, that may limit their clinical use, like poor solubilization, self-aggregation, and lack of specific targeting, which can be addressed with the use of nanomaterials. Herein, a unique type of catansomes (CaSs) was prepared using a gemini imidazolium-based surfactant (1,3-bis[(3-octadecyl-1-imidazolio)methyl]benzene dibromide (GBIB) and a double chain surfactant, diaoctyl sodium sulfosuccinate or Aerosol OT (AOT). The formation of CaS GBIB/AOT was optimized in various ethanol/water (E/W) solvent ratios by employing a facile, quick, and most reliable solution-solution mixing method. The CaS was characterized by dynamic light scattering (DLS) and field emission gun scanning electron microscopy (FEG-SEM) techniques. The experimental results reveal that stable CaSs with a spherical shape were obtained at lower concentration (100 µM). Rose Bengal (RB), a PS of the xanthene family, was incorporated into these prepared CaSs, as proven by fluorescence spectroscopy, UV-visible absorption spectroscopy, and confocal laser scanning microscopy. Singlet oxygen (1O2) generation studies revealed the relevant role of the E/W solvent ratio as there was a 4-fold boost in the 1O2 production for GBIB/AOT in E/W = 50:50 and around 3-fold in E/W = 30:70. Also, the GBIB-rich 80:20 fraction was more efficient in increasing the 1O2 generation as compared to the AOT rich fraction (20:80). Further, their phototoxicity was tested in a water-rich solvent ratio (E/W = 30:70) against MCF-7 cells. Upon irradiation with a 532 nm laser (50 mW) for 5 min, RB@GBIB/AOT(20:80) fraction caused 50% decrease in the metabolic activity of MCF-7 cells, and RB@GBIB/AOT(80:20) fraction produced a maximum 85% decrease in cell viability. Furthermore, the enhancement in intracellular 1O2 generation by RB@GBIB/AOT, as compared to pure RB, was confirmed with singlet oxygen sensor green (SOSG). This new type of CaS based on gemini surfactants exhibiting a large amount of 1O2 generation, holds great interest for several applications, such as use in photomedicine in future.

Metallocatanionic vesicle-mediated enhanced singlet oxygen generation and photodynamic therapy of cancer cells.

In clinics, photodynamic therapy (PDT) is established as a non-invasive therapeutic modality for certain types of cancers and skin disease. However, due to poor water solubility, photobleaching, and the dark toxicity of photosensitizers (PSs), further developments are required to improve the efficiency of PDT. Herein, we report the role of metallocatanionic vesicles (MCVs) in enhancing the phototoxicity of methylene blue (MB) against cancer cells. These MCVs were prepared via a facile and quick solution-solution mixing method using a cationic single-chain metallosurfactant (FeCPC I) in combination with anionic sodium oleate (Na Ol). For singlet oxygen (1O2) generation and PDT studies, two fractions of FeCPC I : Na Ol, i.e., 30 : 70 (V37) and 70 : 30 (V73), were chosen based on their long-term stability in aqueous media. A cationic PS MB was loaded into these vesicles. The MB-loaded MCV 30 : 70 and 70 : 30 fractions enhanced the 1O2 generation by 0.10- and 0.40-fold, respectively, compared with MB alone. Upon illumination using a 650 nm laser, these MB-loaded V73 and V37 MCVs significantly decreased the metabolic activity of MCF-7 cells by ≤50% at a concentration of 0.75 µM. Furthermore, the SOSG assay revealed that the synthesized MCVs enhanced the intracellular 1O2 compared with MB alone. The MB-loaded V73 MCVs showed the highest 1O2-mediated membrane damage and cell-killing effect, as confirmed using the differential nuclear staining assay (DNS), which is attributed to the cellular uptake profile of the different MCV fractions. Altogether, this work shows the advantage of using these biocompatible and dual-charge MCVs as promising delivery vehicles that can enhance the 1O2 generation from the PS. This work suggests the future application of these Fe-MCVs in magnetically guided PDT.

Polysilicon Microchips Functionalized with Bipyridinium-Based Cyclophanes for a Highly Efficient Cytotoxicity in Cancerous Cells.

The use of micrometric-sized vehicles could greatly improve selectivity of cytotoxic compounds as their lack of self-diffusion could maximize their retention in tissues. We have used polysilicon microparticles (SiµP) to conjugate bipyridinium-based compounds, able to induce cytotoxicity under regular intracellular conditions. Homogeneous functionalization in suspension was achieved, where the open-chain structure exhibits a more dense packing than cyclic analogues. The microparticles internalized induce high cytotoxicity per particle in cancerous HeLa cells, and the less densely packed functionalization using cyclophanes promotes higher cytotoxicity per bipy than with open-chain analogues. The self-renewing ability of the particles and their proximity to cell membranes may account for increased lipid peroxidation, achieving toxicity at much lower concentrations than that in solution and in less time, inducing highly efficient cytotoxicity in cancerous cells.

