Ed Luther

Enhanced Cytotoxicity of Folic Acid-Targeted Liposomes Co-Loaded with C6 Ceramide and Doxorubicin: In Vitro Evaluation on HeLa, A2780-ADR, and H69-AR Cells

Current research in cancer therapy is beginning to shift toward the use of combinational drug treatment regimens. However, the efficient delivery of drug combinations is governed by a number of complex factors in the clinical setting. Therefore, the ability to synchronize the pharmacokinetics of the individual therapeutic agents present in combination not only to allow for simultaneous tumor accumulation but also to allow for a synergistic relationship at the intracellular level could prove to be advantageous. In this work, we report the development of a novel folic acid-targeted liposomal formulation simultaneously co-loaded with C6 ceramide and doxorubicin [FA-(C6+Dox)-LP]. In vitro cytotoxicity assays showed that the FA-(C6+Dox)-LP was able to significantly reduce the IC50 of Dox when compared to that after the treatment with the doxorubicin-loaded liposomes (Dox-LP) as well as the untargeted drug co-loaded (C6+Dox)-LP on HeLa, A2780-ADR, and H69-AR cells. The analysis of the cell cycle distribution showed that while the C6 liposomes (C6-LP) did not cause cell cycle arrest, all the Dox-containing liposomes mediated cell cycle arrest in HeLa cells in the G2 phase at Dox concentrations of 0.3 and 1 μM and in the S phase at the higher concentrations. It was also found that this arrest in the S phase precedes the progression of the cells to apoptosis. The targeted FA-(C6+Dox)-LP were able to significantly enhance the induction of apoptotic events in HeLa cell monolayers as compared to the other treatment groups. Next, using time-lapse phase holographic imaging microscopy, it was found that upon treatment with the FA-(C6+Dox)-LP, the HeLa cells underwent rapid progression to apoptosis after 21 h as evidenced by a drastic drop in the average area of the cells after loss of cell membrane integrity. Finally, upon evaluation in a HeLa spheroid cell model, treatment with the FA-(C6+Dox)-LP showed significantly higher levels of cell death compared to those with C6-LP and Dox-LP. Overall, this study clearly shows that the co-delivery of C6 ceramide and Dox using a liposomal platform significantly correlates with an antiproliferative effect due to cell cycle regulation and subsequent induction of apoptosis and thus warrants its further evaluation in preclinical animal models.

Mixed Nanosized Polymeric Micelles as Promoter of Doxorubicin and miRNA-34a Co-Delivery Triggered by Dual Stimuli in Tumor Tissue.

Dual stimuli-sensitive mixed polymeric micelles (MM) are developed for co-delivery of the endogenous tumor suppressor miRNA-34a and the chemotherapeutic agent doxorubicin (Dox) into cancer cells. The novelty of the system resides in two stimuli-sensitive prodrugs, a matrix metalloproteinase 2 (MMP2)-sensitive Dox conjugate and a reducing agent (glutathione, GSH)-sensitive miRNA-34a conjugate, self-assembled in a single particle decorated with a polyethylene glycol corona for longevity, and a cell-penetrating peptide (TATp) for enhanced intracellular delivery. The MMP2-sensitivity of the system results in threefold higher cytotoxicity in MMP2-overexpressing HT1080 cells compared to low MMP2-expressing MCF7 cells. Cellular internalization of Dox increases by more than 70% after inclusion of TATp to the formulation. MMP2-sensitive MM also inhibits proliferation and migration of HT1080 cells. Moreover, GSH-sensitive MM allows for an efficient downregulation of Bcl2, survivin, and notch1 (65%, 55%, and 46%, respectively) in HT1080 cells. Combination of both conjugates in dual sensitive MM reduces HT1080 cell viability to 40% and expression of Bcl2 and survivin. Finally, 50% cell death is observed in 3D models of tumor mass. The results confirm the potential of the MM to codeliver miRNA-34a and doxorubicin triggered by dual stimuli inherent of tumor tissues.

Nanomedicine based curcumin and doxorubicin combination treatment of glioblastoma with scFv-targeted micelles: In vitro evaluation on 2D and 3D tumor models.

