Vladimira Petrakova

Fluorescent Nanodiamonds Embedded in Biocompatible Translucent Shells

High pressure high temperature (HPHT) nanodiamonds (NDs) represent extremely promising materials for construction of fluorescent nanoprobes and nanosensors. However, some properties of bare NDs limit their direct use in these applications: they precipitate in biological solutions, only a limited set of bio-orthogonal conjugation techniques is available and the accessible material is greatly polydisperse in shape. In this work, we encapsulate bright 30-nm fluorescent nanodiamonds (FNDs) in 10-20-nm thick translucent (i.e., not altering FND fluorescence) silica shells, yielding monodisperse near-spherical particles of mean diameter 66 nm. High yield modification of the shells with PEG chains stabilizes the particles in ionic solutions, making them applicable in biological environments. We further modify the opposite ends of PEG chains with fluorescent dyes or vectoring peptide using click chemistry. High conversion of this bio-orthogonal coupling yielded circa 2000 dye or peptide molecules on a single FND. We demonstrate the superior properties of these particles by in vitro interaction with human prostate cancer cells: while bare nanodiamonds strongly aggregate in the buffer and adsorb onto the cell membrane, the shell encapsulated NDs do not adsorb nonspecifically and they penetrate inside the cells.

Automated Neurons Recognition and Sorting for Diamond Based Microelectrode Arrays Recording: A Feasibility Study

Microelectrode arrays (MEA) are extensively used for recording and stimulating neural activity in vitro and in vivo. Depositing nanostructured boron doped diamond (BDD) onto the neuroelectrodes makes it possible to obtain dual mode low-noise neuroelectrical and neurochemical information simultaneously. The signal processing procedure requires finding and distinguishing individual neurons spikes in the recordings. Spike identification is usually done manually which is inaccurate and inappropriate for complex datasets. In this paper, we present a methodology and two algorithms for neurons recognition and evaluation based on unsupervised learning. Forty-five extracellular randomly selected signals from 26 unique measurements of embryonic hippocampal rat neurons (20 kHz, 6 min) were recorded on the commercial 60 TiN channels MEA. The signals were filtered in the 300–3000 Hz band and an amplitude detector (4x std of the background noise) was used for spike detection. WaveClus features were computed and its 3 PCA components were extracted for every spike. The optimal number of clusters were evaluated by an expert rater. K-means + gap criterion (alg. 1) and the Gaussian Mixture Model + Bayesian Information Criterion (alg. 2) were implemented and compared. The total IntraClass Correlation showed a significant inter-rater agreement for all 3 rater procedures (ICC = 0.69, p < 0.001), when post hoc weighted Cohen’s Kappas for 2 raters were 0.85 (expert vs. alg. 1; p < 0.001) and 0.62 (expert vs. alg. 2; p < 0.001). This will contribute to the objective definition of dual mode BDD MEA performance criteria and for a comparison with the current system.

Fluorescent Nanodiamonds are Efficient, Easy-to-Use Cyto-Compatible Vehicles for Monitored Delivery of Non-Coding Regulatory RNAs

MicroRNAs are short molecules of RNA regulating most cellular processes via the mechanism of RNA interference. Their dysregulation leads to a disease burden, making them important therapeutic targets. For the successful development of a therapeutic device, the uptake of a functionalized carrier by live cells and the sufficient release of effector therapeutic molecules are limiting factors. Here for the first time, the inhibition of oncogenic microRNA-21 in CT-26 colon cancer cells is achieved, using an advanced nanosystem consisting of fluorescent nanodiamond and antisense RNA. Stable nanocomplexes efficiently deliver antisense RNA into cell cytoplasm, encouraging further study of microRNA-21 function in target cells. Engaging the fluorescent nanoparticle enables monitoring of transfection and release of the antisense RNA load into cell cytoplasm. Importantly, the internalized antisense RNA effectively destroys target microRNA-21 in CT-26 cancer cells. The absence of oncogenic microRNA-21 liberates tumor suppressor genes Pdcd4 and Timp3 from silencing, and results in a decrease of cell invasion and migration, and in the induction of apoptotic cell death. This study uses a nanodiamond-based imaging and delivery system, and shows that the multidimensional performance of the presented device makes nanodiamond-based complexes promising therapeutic devices.