Can Sarisozen

Intravesical cationic nanoparticles of chitosan and polycaprolactone for the delivery of Mitomycin C to bladder tumors

Cationic nanoparticles of chitosan (CS), poly-epsilon-caprolactone coated with chitosan (CS-PCL) and poly-epsilon-caprolactone coated with poly-L-lysine (PLL-PCL) were developed to encapsulate intravesical chemotherapeutic agent Mitomycin C (MMC) for longer residence time, higher local drug concentration and prevention of drug loss during bladder discharge. Nanoparticle diameters varied between 180 and 340 nm depending on polymer used for preparation and coating. Zeta potential values demonstrated positive charge expected from cationic nanoparticles. MMC encapsulation efficiency depended on hydrophilicity of polymers since MMC is water-soluble. Encapsulation was increased by 2-fold for CS-PCL and 3-fold for PLL-PCL as a consequence of hydrophilic coating. Complete drug release was obtained with only CS-PCL nanoparticles. On the other hand, CS and PLL-PCL nanoparticles did not completely liberate MMC due to strong polymer-drug interactions which were elucidated with DSC studies. As far as cellular interaction was concerned, CS-PCL was the most efficient formulation for uptake of fluorescent markers Nile Red and Rhodamine123 incorporated into nanoparticles. Especially, CS-PCL nanoparticles loaded with Rhodamine123 sharing hydrophilic properties with MMC were selectively incorporated by bladder cancer cell line, but not by normal bladder epithelial cells. CS-PCL nanoparticles seem to be promising for MMC delivery with respect to anticancer efficacy tested against MB49 bladder carcinoma cell line.

The effect of co-delivery of paclitaxel and curcumin by transferrin-targeted PEG-PE-based mixed micelles on resistant ovarian cancer in 3-D spheroids and in vivo tumors.

Multicellular 3D cancer cell culture (spheroids) resemble to in vivo tumors in terms of shape, cell morphology, growth kinetics, gene expression and drug response. However, these characteristics cause very limited drug penetration into deeper parts of the spheroids. In this study, we used multi drug resistant (MDR) ovarian cancer cell spheroid and in vivo tumor models to evaluate the co-delivery of paclitaxel (PCL) and a potent NF-κB inhibitor curcumin (CUR). PCL and CUR were co-loaded into the polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) based polymeric micelles modified with transferrin (TF) as the targeting ligand. Cytotoxicity, cellular association and accumulation into the deeper layers were investigated in the spheroids and compared with the monolayer cell culture. Comparing to non-targeted micelles, flow cytometry and confocal imaging proved significantly deeper and higher micelle penetration into the spheroids with TF-targeting. Both in monolayers and in spheroids, PCL cytotoxicity was significantly increased when co-delivered with CUR in non-targeted micelles or as single agent in TF-targeted micelles, whereas TF-modification of co-loaded micelles did not further enhance the cytotoxicity. In vivo tumor inhibition studies showed good correlation with the 3D cell culture experiments, which suggests the current spheroid model can be used as an intermediate model for the evaluation of co-delivery of anticancer compounds in targeted micelles.

PEG-PE-based micelles co-loaded with paclitaxel and cyclosporine A or loaded with paclitaxel and targeted by anticancer antibody overcome drug resistance in cancer cells

The over-expression of the P-glycoprotein (P-gp) in cancer cells is one of the main reasons of the acquired Multidrug Resistance (MDR). Combined treatment of MDR cancer cells with P-gp inhibitors and chemotherapeutic agents could result in reversal of resistance in P-gp-expressing cells. In this study, paclitaxel (PTX) was co-encapsulated in actively targeted (anticancer mAb 2C5-modified) polymeric lipid-core PEG-PE-based micelles with Cyclosporine A (CycA), which is one of the most effective first generation P-gp inhibitors. Cell culture studies performed using MDCKII (parental and MDR1) cell lines to investigate the potential MDR reversal effect of the formulations. The average size of both empty and loaded PEG2000-PE/Vitamin E mixed micelles was found between 10 and 25 nm. Zeta potentials of the formulations were found between −7 and −35 mV. The percentage of PTX in the micelles was found higher than 3% for both formulations and cumulative PTX release of about 70% was demonstrated. P-gp inhibition with CycA caused an increase in the cytotoxicity of PTX. Dual-loaded micelles demonstrated significantly higher cytotoxicity in the resistant MDCKII-MDR1 cells than micelles loaded with PTX alone. Micelle modification with mAb 2C5 results in the highest cytotoxicity against resistant cells, with or without P-gp modulator, probably because of better internalization bypassing the P-gp mechanism. Our results suggest that micelles delivering a combination of P-gp modulator and anticancer drug or micelles loaded with only PTX, but targeted with mAb 2C5 represent a promising approach to overcome drug resistance in cancer cells.