NF-κB is strongly associated with poor prognosis of different cancer types and an important factor responsible for the malignant phenotype of glioblastoma. Overcoming chemotherapy-induced resistance caused by activation of PI3K/Akt and NF-κB pathways is crucial for successful glioblastoma therapy. We developed an all-in-one nanomedicine formulation for co-delivery of a chemotherapeutic agent (topoisomerase II inhibitor, doxorubicin) and a multidrug resistance modulator (NF-κB inhibitor, curcumin) for treatment of glioblastoma due to their synergism. Both agents were incorporated into PEG-PE-based polymeric micelles. The glucose transporter-1 (GLUT1) is overexpressed in many tumors including glioblastoma. The micellar system was decorated with GLUT1 antibody single chain fragment variable (scFv) as the ligand to promote blood brain barrier transport and glioblastoma targeting. The combination treatment was synergistic (combination index, CI of 0.73) against U87MG glioblastoma cells. This synergism was improved by micellar encapsulation (CI: 0.63) and further so with GLUT1 targeting (CI: 0.46). Compared to non-targeted micelles, GLUT1 scFv surface modification increased the association of micelles (>20%, P<0.01) and the nuclear localization of doxorubicin (∼3-fold) in U87MGcells, which also translated into enhanced cytotoxicity. The increased caspase 3/7 activation by targeted micelles indicates successful apoptosis enhancement by combinatory treatment. Moreover, GLUT1 targeted micelles resulted in deeper penetration into the 3D spheroid model. The increased efficacy of combination nanoformulations on the spheroids compared to a single agent loaded, or to non-targeted formulations, reinforces the rationale for selection of this combination and successful utilization of GLUT1 scFv as a targeting agent for glioblastoma treatment.

Reversal of Chemoresistance in Ovarian Cancer by Co-Delivery of a P-Glycoprotein Inhibitor and Paclitaxel in a Liposomal Platform

The overexpression of permeability-glycoprotein (P-gp), an ABC transporter involved in the cellular exclusion of chemotherapeutic drugs, is a major factor in paclitaxel-resistant ovarian cancer. However, in clinical trials, co-administration of P-gp inhibitors and anticancer drugs has not resulted in the efficient reversal of drug resistance. To improve administration, we encapsulated the third-generation P-gp inhibitor tariquidar (XR-9576, XR), alone or in combination with paclitaxel (PCT) in liposomes (LP). After optimization, the liposomes demonstrated favorable physicochemical properties and the ability to reverse chemoresistance in experiments using chemosensitive/chemoresistant ovarian cancer cell line pairs. Analyzing publicly available datasets, we found that overexpression of P-gp in ovarian cancer is associated with a shorter progression-free and overall survival. In vitro, LP(XR) significantly increased the cellular retention of rhodamine 123, a P-gp substrate. LP(XR,PCT) synergistically inhibited cell viability, blocked proliferation, and caused G2–M arrest in paclitaxel-resistant SKOV3-TR and HeyA8-MDR cell lines overexpressing P-gp. Holographic imaging cytometry revealed that LP(XR,PCT) treatment of SKOV3-TR cells induced almost complete mitotic arrest, whereas laser scanning cytometry showed that the treatment induced apoptosis. In proof-of-concept preclinical studies, LP(XR,PCT), when compared with LP(PCT), significantly reduced tumor weight (43.2% vs. 16.9%, P = 0.0007) and number of metastases (44.4% vs. 2.8%, P = 0.012) in mice bearing orthotopic HeyA8-MDR ovarian tumors. In the xenografts, LP(XR,PCT) efficiently induced apoptosis and impaired proliferation. Our findings suggest that co-delivery of a P-gp inhibitor and paclitaxel using a liposomal platform can sensitize paclitaxel-resistant ovarian cancer cells to paclitaxel. LP(XR,PCT) should be considered for clinical testing in patients with P-gp–overexpressing tumors. Mol Cancer Ther; 15(10); 2282–93. ©2016 AACR.

Cytotoxicity of PEGylated liposomes co-loaded with novel pro-apoptotic drug NCL-240 and the MEK inhibitor cobimetinib against colon carcinoma in vitro.

