Clinical Applications of Carbon Nanomaterials in Diagnostics and Therapy.

Nanomaterials have the potential to improve how patients are clinically treated and diagnosed. While there are a number of nanomaterials that can be used toward improved drug delivery and imaging, how these nanomaterials confer an advantage over other nanomaterials, as well as current clinical approaches is often application or disease specific. How the unique properties of carbon nanomaterials, such as nanodiamonds, carbon nanotubes, carbon nanofibers, graphene, and graphene oxides, make them promising nanomaterials for a wide range of clinical applications are discussed herein, including treating chemoresistant cancer, enhancing magnetic resonance imaging, and improving tissue regeneration and stem cell banking, among others. Additionally, the strategies for further improving drug delivery and imaging by carbon nanomaterials are reviewed, such as inducing endothelial leakiness as well as applying artificial intelligence toward designing optimal nanoparticle-based drug combination delivery. While the clinical application of carbon nanomaterials is still an emerging field of research, there is substantial preclinical evidence of the translational potential of carbon nanomaterials. Early clinically trial studies are highlighted, further supporting the use of carbon nanomaterials in clinical applications for both drug delivery and imaging.

Ex Vivo Expansion of CD34+ CD90+ CD49f+ Hematopoietic Stem and Progenitor Cells from Non-Enriched Umbilical Cord Blood with Azole Compounds.

Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non-enriched mononucleated cells (MNC) using novel azole-based small molecules. Freshly-thawed UCB-MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure-activity-relationship studies of SB203580, a known p38-MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8-fold increase of absolute viable CD45+ CD34+ CD38- CD45RA- progenitors which was at least 3.7-fold higher than control cultures (p < .001). In depth phenotypic analysis revealed >600-fold expansion of CD34+ /CD90+ /CD49f+ rare HSPCs coupled with significant (p < .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p < .001) higher engraftment of human CD45+ and CD45+ CD34+ cells in the PB and BM by day 21 compared to non-expanded and cytokine expanded grafts. The C7 expanded grafts maintained long-term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376-393.

Application of Static Modeling --in the Prediction of In Vivo Drug-Drug Interactions between Rivaroxaban and Antiarrhythmic Agents Based on In Vitro Inhibition Studies.

Rivaroxaban, a direct Factor Xa inhibitor, is indicated for stroke prevention in nonvalvular atrial fibrillation (AF). Studies have revealed that the clearance of rivaroxaban is largely attributed to CYP3A4, CYP2J2 metabolism, and P-glycoprotein (P-gp) efflux pathways. Amiodarone and dronedarone are antiarrhythmic agents employed in AF management. Amiodarone, dronedarone, and their major metabolites, N-desethylamiodarone (NDEA) and N-desbutyldronedarone (NDBD), demonstrate inhibitory effects on CYP3A4 and CYP2J2 with U.S. Food and Drug Administration-recommended probe substrates. In addition, both amiodarone and dronedarone are known P-gp inhibitors. Hence, the concomitant administration of these antiarrhythmic agents has the potential to augment the systemic exposure of rivaroxaban through simultaneous impairment of its clearance pathways. Currently, however, clinical data on the extent of these postulated drug-drug interactions are lacking. In this study, in vitro inhibition assays using rivaroxaban as the probe substrate demonstrated that both dronedarone and NDBD produced reversible inhibition as well as irreversible mechanism-based inactivation of CYP3A4- and CYP2J2-mediated metabolism of rivaroxaban. However, amiodarone and NDEA were observed to cause reversible inhibition as well as mechanism-based inactivation of CYP3A4 but not CYP2J2. In addition, amiodarone, NDEA, and dronedarone, but not NDBD, were determined to inhibit P-gp-mediated rivaroxaban transport. The in vitro inhibition parameters were fitted into a mechanistic static model, which predicted a 37% and 31% increase in rivaroxaban exposure due to the inhibition of hepatic and gut metabolism by amiodarone and dronedarone, respectively. A separate model quantifying the inhibition of P-gp-mediated efflux by amiodarone or dronedarone projected a 9% increase in rivaroxaban exposure.