Synthesis and validation of DOPY: A new gemini dioleylbispyridinium based amphiphile for nucleic acid transfection.

Nucleic acids therapeutics provide a selective and promising alternative to traditional treatments for multiple genetic diseases. A major obstacle is the development of safe and efficient delivery systems. Here, we report the synthesis of the new cationic gemini amphiphile 1,3-bis[(4-oleyl-1-pyridinio)methyl]benzene dibromide (DOPY). Its transfection efficiency was evaluated using PolyPurine Reverse Hoogsteen hairpins (PPRHs), a nucleic acid tool for gene silencing and gene repair developed in our laboratory. The interaction of DOPY with PPRHs was confirmed by gel retardation assays, and it forms complexes of 155 nm. We also demonstrated the prominent internalization of PPRHs using DOPY compared to other chemical vehicles in SH-SY5Y, PC-3 and DF42 cells. Regarding gene silencing, a specific PPRH against the survivin gene delivered with DOPY decreased survivin protein levels and cell viability more effectively than with N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) in both SH-SY5Y and PC-3 cells. We also validated the applicability of DOPY in gene repair approaches by correcting a point mutation in the endogenous locus of the dhfr gene in DF42 cells using repair-PPRHs. All these results indicate both an efficient entry and release of PPRHs at the intracellular level. Therefore, DOPY can be considered as a new lipid-based vehicle for the delivery of therapeutic oligonucleotides.

Enhanced cytotoxicity of highly water-soluble gold nanoparticle-cyclopeptide conjugates in cancer cells.

Conjugation of cytostatic drugs to nanomaterials seeks to improve their low bioavailability and selectivity to overcome the important associated side effects. In this work, we aimed to synthesize water-soluble gold nanoparticles as transporters for synthetic cyclic peptides with a potential anticancer activity but with a limited bioavailability. The highly water-soluble nanoparticles (2.5 nm diameter gold core) are coated with a mixture of polyethylene glycol linkers, one bearing a terminal hydroxyl group for increasing dispersibility in water, and the second bearing a carboxylic acid group for peptide conjugation through amide bond formation. Peptide-functionalized particles have a 9.7 ± 1.8 nm hydrodynamic diameter and are highly water-soluble and stable in solution for at least one year. The morphology of the gold cores as well as their organic coating was studied using Transmission Electron Microscopy, showing that the attachment of a limited number of peptides per nanoparticle leads to a uneven organic coating of two different thicknesses, one of 2.0 ± 0.6 nm formed by polyethylene glycol linkers, and a second of 3.6 ± 0.5 nm which includes the peptide. GNP significantly enhance the internalization of the cyclic peptide BPC734 in cells as compared to peptide in solution, with improved uptake in cancerous HT29 cells. Cytotoxicity studies show that peptide BPC734 in solution is toxic in the micromolar range, whereas peptide-functionalized particles are toxic at nanomolar peptide concentrations and with a significantly higher toxicity for cancerous cells. All these results, besides the stability and expected passive tumor targeting, make these particles a promising option for improving the bioavailability, efficacy, and selectivity in cancer therapy.

π-Donor/π-Acceptor Interactions for the Encapsulation of Neurotransmitters on Functionalized Polysilicon-Based Microparticles.

Bipyridinium salts, commonly known as viologens, are π-acceptor molecules that strongly interact with π-donor compounds, such as porphyrins or amino acids, leading their self-assembling. These properties have promoted us to functionalize polysilicon microparticles with bipyridinium salts for the encapsulation and release of π-donor compounds such as catecholamines and indolamines. In this work, the synthesis and characterization of four gemini-type amphiphilic bipyridinium salts (1·4PF6-4·4PF6), and their immobilization either non-covalently or covalently on polysilicon surfaces and microparticles have been achieved. More importantly, they act as hosts for the subsequent incorporation of π-donor neurotransmitters such as dopamine, serotonin, adrenaline or noradrenaline. Ultraviolet-visible absorption and fluorescence spectroscopies and high-performance liquid chromatography were used to detect the formation of the complex in solution. The immobilization of bipyridinium salts and neurotransmitter incorporation on polysilicon surfaces was corroborated by contact angle measurements. The reduction in the bipyridinium moiety and the subsequent release of the neurotransmitter was achieved using ascorbic acid, or Vitamin C, as a triggering agent. Quantification of neurotransmitter encapsulated and released from the microparticles was performed using high-performance liquid chromatography. The cytotoxicity and genotoxicity studies of the bipyridinium salt 1·4PF6, which was selected for the non-covalent functionalization of the microparticles, demonstrated its low toxicity in the mouse fibroblast cell line (3T3/NIH), the human liver carcinoma cell line (HepG2) and the human epithelial colorectal adenocarcinoma cell line (Caco-2).