The overactivation of signaling pathways, such as the PI3K and MAPK, which are crucial to cell growth and survival, is a common feature in many cancer types. Though a number of advances have been made in the development of molecular agents targeting these pathways, their application as monotherapies has not significantly improved clinical outcome. A novel liposomal preparation was developed, co-loaded with NCL-240, a small-molecule inhibitor of the PI3K/mTOR pathway, along with cobimetinib, a MEK/ERK pathway inhibitor. This combination drug-loaded nanocarrier, (N+C)-LP, was able to significantly enhance the cytotoxicity of these drugs against colon carcinoma cells in vitro demonstrating a clear synergistic effect (combination index of 0.79). The (N+C)-LP was also able to induce cell cycle arrest of the cells, specifically in the G1 phase thereby preventing their progression to the S-phase, typical of the action of MEK inhibitors. Analyzing the apoptotic events, it was found that this effect on cell cycle regulation is followed by the induction of apoptosis. The quantified distribution of apoptotic events showed that the (N+C)-LP induced apoptosis significantly by over 3-4 fold (P<0.001) compared to other treatment groups. The co-loaded liposomal preparation was also targeted to the transferrin receptor of cancer cells by modifying the surface of the liposome with transferrin. FACS analysis showed that transferrin-mediated targeting enhanced the association of liposomes to HCT 116 cells by almost 5-fold. This could potentially allow for cancer cell-specific effects in vivo thereby minimizing any non-specific interactions of the liposomes with non-cancerous cells. Taken together, this study clearly shows that the combined inhibition of the PI3K and MEK pathways correlates with a significant anti-proliferative effect, due to cell-cycle regulation leading to the induction of apoptosis.

Applications of label-free, quantitative phase holographic imaging cytometry to the development of multi-specific nanoscale pharmaceutical formulations

A label‐free, high content, time‐lapse holographic imaging system was applied to studies in pharmaceutical compound development. Multiple fields of cellular images are obtained over typically several day evaluations within standard CO2 incubators. Events are segmented to obtain population data of cellular features, which are displayed in scattergrams and histograms. Cell tracking is accomplished, accompanied by Cartesian plots of cell movement, as well as plots of cell features vs. time in novel 4‐D displays of X position, Y position, time, and cell thickness. Our review of the instrument validation data includes 1) tracking of Giant HeLa cells, which may be undergoing neosis, a process of tumor stem cell generation; 2) tracking the effects of cell cycle related toxic agents on cell lines; 3) using MicroRNAs to reverse the polarization state in macrophages to induce tumor cell killing; 4) development of liposomal nanoformulations to overcome Multi‐Drug Resistance (MDR) in ovarian cancer cells; and 5) development of dual sensitive micelles to specifically target matrix metalloproteinase 2 (MMP2) over‐expressing cell lines.

Abstract 202: Four dimensional quantitative label-free holographic imaging of the cell cycle in tumor cell lines

Introduction: Quantitative analytical systems have the ability to quantify the amount of constituents on a per entity basis. For example, flow cytometers use stoichiometric DNA stains to quantify the amount of DNA on a per cell basis, and allow discrimination of the cell cycle phases G0/G1, S-Phase, and G2/M. Quantitative imaging cytometers employ morphometric features (total DNA content, DNA maximum brightness, and nuclear area) to track the cell cycle during the mitotic phases. As cells enter mitosis, the condensation of the chromatin to form chromosomes increases the DNA density, while the nuclear area and volume decrease. Unfortunately, DNA dyes are harmful for live cells and can interfere with the results of toxicological assays. It is desirable to have a method for label-free long-term evaluation of the cell cycle in living cells. Methods: The Holomonitor®M4 (Phase Holographic Imaging, Lund, Sweden) is an incubator-adapted time-lapse imaging system that records interference patterns from a low power 635nm diode laser. Computer software is used to de-convolve the interference pattern at a plurality of heights to form 3-dimensional and 2-dimensional images of the sample thickness. These images are segmented and features (cellular area, optical thickness and volume, etc.) are calculated on a per cell basis. Positional information is recorded for each cell. Data can be presented as positional track charts, position vs. feature graphs, and feature vs. feature graphs. We obtained time-lapse videos using proprietary PHI software and also generated 4-dimensional plots (X-position, Y- position, optical thickness coded as the pixel intensity, and time) in ImageJ software. Results: In our studies of SKOV3 tumor cell lines, we found a set of cellular based features (cell volume, optical thickness, and cell area) that correlate with the DNA-based features we are accustomed to using in our traditional label-based systems. In the 4D plots, the history of each cell within the imaging area is presented for the entire evaluation time - typically 18-24 hours with 5 minute sampling increments. With a low optical thickness threshold, tracks of all of the cells are displayed. The image intensity threshold can be raised, so only mitotic events are displayed. Mitotic cells have a characteristic Y shaped tracks, appearing suddenly as the chromatin condenses, bifurcating as the daughter cells move away from each other, and then disappearing as the chromatin de-condenses. Cells blocked in mitosis present as persistent long tracks. The rapid degradation of apoptotic cells is often visible, while cells undergoing mitotic catastrophe suddenly disappear. Conclusions: Time-lapse label-free holographic imaging combining traditional analysis and novel display techniques is well suited for toxicological studies and visualization of drug effects on the cell cycle. Citation Format: Ed Luther, Jeffrey Agar, Mansoor Amiji. Four dimensional quantitative label-free holographic imaging of the cell cycle in tumor cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 202. doi:10.1158/1538-7445.AM2015-202