Lipid-dendrimer hybrid nanosystem as a novel delivery system for paclitaxel to treat ovarian cancer.

Combining lipids and dendrimers into one formulation is an emerging platform in the drug delivery field. This study aims to (i) develop and characterize a lipid-dendrimer hybrid (LDH) nanosystem for the hydrophobic anticancer drug paclitaxel, and (ii) evaluate its in vitro and in vivo anti-cancer activity in ovarian cancer models. The LDH nanosystems were prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and poly (amidoamine) (PAMAM) G4.0. The size and zeta potential of the LDH nanosystem were 37.6 ± 6.1n m and +2.9 ± 0.1 mV, respectively, with vesicular morphology observed under cryo-TEM. The encapsulation efficiency of paclitaxel in the LDH system was 78.0 ± 2.1%. The potency of paclitaxel could be significantly improved by 37-fold when presented in the LDH nanosystem as compared to free drug, whereby paclitaxel and PAMAM G4.0 acted synergistically in killing the ovarian cancer cells. As shown by fluorescence confocal microscopy, majority of the lipids in the LDH nanosystem were located in the plasma membrane, while the dendrimers were distributed intracellularly upon uptake. Despite the use of a 10-fold lower paclitaxel dose, the survival of IGROV-1 ovarian tumor-bearing animals could be significantly prolonged by the paclitaxel-loaded LDH nanosystem, as reflected by a 50% increase in the median survival time. Such hybrid nanosystem emerged from combining two established drug delivery platforms could pave way for the development of multifunctional delivery systems for potential theranostic applications.

Dual-functionalized poly(amidoamine) dendrimers with poly(ethylene glycol) conjugation and thiolation improved blood compatibility.

OBJECTIVES: This study aims to examine the blood compatibility of dual-functionalized poly(amidoamine) (PAMAM) dendrimers. METHODS: The cationic PAMAM dendrimer of generation 4.0 (PM4.0) were functionalized by poly(ethylene glycol) (PEG) conjugation or by thiolation or the combination of both methods. Various in-vitro assays including immune cell cytotoxicity, haemoglobin release, serum albumin binding, complement activation and coagulation times were used to characterize the compatibility with blood components. KEY FINDINGS: Although thiolation of polymers has been reported as a strategy to reduce platelet activation or aggregation, thiolation of PM4.0 alone did not offer any protective effect against the dendrimer toxicity on blood components or functions. PEGylation was able to reduce the toxic effect and interactions of the unmodified and thiolated PM4.0 on various blood components and functions; yet, PEGylated PM4.0 displayed prolonged prothrombin times and activated partial thromboplastin times. Among various PM4.0 derivatives, dual-functionalized PM4.0 with PEG and thiol groups displayed the least toxicity to various blood components and functions. CONCLUSIONS: Our findings suggested that comprehensive studies of dendrimer biocompatibility should be performed so as to establish the safe dose window for systemic administration.

Mitochondrial superoxide reduction and cytokine secretion skewing by carbon nanotube scaffolds enhance ex vivo expansion of human cord blood hematopoietic progenitors.

In this study, we report that surface functional groups of single walled carbon nanotubes (f-SWCNT) are critical for mediating survival and ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) in human umbilical cord blood (UCB). In comparison to amide (-O-NH2) and polyethylene-glycol (-PEG) functionalized SWCNT, carboxylic acid (-COOH) variants gave optimal viability support which correlated with maximal reduction of lethal mitochondrial superoxides in HSPC. Cytokine array illustrated that f-SWCNT-COOH maintained higher proportion of HSPC associated cytokines and minimal level of differentiation promoting factors. Transplantation of f-SWCNT-COOH expanded grafts in sub-lethally irradiated immunodeficient mice resulted in higher engraftment of HSPC in bone marrow compared to control when they were co-transplanted with non-expanded cells from the same UCB. Expanded grafts mediated higher survival rate of mice compared to non-expanded grafts due to lower graft-versus-host-disease which is likely a consequence of proportion of immune cells in the grafts. FROM THE CLINICAL EDITOR: Umbilical cord blood (UCB) is a potential source of hematopoietic stem and progenitor (HSPC) cells. One major hurdle for its clinical use is the insufficient yield of cell number. The authors in this study elegantly demonstrated the importance of various functional groups on single-walled carbon nanotubes (f-SWCNT) in enhancing ex vivo expansion of HSPC in UCB. The findings may pave a way for having UCB as a source for HSPC for clinical use in the future.

