Shravan Kumar Sriraman

Polyethyleneimine-lipid conjugate-based pH-sensitive micellar carrier for gene delivery.

A low molecular weight polyethyleneimine (PEI 1.8 kDa) was modified with dioleoylphosphatidylethanolamine (PE) to form the PEI-PE conjugate investigated as a transfection vector. The optimized PEI-PE/pDNA complexes at an N/P ratio of 16 had a particle size of 225 nm, a surface charge of +31 mV, and protected the pDNA from the action of DNase I. The PEI-PE conjugate had a critical micelle concentration (CMC) of about 34 μg/ml and exhibited no toxicity compared to a high molecular weight PEI (PEI 25 kDa) as tested with B16-F10 melanoma cells. The B16-F10 cells transfected with PEI-PE/pEGFP complexes showed protein expression levels higher than with PEI-1.8 or PEI-25 vectors. Complexes prepared with YOYO 1-labeled pEGFP confirmed the enhanced delivery of the plasmid with PEI-PE compared to PEI-1.8 and PEI-25. The PEI-PE/pDNA complexes were also mixed with various amounts of micelle-forming material, polyethylene glycol (PEG)-PE to improve biocompatibility. The resulting particles exhibited a neutral surface charge, resistance to salt-induced aggregation, and good transfection activity in the presence of serum in complete media. The use of the low-pH-degradable PEG-hydrazone-PE produced particles with transfection activity sensitive to changes in pH consistent with the relatively acidic tumor environment.

Barriers to drug delivery in solid tumors.

Over the last decade, significant progress has been made in the field of drug delivery. The advent of engineered nanoparticles has allowed us to circumvent the initial limitations to drug delivery such as pharmacokinetics and solubility. However, in spite of significant advances to tumor targeting, an effective treatment strategy for malignant tumors still remains elusive. Tumors possess distinct physiological features which allow them to resist traditional treatment approaches. This combined with the complexity of the biological system presents significant hurdles to the site-specific delivery of therapeutic drugs. One of the key features of engineered nanoparticles is that these can be tailored to execute specific functions. With this review, we hope to provide the reader with a clear understanding and knowledge of biological barriers and the methods to exploit these characteristics to design multifunctional nanocarriers, effect useful dosing regimens and subsequently improve therapeutic outcomes in the clinic.

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.

Anti-cancer activity of doxorubicin-loaded liposomes co-modified with transferrin and folic acid

Cancer-specific drug delivery represents an attractive approach to prevent undesirable side-effects and increase the accumulation of the drug in the tumor. Surface modification of nanoparticles such as liposomes with targeting moieties specific to the up-regulated receptors on the surface of tumor cells thus represents an effective strategy. Furthermore, since this receptor expression can be heterogeneous, using a dual-combination of targeting moieties may prove advantageous. With this in mind, the anti-cancer activity of PEGylated doxorubicin-loaded liposomes targeted with folic acid (F), transferrin (Tf) or both (F+Tf) was evaluated. The dual-targeted liposomes showed a 7-fold increase in cell association compared to either of the single-ligand targeted ones in human cervical carcinoma (HeLa) cell monolayers. The increased penetration and cell association of the dual-targeted liposomes were also demonstrated using HeLa cell spheroids. The in vitro cytotoxicity of the doxorubicin liposomes (LD) was then evaluated using HeLa and A2780-ADR ovarian carcinoma cell monolayers. In both these cell lines, the (F+Tf) LD showed significantly higher cytotoxic effects than the untargeted, or single-ligand targeted liposomes. In a HeLa xenograft model in nude mice, compared to the untreated group, though the untargeted LD showed 42% tumor growth inhibition, both the (F) LD and (F+Tf) LD showed 75% and 79% tumor growth inhibition respectively. These results thus highlight that though the dual-targeted liposomes represent an effective cytotoxic formulation in the in vitro setting, they were equally effective as the folic acid-targeted liposomes in reducing tumor burden in the more complex in vivo setting in this particular model.

Ceramide limits phosphatidylinositol-3-kinase C2β-controlled cell motility in ovarian cancer: potential of ceramide as a metastasis-suppressor lipid.

Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. Short interfering RNA screening identified class II phosphatidylinositol 3-kinase C2β (PI3KC2β) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2β-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2β and affect its compartmentalization, thereby suppressing PI3KC2β activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2β knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2β in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2β-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for metastatic diseases.