Long-circulating PEG-PE micelles co-loaded with paclitaxel and elacridar (GG918) overcome multidrug resistance

Overexpression of drug efflux pump P-gp is one of the major reasons to cause multidrug resistance (MDR). To overcome P-gp mediated MDR, modulators, so called P-gp inhibitors, can be used to block efflux pump activity. Elacridar is one of the most potent P-gp inhibitors, which can cause irreversible and total P-gp blockage. Elacridar, among with other P-gp inhibitors, can be used in combination with anticancer drugs to enhance the effectiveness of chemotherapy against resistant tumor cells. On the other hand, P-gp is presented in normal tissues, thus non-selective blockage of P-gp can cause undesired side effects. Therefore, it is important to deliver P-gp inhibitor only to the tumor cells (along with anticancer drug) and limit its distribution in the body. In this study, we have developed PEG-PE-based long-circulating ca. 15 nm micelles co-loaded with elacridar and paclitaxel, and investigated their ability to overcome paclitaxel resistance in two cancer cell lines. Vitamin E, a common solubility enhancer for PEG-PE micelles, was found to have a negative effect on both particle size and encapsulation efficiencies. The human MDR1 gene-transfected and thus paclitaxel-resistant MDCKII-MDR1 P-gp overexpressing cells were used for cytotoxicity evaluation. Even though PEG-PE based micelles itself have a potential to enhance the cytotoxicity of paclitaxel, elacridar/paclitaxel-co-loaded micelles demonstrated the highest cytotoxicity compared to both free and micellar paclitaxel. The obtained results suggest that co-loading of paclitaxel and elacridar into micellar drug carriers results in promising preparations capable of overcoming paclitaxel resistance.

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.

Rosuvastatin induces apoptosis in cultured human papillary thyroid cancer cells

Statins show antiproliferative activity in various cancer cells. The aim of this study was to evaluate the effects of rosuvastatin treatment on papillary thyroid carcinoma. The papillary thyroid carcinoma (B-CPAP) and normal (Nthy-ori 3-1) thyroid cell lines were treated with rosuvastatin at 12.5, 18.5, 25, 50, 100, and 200 μM concentrations. After 48 and 72 h of rosuvastatin treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Ki-67 immunolabeling, FACS analysis, electron microscopy, caspase-3, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) analysis were performed. Decreased cell viability and G1 phase arrest were detected in papillary thyroid cell line treated with rosuvastatin. Positive immunoreactivity of Ki-67 and dose-dependent increase in S phase on Nthy-ori 3-1 cells were also detected. B-CPAP cells showed intense vacuolisation and autophagosomes with low concentrations and 48 h incubations, while Nthy-ori 3-1 cells showed these changes at higher concentrations. A decrease in the percentage of cells showing autophagy was determined with increasing concentrations of rosuvastatin in B-CPAP cells. Rosuvastatin treatment also caused a dose- and time-dependent increase in caspase-3 activity and apoptotic index by TUNEL assay in B-CPAP cells compared with the Nthy-ori 3-1 cells. Apoptotic cells with nuclear condensation and fragmentation were observed in B-CPAP cell line. Rosuvastatin induced autophagic changes in B-CPAP papillary thyroid cancer cells in lower doses and caused a shift from autophagy to apoptosis. Rosuvastatin may be an alternative treatment for refractory papillary thyroid cancer. Further in vivo studies are necessary to clarify the effects of rosuvastatin in papillary thyroid carcinoma and the clinical implications of rosuvastatin treatment.

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.

Polymers in the co-delivery of siRNA and anticancer drugs to treat multidrug-resistant tumors

Traditional chemotherapeutic agents have long been associated with serious drawbacks due to the heterogeneity and complexity of human cancer. One of the major hurdles regarding chemotherapy is the development of multidrug resistance by the cancer cells. Combination therapy of cancer using different anticancer vectors holds great promise in clinical settings and has been accepted as a current treatment standard for cancer. Recently, siRNA-mediated RNA interference has gained significant attention for cancer treatment due to its ability to knock down genes and proteins with great specificity. Combination of this fast evolving approach to downregulation of genes associated with multidrug resistance in cancer with the simultaneous delivery of traditional chemotherapeutics is fast becoming an effective approach in the cancer therapy. This article provides a view focused on the polymers used to develop multifunctional co-delivery systems that specifically encapsulate siRNAs targeted against MDR and conventional chemotherapeutics. The many studies reviewed here that reported successful in vitro and in vivo results emphasize the promise of this combination therapy approach to overcome multidrug resistance in cancer.