Dendrimers in oral drug delivery application: current explorations, toxicity issues and strategies for improvement.

Dendrimers are emerging as potential novel nano-scaled material in drug delivery applications. An interesting area of application is oral drug delivery. In oral drug delivery, many drugs suffer from low bioavailability due to the presence of various biological barriers. Dendrimers have been shown to modulate tight junctions and the integrity of cellular membranes. This effect gives hope for dendrimer to be applied in oral drug delivery. Based on such properties, dendrimers are further surface-modified so that the system will be more suitable for oral delivery applications. Cationic dendrimers are commonly conjugated with neutral or negatively charged ligands, such as polyethylene glycol (PEG), to reduce potential toxicity in gastrointestinal (G.I.) tract. Dendrimers are also surfacemodified to inhibit the efflux effect of P-glycoprotein, which is one of the major drug efflux pumps in G.I. tract. Another interesting strategy is to directly conjugate or mix dendrimer with drugs either to form a dendrimer-drug conjugation or complex to deliver the drug. In this review, application of dendrimers in oral drug delivery will be discussed. The main focus is on the various surface modification strategies to design a more desirable dendrimer-based delivery system that fits the need in oral drug delivery.

Discovery of mixed type thymidine phosphorylase inhibitors endowed with antiangiogenic properties: synthesis, pharmacological evaluation and molecular docking study of 2-thioxo-pyrazolo[1,5-a][1,3,5]triazin-4-ones. Part II.

In our drug discovery program, a series of 2-thioxo-pyrazolo[1,5-a][1,3,5]triazin-4-ones were designed, synthesized and evaluated for their TP inhibitory potential. All the synthesized analogues conferred a varying degree of TP inhibitory activity, comparable or better than positive control, 7-deazaxanthine (7-DX, 2) (IC50 value = 42.63 µM). A systematic approach to the lead optimization identified compounds 3c and 4a as the most promising TP inhibitors, exhibiting mixed mode of enzyme inhibition. Moreover, selected compounds demonstrated the ability to attenuate the expression of the angiogenic markers (viz. MMP-9 and VEGF) in MDA-MB-231 cells at sublethal concentrations. In addition, molecular docking studies revealed the plausible binding orientation of these inhibitors towards TP, which was in accordance with the experimental results. Taken as a whole, these compounds would constitute a new direction for the design of novel TP inhibitors with promising antiangiogenic properties.

Liposomal codelivery of a synergistic combination of bioactive lipids in the treatment of acute myeloid leukemia.

AIM: The aim of this work was to develop a liposomal formulation to facilitate delivery of a synergistic safingol/C2-ceramide combination in the treatment of acute myeloid leukemia (AML). MATERIALS & METHODS: Liposomes were prepared using the extrusion method and the bioactive lipids were encapsulated passively. Drug concentrations were determined by liquid chromatography tandem mass spectrometry. Antileukemic activity was evaluated using human leukemic cell lines, patient samples and U937 leukemic xenograft models. RESULTS: A stable liposome formulation was developed to coencapsulate safingol and C2-ceramide at 1:1 molar ratio with >90% encapsulation efficiency. The liposomal safingol/C2-ceramide was effective in AML cell lines, patient samples and murine xenograft models of AML, compared with liposomal safingol or liposomal C2-ceramide alone despite a dose reduction of 33%. CONCLUSION: Our study provided proof-of-concept evidence to deliver synergistic combination of bioactive lipid to achieve complete remission in AML.

Dual-functionalized PAMAM dendrimers with improved P-glycoprotein inhibition and tight junction modulating effect.