Applications of quantitative time lapse holographic imaging to the development of complex pharmaceutical nano formulations

We rely on in vitro cellular cultures to evaluate the effects of the components of multifunctional nano-based formulations under development. We employ an incubator-adapted, label-free holographic imaging cytometer HoloMonitor M4® (Phase Holographic Imaging, Lund, Sweden) to obtain multi-day time-lapse sequences at 5- minute intervals. An automated stage allows hand-free acquisition of multiple fields of view. Our system is based on the Mach-Zehnder interferometry principle to create interference patterns which are deconvolved to produce images of the optical thickness of the field of view. These images are automatically segmented resulting in a full complement of quantitative morphological features, such as optical volume, thickness, and area amongst many others. Precise XY cell locations and the time of acquisition are also recorded. Visualization is best achieved by novel 4-Dimensional plots, where XY position is plotted overtime time (Z-directions) and cell-thickness is coded as color or gray scale brightness. Fundamental events of interest, i.e., cells undergoing mitosis or mitotic dysfunction, cell death, cell-to-cell interactions, motility are discernable. We use both 2D and 3D models of the tumor microenvironment. We report our new analysis method to track feature changes over time based on a 4-sample version of the Kolmogorov-Smirnov test. Feature A is compared to Control A, and Feature B is compared to Control B to give a 2D probability plot of the feature changes over time. As a result, we efficiently obtain vectors quantifying feature changes over time in various sample conditions, i.e., changing compound concentrations or multi-compound combinations.

Abstract B18: A label-free, high content, moderate throughput analytical platform for quantitative kinetic analysis of cell behavior upon drug activation in cell-culture models based on the Kolmogorov-Smirnov test

Our objective is to develop multi-functional nanotechnology-based anti-tumor drug delivery systems for improving efficacy of treatments and reducing undesirable side effects. The essential part of this process is the development of un-biased quantitative analytical techniques. We are reporting a successful validation of a very high content, medium throughput system in multi-well plates. We employed a newly developed holographic imaging cytometry system HoloMonitor® M4 for label-free time-lapse cellular analysis (Phase Holographic Imaging, Sweden), typically at 5 minute intervals for 48–72 hours. A low power red laser is split into sample and reference beams to obtain holograms of cells. The holograms are unwrapped by proprietary software into quantitative dark field images, with very precise calculation of the cell optical thickness. These images are segmented, and a vast variety of features are extracted for cellular events. In our evaluations we found that cell optical thickness, volume and area have high correlation with features used in traditional fluorescent DNA-stained analysis. We obtain full cell cycle profiles, including the separation of mitotic cells, cells undergoing mitotic dysfunction, and apoptotic cells—all in label-free environment. We recently developed a novel 4D image display of evaluated fields of view: X position, Y position, cell thickness coded as brightness over time in the Z direction. In these images, the history of the viewing area over time is displayed, and salient features such as cell proliferation, cell thickness, and cell motility, contact inhibition, and cell death are discernable. As an example, a combinatorial liposomal formulation containing paclitaxel and a P-gp inhibitor tariquidar was compared to free paclitaxel in SKOV3 TR (taxol resistant) cells seeded in Petri dishes. TR cells treated with free paclitaxel presented increased mitoses, while TR cells treated with the combinatorial preparation exhibited a complete abrogation of mitotic division. Cells were frozen in mitosis due to the polymerization of tubulin, the mechanism of paclitaxel toxicity. In this study Hela cells were seeded in a 6-well plate and allowed to acclimate overnight. Free doxorubicin (DOX) was added to 5 wells at concentrations ranging from 1.6 nM to 1 uM in 5-fold increments. Three fields (0.56 mm2) in each well were imaged for 48 hours at 5 min. intervals. We developed a modified version of the Kolmogorov-Smirnov 2-sample test to analyze the data. The classic test takes control and test frequency distributions (histograms), and converts them to probability functions. The maximum vertical displacement between the two is reported as the D-value, which quantifies the amount of difference between the two. In our version, we obtain histograms of the D-values. We set up our analysis so the X-axis is time, and the feature is any of the reported metrics from the HoloMonitorM4 software. Our results are briefly summarized in the following table indicating where the sample is greater than the control (+), less than the control (-), and a transition (/). Dox Con......(1.6 nM).(8 nM).....(40 nM)..(200 nM)..(1 uM) Cell Count.........(+).......(-)........(---).......(---).......(---) Area (-)......(+)....(+++++)..(++/-)...(++/-) Max. Thick.........(+)....(++)...(+++++)...(-/+)......(-/+) Volume (+)......(+)....(+++++)...(++)......(++) Perimeter........ (+/-)...(+/-).......(++)....(--/++)...(-/++) Roughness.........(-).......(-).......(++/-)....(++/-)......(--) The results are consistent with the known effects of Dox. At low concentration, unrepaired DNA strand breaks start to accumulate and cause slight increases in the volume and thickness of the cells. At mid-concentration the DNA repair process is overwhelmed, proliferation is halted, and vast changes in cellular morphology are evident. At the higher concentration, changes in morphology correspond with cell death followed by eventual deterioration. Citation Format: Ed Luther, Livia Mendes, Daniel Costa, Jiayi Pan, Elena Holden, Vladimir Torchilin. A label-free, high content, moderate throughput analytical platform for quantitative kinetic analysis of cell behavior upon drug activation in cell-culture models based on the Kolmogorov-Smirnov test. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B18.