Photosensitiser-gold nanoparticle conjugates for photodynamic therapy of cancer.

Gold nanoparticles (AuNPs) have been extensively studied within biomedicine due to their biocompatibility and low toxicity. In particular, AuNPs have been widely used to deliver photosensitiser agents for photodynamic therapy (PDT) of cancer. Here we review the state-of-the-art for the functionalisation of the gold nanoparticle surface with both photosensitisers and targeting ligands for the active targeting of cancer cell surface receptors. From the initial use of the AuNPs as a simple carrier of the photosensitiser for PDT, the field has significantly advanced to include: the use of PEGylated modification to provide aqueous compatibility and stealth properties for in vivo use; gold metal-surface enhanced singlet oxygen generation; functionalisation of the AuNP surface with biological ligands to specifically target over-expressed receptors on the surface of cancer cells and; the creation of nanorods and nanostars to enable combined PDT and photothermal therapies. These versatile AuNPs have significantly enhanced the efficacy of traditional photosensitisers for both in vitro and in vivo cancer therapy. From this review it is apparent that AuNPs have an important future in the treatment of cancer.

Multifunctional Serine Protease Inhibitor-Coated Water-Soluble Gold Nanoparticles as a Novel Targeted Approach for the Treatment of Inflammatory Skin Diseases.

The overexpression and increased activity of the serine protease Kallikrein 5 (KLK5) is characteristic of inflammatory skin diseases such as Rosacea. The use of inhibitors of this enzyme-such as 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF·HCl) or the anti-human recombinant Kallikrein 5 (anti-KLK5) antibody-in the treatment of the disease has been limited due to their low bioavailability, for which their immobilization in drug delivery agents can contribute to making serine protease inhibitors clinically useful. In this work, we synthesized gold nanoparticles (GNP) coated with a mixture of hydroxyl- and carboxyl-terminated thiolates (GNP.OH/COOH), whose carboxyl groups were used to further functionalize the nanoparticles with the serine protease inhibitor AEBSF·HCl either electrostatically or covalently (GNP.COOH AEBSF and GNP.AEBSF, respectively), or with the anti-KLK5 antibody (GNP.antiKLK5). The synthesized and functionalized GNP were highly water-soluble, and they were extensively characterized using UV-vis absorption spectroscopy, Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and Thermogravimetric Analysis (TGA). GNP.OH/COOH and their subsequent functionalizations effectively inhibited KLK5 in vitro. Internalization of fluorophore-coated GNP.OH/COOH in human keratinocytes (HaCaT cells) was proven using confocal fluorescence microscopy. Cell viability assays revealed that the cytotoxicity of free AEBSF is importantly decreased when it is incorporated in the nanoparticles, either ionically (GNP.COOH AEBSF) or, most importantly, covalently (GNP.AEBSF). The functionalized nanoparticles GNP.AEBSF and GNP.antiKLK5 inhibited intracellular KLK5 activity in HaCaT cells and diminished secretion of IL-8 under inflammatory conditions triggered by TLR-2 ligands. This study points to the great potential of these GNP as a new intracellular delivery strategy for both small drugs and antibodies in the treatment of skin diseases such as Rosacea.

Amphiphilic gemini pyridinium-mediated incorporation of Zn(II)meso-tetrakis(4-carboxyphenyl)porphyrin into water-soluble gold nanoparticles for photodynamic therapy.

Zn-containing porphyrins are intensely investigated for their ability to form reactive oxygen species and thereby being potent photosensitizers for use in photodynamic therapy (PDT). Some of the drawbacks of the PDT approach, such as unspecific distribution, could be addressed by means of photosensitizer drug delivery systems. In this work, we synthesize and characterize new water-soluble gold nanoparticles (GNP) stabilized by a mixture of a polyethyleneglycol-containing thiol (to improve water solubility) and a new amphiphilic gemini-type pyridinium salt, which also acts as promotor of the incorporation of the anionic photosensitizer Na-ZnTCPP into the GNP. The obtained GNP have sizes between 7 and 10nm, as observed by Transmission Electron Microscopy. The incorporation of the photosensitizer caused an increase in the hydrodynamic size, detected by Dynamic Light Scattering, as well as a shift in the Surface Plasmon Resonance peak on the GNP UV-vis absorption spectra. The presence of the photosensitizer in the GNP was corroborated using Fluorescence Spectroscopy. The amount of Na-ZnTCPP was found to be 327 molecules per GNP. The porphyrin-containing Na-ZnTCPP-1·GNP showed good enhanced ability to produce singlet oxygen, compared to free Na-ZnTCPP. Their cytotoxicity and phototoxicity were investigated in vitro using two different human breast cell lines, one of tumoral origin (SKBR-3) and another of normal epithelium origin (MCF-10A). SKBR-3 cells showed higher sensitivity to Na-ZnTCCP and Na-ZnTCPP-1·GNP in dark conditions. After irradiation, no significant differences were observed between both cell lines except for 1µM Na-ZnTCCP-1·GNP where SKBR-3 cells were also more sensitive.