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.

Drug Delivery to the Central Nervous System

The brain is a very delicate environment where its homeostasis is tightly regulated by the complex central nervous system components. The presence of the blood–brain barrier (BBB) represents a significant hurdle to the effective therapeutic delivery of pharmaceutical agents into the central nervous system for the treatment of neurological conditions, cerebral diseases, as well as brain tumors. To overcome this BBB hurdle, two main strategies have been used: first to traverse the BBB directly, and second, to bypass the BBB altogether. Agents can be allowed to traverse the BBB by directly affecting BBB function either due to its temporary disruption by physical means such as focused ultrasound, or by specifically targeting one or more of its biological components such as cell-surface receptors as well as drug efflux proteins. This chapter provides significant insight into the use of these strategies by comprehensively reviewing current research methodologies in preclinical and clinical settings.

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.

Abstract 4416: Reversal of chemoresistance in ovarian cancer cells by the liposomal co-delivery of MDR inhibitors and paclitaxel

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Ovarian cancer is one of the most lethal gynaecologic malignancies. This can be attributed to the emergence of multidrug resistance in the clinic due to the over-expression of drug-efflux pumps such as P-gp. To overcome this, we have developed a liposomal platform for the the co-delivery of potent P-gp inhibitors with chemotherapeutic drugs. Purpose: To allow for the effective reversal of chemo-resistance in ovarian cancer cells using liposomes co-loaded with tariquidar and paclitaxel. Methods: PEGylated liposomes composed of eggphosphatidylcholine, cholesterol and DOTAP loaded with equimolar amounts of tariquidar (XR) and paclitaxel (PCT) were prepared by the thin film hydration method followed by extrusion. Evaluation of drug resistance of ovarian cancer lines SKOV3, HeyA8 and Tynku was carried out by quantifying MDR1 and MRP1 receptor expression followed by a rhodamine123 exclusion assay. The cytotoxicity of the formulations on the above cancer cell lines was evaluated using the Promega cell viability assay. The effects of the formulations were evaluated in fluorescently stained cells using the iCyte® imaging cytometer (Thorlabs, USA). Long-term kinetic analysis of unstained live cultures was perfomed by holographic imaging cytometer Holomonitor ® M4 (Phase Holographic Imaging, Sweden) and fluorescence microscopy. Results: The liposomes had a particle size distribution of about 200 nm with a zeta potential of 30 mV. As compared to MRP1, a 2.4 fold, 1.9-fold and 1.6-fold higher expression of MDR1 was seen in the HeyA8-MDR, SKOV3-TR and Tynku-R cells respectively over their drug-sensitive counterparts. Using the rhodamine exclusion assay, 14 nM of liposomal XR showed better rhodamine inclusion than 140 nM free XR. In vitro cytotoxicity experiments showed that the drug co-loaded formulation was able to effectively overcome resistance to PCT even at low PCT doses. Using live-cell imaging, paclitaxel-induced arrest of cell cycle progression and onset of apoptosis following this mitotic catastrophe was visible from 1.5 μM to 50 nM only with the co-loaded liposomes. The cell cycle arrest was further visualized using time-lapse Holomonitor M4 which showed the increasing incidence of cells arrested in mitosis over time. Using fluorescence microscopy, we were able to clearly visualize the presence of polymerized microtubules around the cell nucleus as a result of the paclitaxel-mediated microtubule dysfunction. Conclusions: Uniform liposomes co-loaded with tariquidar and paclitaxel were prepared and characterized. The co-loaded liposomes were able to effectively reverse chemoresistance and induce cytotoxicity in a variety of drug-resistant ovarian cancers. The formulations were shown to arrest cell cycle, preventing its progression to the G1 phase. This arrest of cell cycle was shown to be due to the paclitaxel-mediated micro-tubule dysfunction. Note: This abstract was not presented at the meeting. Citation Format: Shravan K. Sriraman, Yilin Zhang, Ed Luther, Ernst Lengyel, Vladimir Torchilin, Vladimir Torchilin. Reversal of chemoresistance in ovarian cancer cells by the liposomal co-delivery of MDR inhibitors and paclitaxel. [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 4416. doi:10.1158/1538-7445.AM2015-4416