Computational analysis of drug transport in tumor microenvironment as a critical compartment for nanotherapeutic pharmacokinetics

<p>Over the last decade, nanotherapeutics gained increasingly important role in drug delivery because of their frequently beneficial pharmacokinetics (PK) and lower toxicity when compared to classical systemic drug delivery. In view of therapeutic payload delivery, convective transport is crucial for systemic distribution via circulatory system, but the target domain is tissue outside vessels where transport is governed by diffusion. Here, we have computationally investigated the understudied interplay of physical transports to characterize PK of payload of nanotherapeutics. The analysis of human vasculature tree showed that convective transport is still 5 times more efficient than diffusion suggesting that circulating and payload releasing drug vectors can contribute mostly to systemic delivery. By comparing payload delivery using systemic circulation and drug vectors to microenvironment, internalized vectors were the most efficient and showed Area under the Curve almost 100 higher than in systemic delivery. The newly introduced zone of influence parameter indicated that vectors, especially internalized, lead to the largest tissue fraction covered with therapeutically significant payload concentration. The internalization to microenvironment minimizes effects of plasma domain on payload extravasation from nanotherapeutics. The computed results showed that classical PK, which mostly relies on concentration profiles in plasma, sometimes might be inadequate or not sufficient in explaining therapeutic efficacy of nanotherapeutics. These results provide a deeper look into PK of drug vectors and can help in the design of better drug delivery strategies.</p

Label-Free Isothermal Amplification Assay for Specific and Highly Sensitive Colorimetric miRNA Detection

We describe a new method for the detection of miRNA in biological samples. This technology is based on the isothermal nicking enzyme amplification reaction and subsequent hybridization of the amplification product with gold nanoparticles and magnetic microparticles (barcode system) to achieve naked-eye colorimetric detection. This platform was used to detect a specific miRNA (miRNA-10b) associated with breast cancer, and attomolar sensitivity was demonstrated. The assay was validated in cell culture lysates from breast cancer cells and in serum from a mouse model of breast cancer

Human Equilibrative Nucleoside Transporter-1 Knockdown Tunes Cellular Mechanics through Epithelial-Mesenchymal Transition in Pancreatic Cancer Cells

<div><p>We report cell mechanical changes in response to alteration of expression of the human equilibrative nucleoside transporter-1 (hENT1), a most abundant and widely distributed plasma membrane nucleoside transporter in human cells and/or tissues. Modulation of hENT1 expression level altered the stiffness of pancreatic cancer Capan-1 and Panc 03.27 cells, which was analyzed by atomic force microscopy (AFM) and correlated to microfluidic platform. The hENT1 knockdown induced reduction of cellular stiffness in both of cells up to 70%. In addition, cellular phenotypic changes such as cell morphology, migration, and expression level of epithelial-mesenchymal transition (EMT) markers were observed after hENT1 knockdown. Cells with suppressed hENT1 became elongated, migrated faster, and had reduced E-cadherin and elevated N-cadherin compared to parental cells which are consistent with epithelial-mesenchymal transition (EMT). Those cellular phenotypic changes closely correlated with changes in cellular stiffness. This study suggests that hENT1 expression level affects cellular phenotype and cell elastic behavior can be a physical biomarker for quantify hENT1 expression and detect phenotypic shift. Furthermore, cell mechanics can be a critical tool in detecting disease progression and response to therapy.</p></div

Quantify cell roundness.

<p>(A) Representative confocal micrographs of pancreatic cells (Top panel: Capan-1, Bottom panel: Panc 03.27) showing F-actin distribution, (B) form factor (f = 4πa/p²; a: area; p: perimeter) analyzed by Image J (Capan-1 cells, ctrl: n = 37, scrl: n = 59, hENT1↓: n = 29; Panc 03.27, ctrl: n = 36, scrl: n = 28, hENT1↓:n = 28, N.S: statistically not significant).</p

Compare deformability of control and hENT1 knockdown cells using a microfluidic separation chip.