Water soluble, multifunctional antibody-porphyrin gold nanoparticles for targeted photodynamic therapy.

Photodynamic therapy (PDT) is a treatment of cancer by which tumour cells are destroyed using reactive oxygen species produced by photosensitizers following activation with visible or near infrared light. Successful PDT depends on the solubility and the targeting ability of the photosensitizers. In this work, the synthesis of a porphyrin-based water soluble nanoparticle conjugate containing a targeting agent that recognizes the erbB2 receptor overexpressed on the surface of particular cancer cells is reported. The nanoparticle conjugates were synthesized following two different protocols, viz. a biphasic and a monophasic method, with the aim to determine which method yielded the optimal nanosystem for potential PDT applications. The nanoparticles were characterized using UV-Vis absorption and fluorescence spectroscopies together with transmission electron microscopy and zeta potential measurements; and their ability to produce singlet oxygen following irradiation was investigated following the decay in absorption of a singlet oxygen probe. The nanoparticles synthesized using the monophasic method were shown to produce the highest amount of singlet oxygen and were further functionalized with anti-erbB2 antibody to target the erbB2 receptors expressed on the surface of SK-BR-3 human breast cancer cells. The water soluble, antibody-porphyrin nanoparticle conjugates were shown to elicit targeted PDT of the breast cancer cells.

Polysilicon-chromium-gold intracellular chips for multi-functional biomedical applications.

The development of micro- and nanosystems for their use in biomedicine is a continuously growing field. One of the major goals of such platforms is to combine multiple functions in a single entity. However, achieving the design of an efficient and safe micro- or nanoplatform has shown to be strongly influenced by its interaction with the biological systems, where particle features or cell types play a critical role. In this work, the feasibility of using multi-material pSi-Cr-Au intracellular chips (MMICCs) for multifunctional applications by characterizing their interactions with two different cell lines, one tumorigenic and one non-tumorigenic, in terms of biocompatibility, internalization and intracellular fate, has been explored. Moreover, the impact of MMICCs on the induction of an inflammatory response has been assessed by evaluating TNFα, IL1b, IL6, and IL10 human inflammatory cytokines secretion by macrophages. Results show that MMICCs are biocompatible and their internalization efficiency is strongly dependent on the cell type. Finally as a proof-of-concept, MMICCs have been dually functionalized with transferrin and pHrodo™ Red, SE to target cancer cells and detect intracellular pH, respectively. In conclusion, MMICCs can be used as multi-functional devices due to their high biocompatibility, non-inflammatory properties and the ability of developing multiple functions.

Iron oxide nanoparticles functionalized with novel hydrophobic and hydrophilic porphyrins as potential agents for photodynamic therapy.

The preparation of novel porphyrin derivatives and their immobilization onto iron oxide nanoparticles to build up suitable nanotools for potential use in photodynamic therapy (PDT) has been explored. To achieve this purpose, a zinc porphyrin derivative, ZnPR-COOH, has been synthesized, characterized at the molecular level and immobilized onto previously synthesized iron oxide nanoparticles covered with oleylamine. The novel nanosystem (ZnPR-IONP) has been thoroughly characterized by a variety of techniques such as UV-Vis absorption spectroscopy, fluorescence spectroscopy, X-ray photoloectron spectroscopy (XPS) and transmission electron microscopy (TEM). In order to probe the capability of the photosensitizer for PDT, the singlet oxygen production of both ZnPR-IONP and the free ligand ZnPR-COOH have been quantified by measuring the decay in absorption of the anthracene derivative 9,10-anthracenedipropionic acid (ADPA), showing an important increase on singlet oxygen production when the porphyrin is incorporated onto the IONP (ZnPR-IONP). On the other hand, the porphyrin derivative PR-TRIS3OH, incorporating several polar groups (TRIS), was synthesized and immobilized with the intention of obtaining water soluble nanosystems (PR-TRIS-IONP). When the singlet oxygen production ability was evaluated, the values obtained were similar to ZnPR-COOH/ZnPR-IONP, again much higher in the case of the nanoparticles PR-TRIS-IONP, with more than a twofold increase. The efficient singlet oxygen production of PR-TRIS-IONP together with their water solubility, points to the great promise that these new nanotools represent for PDT.