Study of PEG-b-PLA/Eudragit S100 Blends on the Nanoencapsulation of Indigo Carmine Dye and Application in Controlled Release.

A nanocapsule shell of poly(ethylene glycol)-block-poly(d,l-lactic acid) (PEG-b-PLA) mixed with anionic Eudragit S100 (90/10% w/w) was previously used to entrap and define the self-assembly of indigo carmine (IC) within the hydrophilic cavity core. In the present work, binary blends were prepared by solution mixing at different PEG-b-PLA/Eudragit S100 ratios (namely, 100/0, 90/10, 75/25, and 50/50% w/w) to elucidate the role of the capsule shell in tuning the encapsulation of the anionic dye (i.e., IC). The results showed that the higher content of Eudragit S100 in the blend decreases the miscibility of the two polymers due to weak intermolecular interactions between PEG-b-PLA and Eudragit S100. Moreover, with an increase in the amount of Eudragit S100, a higher thermal stability was observed related to the mobility restriction of PEG-b-PLA chains imposed by Eudragit S100. Formulations containing 10 and 25% Eudragit S100 exhibited an optimal interplay of properties between the negative surface charge and the miscibility of the polymer blend. Therefore, the anionic character of the encapsulating agent provides sufficient accumulation of IC molecules in the nanocapsule core, leading to dye aggregates following the self-assembly. At the same time, the blending of the two polymers tunes the IC release properties in the initial stage, achieving slow and controlled release. These findings give important insights into the rational design of polymeric nanosystems containing organic dyes for biomedical applications.

Ultrasound-induced and MRI-monitored CuO nanoparticles release from micelle encapsulation.

Copper oxide nanoparticles (CuO NPs) have anticancer and antimicrobial activities. Moreover, they have a contrast enhancing effect in both MRI and ultrasound. Nonetheless, encapsulation is needed to control their toxic side effects and a mechanism for release on demand is required. A methodology is introduced herein for encapsulating and releasing CuO NPs from micelles by ultrasound induced hyperthermia and monitoring the process by MRI. For this aim, CuO NPs loaded poly(ethylene glycol)-block-poly(D,L-lactic acid) (PEG-b-PLA) micelles were prepared. Then, the profile of copper release with application of ultrasound was examined as a function of time and temperature using a colorimetric method. Finally, T1 weighted MRI images of suspensions and ex vivo poultry liver samples containing the CuO NPs loaded micelles were acquired before and after ultrasound application. The results confirmed that: (i) encapsulated NPs are detectible by MRI T1 mapping, depicting substantial T1 shortening from 1872 ± 62 ms to 683 ± 20 ms. (ii) Ultrasonic hyperthermia stimulated the NPs release with an about threefold increase compared to non-treated samples. (iii) Releasing effect was clearly visible by T1-weighted imaging (mean signal increase ratio of 2.29). These findings can potentially lead to the development of a new noninvasive methodology for CuO NPs based theranostic process.

Copper oxide nanoparticles inhibit pancreatic tumor growth primarily by targeting tumor initiating cells.

Cancer stem cells, also termed tumor initiating cells (TICs), are a rare population of cells within the tumor mass which initiate tumor growth and metastasis. In pancreatic cancer, TICs significantly contribute to tumor re-growth after therapy, due to their intrinsic resistance. Here we demonstrate that copper oxide nanoparticles (CuO-NPs) are cytotoxic against TIC-enriched PANC1 human pancreatic cancer cell cultures. Specifically, treatment with CuO-NPs decreases cell viability and increases apoptosis in TIC-enriched PANC1 cultures to a greater extent than in standard PANC1 cultures. These effects are associated with increased reactive oxygen species (ROS) levels, and reduced mitochondrial membrane potential. Furthermore, we demonstrate that CuO-NPs inhibit tumor growth in a pancreatic tumor model in mice. Tumors from mice treated with CuO-NPs contain a significantly higher number of apoptotic TICs in comparison to tumors from untreated mice, confirming that CuO-NPs target TICs in vivo. Overall, our findings highlight the potential of using CuO-NPs as a new therapeutic modality for pancreatic cancer.

Copper oxide loaded PLGA nanospheres: towards a multifunctional nanoscale platform for ultrasound-based imaging and therapy.

Copper oxide nanoparticles (CuO-NPs) are increasingly becoming the subject of investigation exploring their potential use for diagnostic and therapeutic purposes. Recent work has demonstrated their anticancer potential, as well as contrast agent capabilities for magnetic resonance imaging (MRI) and through-transmission ultrasound. However, no capability of CuO-NPs has been demonstrated using conventional ultrasound systems, which, unlike the former, are widely deployed in the clinic. Furthermore, in spite of their potential as multifunctional nano-based materials for diagnosis and therapy, CuO-NPs have been delayed from further clinical application due to their inherent toxicity. Herein, we present the synthesis of a novel nanoscale system, composed of CuO-loaded PLGA nanospheres (CuO-PLGA-NS), and demonstrate its imaging detectability and augmented heating effect by therapeutic ultrasound. The CuO-PLGA-NS were prepared by a double emulsion (W/O/W) method with subsequent solvent evaporation. They were characterized as sphere-shaped, with size approximately 200 nm. Preliminary results showed that the viability of PANC-1, human pancreatic adenocarcinoma cells was not affected after 72 h exposure to CuO-PLGA-NS, implying that PLGA masks the toxic effects of CuO-NPs. A systematic ultrasound imaging evaluation of CuO-PLGA-NS, using a conventional system, was performed in vitro and ex vivo using poultry heart and liver, and also in vivo using mice, all yielding a significant contrast enhancement. In contrast to CuO-PLGA-NS, neither bare CuO-NPs nor blank PLGA-NS possess these unique advantageous ultrasonic properties. Furthermore, CuO-PLGA-NS accelerated ultrasound-induced temperature elevation by more than 4 °C within 2 min. The heating efficiency (cumulative equivalent minutes at 43 °C) was increased approximately six-fold, demonstrating the potential for improved ultrasound ablation. In conclusion, CuO-PLGA-NS constitute a versatile platform, potentially useful for combined imaging and therapeutic ultrasound-based procedures.