<p>(A) Photograph (left) of microfluidic separation chip (MS-chip) visualized by red dye inside and a representative scanning electron microscopic (right) image of the post array (diameter of pillar: 35 µm, height of pillar: 30 µm). (B) Bright field image and scheme of cells flowing in MS-chip when the cell diameter is smaller than pillar gap size (red arrow indicates cells). It is assumed here that friction is due only to shear stress (F_p: pressure force against the applied pressure (10 psi in this study), F_f: friction force). (C) The fluorescence image of one of channels among eight shows retained Panc 03.27 cells in the MS-chip after separation of Panc 03.27-ctrl (red fluorescence) and Panc 03.27-hENT1 knockdown (green fluorescence). Representative higher magnification confocal microscopic images of the MS chip (gap size from 12 µm to 6 µm) show the efficiency of separation through gaps. Size distribution of cells (D: Capan-1, G: Panc 03.27 cells). Statistical analysis of cells (E: Capan-1, H: Panc 03.27) and fraction ratio of cells (F: Capan-1, I: Panc 03.27) retained on chip. Values represent mean ± standard deviation.</p

hENT1 knockdown induces changes in EMT markers.

<p>Western blots and relative expression levels of E-cadherin (110 kDa), N-cadherin (140 kDa) in (A) Capan-1 and (C) Panc 03.27 cells after treatment with hENT1 siRNA. Columns in the histograms are the mean of two independent experiments (intensity ratio of GAPDH to E-cadherin or N-cadherin). Representative confocal images of E-cadherin (red fluorescence) and N-cadherin (green fluorescence) expressions in (B) Capan-1 and (D) Panc 03.27 cells.</p

Establish hENT1 knockdown pancreatic cancer cell lines.

<p>(A) Western blots of hENT1 (55 kDa) and GAPDH (37 kDa) in Capan-1 (left) and Panc 03.27 cells (right) without treatment (ctrl) or after treatment of negative siRNA (scrl) or hENT1 siRNA (hENT1↓), (B) Bar histograms show Young's modulus of control, negative siRNA transfected, and hENT1 knockdown cells. (N.S: statistically not significant).</p

Alterations in tumor perfusion after MHT treatment.

<p>Concurrent reduction in arterial and venous flow after with MHT treatment which contributed to decreased half-life AVTT. Tabulated data represents average of ∼30 ROIs per treatment group (n = 5) where * denotes p<0.03.</p

Schematic illustrations describing overall flow of this study.

<p>The hENT1 knockdown induces changes in cellular mechanics via EMT accompanied by alterations in E-cadherin and N-cadherin expression levels, cellular morphology, and motility of pancreatic cancer cells. Further, hENT1 knockdown induces decrease in cell stiffness as demonstrated on representative force separation curves obtained from Panc 03.27 cells (upper graph from a parent cell; the second graph from a hENT1 knockdown cell) using AFM.</p

PLGA-Mesoporous Silicon Microspheres for the <i>in Vivo</i> Controlled Temporospatial Delivery of Proteins

In regenerative medicine, the temporospatially controlled delivery of growth factors (GFs) is crucial to trigger the desired healing mechanisms in the target tissues. The uncontrolled release of GFs has been demonstrated to cause severe side effects in the surrounding tissues. The aim of this study was to optimize a translational approach for the fine temporal and spatial control over the release of proteins, <i>in vivo</i>. Hence, we proposed a newly developed multiscale composite microsphere based on a core consisting of the nanostructured silicon multistage vector (MSV) and a poly­(dl-lactide-<i>co</i>-glycolide) acid (PLGA) outer shell. Both of the two components of the resulting composite microspheres (PLGA-MSV) can be independently tailored to achieve multiple release kinetics contributing to the control of the release profile of a reporter protein <i>in vitro</i>. The influence of MSV shape (hemispherical or discoidal) and size (1, 3, or 7 μm) on PLGA-MSV’s morphology and size distribution was investigated. Second, the copolymer ratio of the PLGA used to fabricate the outer shell of PLGA-MSV was varied. The composites were fully characterized by optical microscopy, scanning electron microscopy, ζ potential, Fourier transform infrared spectroscopy, and thermogravimetric analysis–differential scanning calorimetry, and their release kinetics over 30 days. PLGA-MSV’s biocompatibility was assessed <i>in vitro</i> with J774 macrophages. Finally, the formulation of PLGA-MSV was selected, which concurrently provided the most consistent microsphere size and allowed for a zero-order release kinetic. The selected PLGA-MSVs were injected in a subcutaneous model in mice, and the <i>in vivo</i> release of the reporter protein was followed over 2 weeks by intravital microscopy, to assess if the zero-order release was preserved. PLGA-MSV was able to retain the payload over 2 weeks, avoiding the initial burst release typical of most drug delivery systems. Finally, histological evaluation assessed the biocompatibility of the platform <i>in vivo</i>