Solid-State Formation of a Potential Melphalan Delivery Nanosystem Based on ß-Cyclodextrin and Silver Nanoparticles.

Melphalan (Mel) is an antineoplastic widely used in cancer and other diseases. Its low solubility, rapid hydrolysis, and non-specificity limit its therapeutic performance. To overcome these disadvantages, Mel was included in ß-cyclodextrin (ßCD), which is a macromolecule that increases its aqueous solubility and stability, among other properties. Additionally, the ßCD-Mel complex has been used as a substrate to deposit silver nanoparticles (AgNPs) through magnetron sputtering, forming the ßCD-Mel-AgNPs crystalline system. Different techniques showed that the complex (stoichiometric ratio 1:1) has a loading capacity of 27%, an association constant of 625 M-1, and a degree of solubilization of 0.034. Added to this, Mel is partially included, exposing the NH2 and COOH groups that stabilize AgNPs in the solid state, with an average size of 15 ± 3 nm. Its dissolution results in a colloidal solution of AgNPs covered by multiple layers of the ßCD-Mel complex, with a hydrodynamic diameter of 116 nm, a PDI of 0.4, and a surface charge of 19 mV. The in vitro permeability assays show that the effective permeability of Mel increased using ßCD and AgNPs. This novel nanosystem based on ßCD and AgNPs is a promising candidate as a Mel nanocarrier for cancer therapy.

Evidence of the Disassembly of α-Cyclodextrin-octylamine Inclusion Compounds Conjugated to Gold Nanoparticles via Thermal and Photothermal Effects.

Cyclodextrin (CD) molecules form inclusion compounds (ICs), generating dimers that are capable of encapsulating molecules derived from long-chain hydrocarbons. The aim of this study is to evaluate the structural changes experienced by ICs in solution with increasing temperatures. For this, a nuclear magnetic resonance (¹H-NMR) titration was performed to determinate the stoichiometric α-cyclodextrin (α-CD):octylamine (OA) 2:1 and binding constant (k = 2.16 M-2) of ICs. Solution samples of α-CD-OA ICs conjugated with gold nanoparticles (AuNPs) were prepared, and ¹H-NMR spectra at different temperatures were recorded. Comparatively, ¹H-NMR spectra of the sample irradiated with a laser with tunable wavelengths, with plasmons of conjugated AuNPs, were recorded. In this work, we present evidence of the disassembly of ICs conjugated with AuNPs. Thermal studies demonstrated that, at 114 °C, there are reversible rearrangements of the host and guests in the ICs in a solid state. Migration movements of the guest molecules from the CD cavity were monitored via temperature-dependent ¹H-NMR, and were verified comparing the chemical shifts of octylamine dissolved in deuterated dimethylsulfoxide (DMSO-d6) with the OA molecule included in α-CD dissolved in the same solvent. It was observed that, at 117 °C, OA exited the α-CD cavity. CD IC dimer disassembly was also observed when the sample was irradiated with green laser light.

Quantitative analysis of the mitochondrial and plastid proteomes of the moss Physcomitrella patens reveals protein macrocompartmentation and microcompartmentation.

Extant eukaryotes are highly compartmentalized and have integrated endosymbionts as organelles, namely mitochondria and plastids in plants. During evolution, organellar proteomes are modified by gene gain and loss, by gene subfunctionalization and neofunctionalization, and by changes in protein targeting. To date, proteomics data for plastids and mitochondria are available for only a few plant model species, and evolutionary analyses of high-throughput data are scarce. We combined quantitative proteomics, cross-species comparative analysis of metabolic pathways, and localizations by fluorescent proteins in the model plant Physcomitrella patens in order to assess evolutionary changes in mitochondrial and plastid proteomes. This study implements data-mining methodology to classify and reliably reconstruct subcellular proteomes, to map metabolic pathways, and to study the effects of postendosymbiotic evolution on organellar pathway partitioning. Our results indicate that, although plant morphologies changed substantially during plant evolution, metabolic integration of organelles is largely conserved, with exceptions in amino acid and carbon metabolism. Retargeting or regulatory subfunctionalization are common in the studied nucleus-encoded gene families of organelle-targeted proteins. Moreover, complementing the proteomic analysis, fluorescent protein fusions revealed novel proteins at organelle interfaces such as plastid stromules (stroma-filled tubules) and highlight microcompartments as well as intercellular and intracellular heterogeneity of mitochondria and plastids. Thus, we establish a comprehensive data set for mitochondrial and plastid proteomes in moss, present a novel multilevel approach to organelle biology in plants, and place our findings into an evolutionary context.

Optimizing Dacarbazine Therapy: Design of a Laser-Triggered Delivery System Based on ß-Cyclodextrin and Plasmonic Gold Nanoparticles.