Abstract LB-A22: Comparison of the effect of pharmaceutical compounds on tumor cells in 2D and 3D in vitro models using label-free, quantitative 4 dimensional holographic imaging

Introduction Development of in vitro models for the evaluation of drugs represents a useful approach as in vivo studies may be costly and time consuming. Ideal models should take into account the effects of the cellular microenvironment, which includes the extra-cellular matrix, stroma and neighboring cells. Phase Holographic Imaging The Holomonitor®M4 (Phase Holographic Imaging, Lund, Sweden) is an incubator-adapted time-lapse holographic imaging cytometry system that enables quantitative, label-free analysis of living cells. A low-power red laser creates an interference pattern (hologram) which is reconstructed by software into images. Software modules are available for cellular segmentation, calculating quantitative features (optical thickness and volume), and individual cell tracking. Cells were plated in Petri dishes and imaged in 5 minute intervals for 2 to 3 days. The field of view with a 20X objective is 538 μm 2 with an effective depth of field in the mm range, allowing monitoring elongated cubes of the tumor microenvironment. We export 2D projections of the holograms into Image J. We then produce 4-dimensional plots of the tumor microenvironment (X and Y position, cell thickness coded as brightness, and time in the Z direction). 2D models of non-motile adherent cells. When untreated HeLa cells are seeded at low density, colonies manifest as inverted cones increasing in diameter as the cell number increases. Mitotic cells appear as short duration bright spots due to rounding of the cells and increase in optical thickness. Treatment with colchicine at concentrations sufficient to block the completion of mitosis caused the brightness of the tracks is persistent. When treated with doxorubicin (dox) at concentrations sufficient to induce apoptosis, cell tracks briefly become bright, but then decrease in diameter and brightness as the cells gradually disintegrate. 2D models of motile cells. HT1080 fibroblasts present two distinct morphological types, an amoeboid form that move via extensions of lamellipodia from a leading edge that adhere to a substrate and a mesenchymal form, where small protrusions termed lobopodia propel cells by attaching to ECM components. In untreated samples, cells are predominately in the amoeboid form, with the cell tracks moving upward. Low concentrations of dox (50nM) abolish cell proliferation, and a shuttling motion is seen, caused by dysfunctional lamellipodia attachment to the substrate prior to eventual cell death. 3D models of motile cells. We plated HT1080 cells on the dishes, treated them with compounds, and then overlaid the cells with 1 mg/ml collagen type 1. With 50 nM dox there is a high degree of proliferation and vastly increased planar motion. In videos it can be seen that cells are following pathways created by other cells, consistent with cells burrowing through the ECM. Conclusion We developed a novel 4-D holographic imaging method using the Holomonitor HM4 and Image J. Here, we present methods for comparing traditional 2D and 3D in vitro models. Our example of HT1080 cells treated with dox clearly shows the superiority of the 3D model, an important step in developing assays that better emulate multi-dimensional biological processes and offer the possibility of evaluating effects of drugs at lower cost and experimental complexity than those of in vivo assays. Citation Format: Ed Luther, Giuseppina Salzano, Shravan K. Sriraman, Daniel Costa, Vladimir P. Torchilin. Comparison of the effect of pharmaceutical compounds on tumor cells in 2D and 3D in vitro models using label-free, quantitative 4 dimensional holographic imaging. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A22.