Target visualisation and microwave hyperthermia monitoring using nanoparticle-enhanced transmission ultrasound (NETUS).

PURPOSE: The aim of this study was to examine the feasibility of using nanoparticle-enhanced transmission ultrasound (NETUS) as an image-based monitoring modality for microwave hyperthermia treatment. METHODS: A dedicated transmission ultrasound imaging system was used to obtain acoustic projections and ultrasound computed tomography images. Initially, speed-of-sound based images were used to non-invasively monitor temperature changes in in vitro and ex vivo specimens, induced by a microwave needle-type applicator. Next, the hyperthermia acceleration ability of two ultrasound nanoparticles based contrast agents (iron oxide and copper oxide) was examined and visualised. Finally, a two-step image guided microwave therapeutic procedure using NETUS was investigated in a realistic breast mimicking phantom. First, the pathology simulating region borders were detected. Then, a microwave-induced temperature elevation was non-invasively monitored. RESULTS: The transmission ultrasound scanning system was able to detect temperature changes with a resolution of less than 0.5 °C, both in vitro and ex vivo. In accordance with previous studies, it was visually demonstrated that iron oxide nanoparticles expedite the heating process (p < 0.05). Copper oxide nanoparticles, however, did not alter the hyperthermia profile significantly. In the breast mimicking phantom, NETUS yielded accurate detection of the target region as well as thermal monitoring of the microwave heating procedure. CONCLUSIONS: NETUS can combine enhanced target visualisation with non-invasive thermometry and accelerated heating effect. Quantitative feedback, however, requires a tissue-specific calibration-curve. A proof of concept for microwave hyperthermia treatment monitoring using NETUS was established. The suggested methodology may potentially provide a non-invasive cost-effective means for monitoring thermal treatment of the breast.

Copper oxide nanoparticles as contrast agents for MRI and ultrasound dual-modality imaging.

Multimodal medical imaging is gaining increased popularity in the clinic. This stems from the fact that data acquired from different physical phenomena may provide complementary information resulting in a more comprehensive picture of the pathological state. In this context, nano-sized contrast agents may augment the potential sensitivity of each imaging modality and allow targeted visualization of physiological points of interest (e.g. tumours). In this study, 7 nm copper oxide nanoparticles (CuO NPs) were synthesized and characterized. Then, in vitro and phantom specimens containing CuO NPs ranging from 2.4 to 320 µg · mL(-1) were scanned, using both 9.4 T MRI and through-transmission ultrasonic imaging. The results show that the CuO NPs induce shortening of the magnetic T1 relaxation time on the one hand, and increase the speed of sound and ultrasonic attenuation coefficient on the other. Moreover, these visible changes are NP concentration-dependent. The change in the physical properties resulted in a substantial increase in the contrast-to-noise ratio (3.4-6.8 in ultrasound and 1.2-19.3 in MRI). In conclusion, CuO NPs are excellent candidates for MRI-ultrasound dual imaging contrast agents. They offer radiation-free high spatial resolution scans by MRI, and cost-effective high temporal resolution scans by ultrasound.

Synthesis of a Trisubstituted 1,4-Diazepin-3-one-Based Dipeptidomimetic as a Novel Molecular Scaffold.

We describe two routes for the synthesis of a trisubstituted 1,2,5-hexahydro-3-oxo-1H-1,4-diazepine ring (DAP), a novel, conformationally constrained, seven-membered dipeptidomimetic ring system. The linear precursor for the model DAPs, targeted for conformational analysis studies, was obtained by reductive alkylation of tert-butyl alaninate or phenylalaninate by N-Boc-alpha-amino-gamma-oxo-N,N-dimethylbutyramide. Acetylation of the newly formed secondary amine followed by acidolytic deprotection of the amino and carboxyl terminal protecting groups and subsequent diphenylphosphorazidate-mediated ring formation yielded the blocked model DAPs. The synthesis of the DAP synthon started with 1-tert-butyl hydrogen N-(benzyloxycarbonyl)aspartate. The aldehyde obtained from the beta-carboxyl was used to reductively alkylate benzyl phenylalaninate, generating a secondary amine. Hydrogenolytic deprotection of the end-groups yielded the linear precursor which was cyclized via lactam formation mediated by 1-hydroxy-7-azabenzotriazolyl-N,N,N',N'-tetramethyluronium hexafluorophosphate. This route yielded the reversibly protected hexahydro-1H-3-oxo-2(S)-benzyl-5(S)-(tert-butyloxycarbonyl)-1,4-diazepine. This synthon unit can be subsequently elaborated by substituting the functional groups (secondary amine and carboxyl). Therefore, the DAPs may serve as novel molecular scaffolds to reproduce a biologically relevant topology or as a dipeptido-conformation-mimetic that can be incorporated into bioactive peptides. In addition, these synthetic routes will allow the introduction of different chiralities at positions 2 and 5 as well as the diversification of the side chains at position 2. Furthermore, the synthetic routes described here can be easily modified to obtain larger ring systems with variable degrees of conformational flexibility.