Dacarbazine (DB) is an antineoplastic drug extensively used in cancer therapy. However, present limitations on its performance are related to its low solubility, instability, and non-specificity. To overcome these drawbacks, DB was included in ß-cyclodextrin (ßCD), which increased its aqueous solubility and stability. This new ßCD@DB complex has been associated with plasmonic gold nanoparticles (AuNPs), and polyethylene glycol (PEG) has been added in the process to increase the colloidal stability and biocompatibility. Different techniques revealed that DB allows for a dynamic inclusion into ßCD, with an association constant of 80 M-1 and a degree of solubilization of 0.023, where ßCD showed a loading capacity of 16%. The partial exposure of the NH2 group in the included DB allows its interaction with AuNPs, with a loading efficiency of 99%. The PEG-AuNPs-ßCD@DB nanosystem exhibits an optical plasmonic absorption at 525 nm, a surface charge of -29 mV, and an average size of 12 nm. Finally, laser irradiation assays showed that DB can be released from this platform in a controlled manner over time, reaching a concentration of 56 µg/mL (43% of the initially loaded amount), which, added to the previous data, validates its potential for drug delivery applications. Therefore, the novel nanosystem based on ßCD, AuNPs, and PEG is a promising candidate as a new nanocarrier for DB.

Simultaneous isolation of pure and intact chloroplasts and mitochondria from moss as the basis for sub-cellular proteomics.

The moss Physcomitrella patens is increasingly being used as a model for plant systems biology studies. While genomic and transcriptomic resources are in place, tools and experimental conditions for proteomic studies need to be developed. In the present study we describe a rapid and efficient protocol for the simultaneous isolation of chloroplasts and mitochondria from moss protonema. Routinely, 60-100 µg mitochondrial and 3-5 mg chloroplast proteins, respectively, were obtained from 20 g fresh weight of green moss tissue. Using 14 plant compartment marker antibodies derived from seed plant and algal protein sequences, respectively, the evolutionary conservation of the compartment marker proteins in the moss was demonstrated and purity and intactness of the extracted organelles confirmed. This isolation protocol and these validated compartment markers may serve as basis for sub-cellular proteomics in P. patens and other mosses.

A surface functionalized with per-(6-amino-6-deoxy)-ß-cyclodextrin for potential organic pollutant removal from water.

In this work, we present a solid silicon substrate functionalized with modified ß-cyclodextrin monolayers as an optimal surface for organic contaminant uptake. The inclusion and capture of three potential pollutants, 4-chlorophenoxyacetic acid, 4-aminobenzoic acid and phenylethylamine, were studied. 1H-NMR and ROESY studies revealed the complete inclusion and details of the conformational orientation of the three guests in the per-(6-amino-6-deoxy)-ß-cyclodextrin matrix, forming three new inclusion complexes that have not yet been reported. Capture assays for the guests were carried out by immersing the substrates in an aqueous pollutant solution and by measuring the UV-vis spectra. This substrate showed a high sorption capacity at equilibrium, between 2.5 × 10-5 and 6.0 × 10-5 mmol/substrate, for the studied pollutants. In addition, this surface can be reused four times with an efficiency equal to the initial use. Therefore, it could be a versatile platform that could be applied for the capture of other organic pollutants from water.

Photothermally Controlled Methotrexate Release System Using ß-Cyclodextrin and Gold Nanoparticles.

The inclusion compound (IC) of cyclodextrin (CD) containing the antitumor drug Methotrexate (MTX) as a guest molecule was obtained to increase the solubility of MTX and decrease its inherent toxic effects in nonspecific cells. The IC was conjugated with gold nanoparticles (AuNPs), obtained by a chemical method, creating a ternary intelligent delivery system for MTX molecules, based on the plasmonic properties of the AuNPs. Irradiation of the ternary system, with a laser wavelength tunable with the corresponding surface plasmon of AuNPs, causes local energy dissipation, producing the controlled release of the guest from CD cavities. Finally, cell viability was evaluated using MTS assays for ß-CD/MTX and AuNPs + ß-CD/MTX samples, with and without irradiation, against HeLa tumor cells. The irradiated sample of the ternary system AuNPs + ß-CD/MTX produced a diminution in cell viability attributed to the photothermal release of MTX.

Gold nanoparticles interacting with ß-cyclodextrin-phenylethylamine inclusion complex: a ternary system for photothermal drug release.

We report the synthesis of a 1:1 ß-cyclodextrin-phenylethylamine (ßCD-PhEA) inclusion complex (IC) and the adhesion of gold nanoparticles (AuNPs) onto microcrystals of this complex, which forms a ternary system. The formation of the IC was confirmed by powder X-ray diffraction and NMR analyses ((1)H and ROESY). The stability constant of the IC (760 M(-1)) was determined using the phase solubility method. The adhesion of AuNPs was obtained using the magnetron sputtering technique, and the presence of AuNPs was confirmed using UV-vis spectroscopy (surface plasmon resonance effect), which showed an absorbance at 533 nm. The powder X-ray diffractograms of ßCD-PhEA were similar to those of the crystals decorated with AuNPs. A comparison of the one- and two-dimensional NMR spectra of the IC with and without AuNPs suggests partial displacement of the guest to the outside of the ßCD due to attraction toward AuNPs, a characteristic tropism effect. The size, morphology, and distribution of the AuNPs were analyzed using TEM and SEM. The average size of the AuNPs was 14 nm. Changes in the IR and Raman spectra were attributed to the formation of the complex and to the specific interactions of this group with the AuNPs. Laser irradiation assays show that the ternary system ßCD-PhEA-AuNPs in solution enables the release of the guest.