This study aims to surface modify poly(amido amine) or PAMAM dendrimers by sequentially grafting poly(ethylene glycol) or PEG and 4-thiobutylamidine (TBA) so as to reduce PAMAM cytotoxicity while improving the ability of PAMAM to modulate P-glycoprotein (P-gp) efflux and tight junction integrity. Conjugation of functional groups was determined by NMR spectroscopy, FT-IR, thiol group quantification and molecular weight estimation. The yield of the dual-functionalized dendrimers was >80%. The dual-functionalized dendrimer could significantly reduce PAMAM cytotoxicity to <15% as reflected by LDH release in Caco-2 and MDCK/MDR1 cells after 72 h of exposure. Thiolated dendrimers could increase cellular accumulation and permeation of the P-gp substrate R-123, and such effect could be affected by the extent of PEGylation of the dendrimer. Surface-modified PAMAM dendrimers, either by single or dual functionalization, could better modulate tight junction integrity in comparison with unmodified PAMAM, as demonstrated through immunostaining of the tight junction marker ZO-1, permeation of the model compound Lucifer Yellow (LY) and transepithelial electrical resistance (TEER). Of importance, reversible tight junction modulating effect was only observed in the dual-functionalized dendrimers. Collectively, dual functionalization with PEG and TBA represented a promising approach in altering PAMAM dendrimer surface for potential application in oral drug delivery.

Fragment-based approach to the design of 5-chlorouracil-linked-pyrazolo[1,5-a][1,3,5]triazines as thymidine phosphorylase inhibitors.

5-Chlorouracil-linked-pyrazolo[1,5-a][1,3,5]triazines were designed as new thymidine phosphorylase inhibitors based on the fragment based drug design approach. Multiple-step convergent synthetic schemes were devised to generate the target compounds. The intermediate 5-chloro-6-chloromethyluracil was synthesized by a 4-step reaction. A series of the second bicyclic intermediates, namely pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-one, was obtained from various substituted 3-aminopyrazoles. These two intermediates were coupled finally in the presence of sodium ethoxide and methanol to yield the desirable target compounds. The methylthio coupling spacer was found to be suitable in enabling the interaction of the two fragments at the active site and allosteric site of the enzyme. The best coupled compound (9q) inhibited the thymidine phosphorylase with an IC50 value as low as 0.36 ± 0.1 µM. In addition, 9q demonstrated a mixed-type of enzyme inhibition kinetics, thus suggesting that it might indeed potentially bind at two different sites on the enzyme.

A structure-activity relationship study of 1,2,4-triazolo[1,5-a][1,3,5]triazin-5,7-dione and its 5-thioxo analogues on anti-thymidine phosphorylase and associated anti-angiogenic activities.

Thirty-three 1,2,4-triazolo[1,5-a][1,3,5]triazin-5,7-dione and its 5-thioxo analogues were designed and synthesized which contained different substituents at meta- and/or para-positions of 2-phenyl or 2-benzyl ring attached to the fused ring structure. The preliminary pharmacological evaluation demonstrated that the 5-thioxo analogues of 1,2,4-triazolo[1,5-a][1,3,5]triazine exhibited a varying degree of inhibitory activity towards thymidine phosphorylase, comparable or better than reference compound, 7-Deazaxanthine (7-DX, 2) (IC50 value = 42.63 µM). Moreover, compounds 5q and 6i displayed a mixed-type of inhibitory mechanism in the presence of variable concentrations of thymidine (dThd). In addition, selected compounds were found to have a noticeable inhibitory effect on the expression of angiogenesis markers, including VEGF and MMP-9 in MDA-MB-231 breast cancer cells.

Protective role of functionalized single walled carbon nanotubes enhance ex vivo expansion of hematopoietic stem and progenitor cells in human umbilical cord blood.

In this study, carboxylic acid functionalized single walled carbon nanotubes (f-SWCNT-COOH) was shown to support the viability and ex vivo expansion of freeze-thawed, non-enriched hematopoietic stem and progenitor cells (HSPC) in human umbilical cord blood-mononucleated cells (UCB-MNC). Our in vitro experiments showed that f-SWCNT-COOH increased the viability of the CD45(+) cells even without cytokine stimulation. It also reduced mitochondrial superoxides and caspase activity in CD45(+) cells. f-SWCNT-COOH drastically reduced the proportions of CD45(-) cells in the non-enriched UCB-MNC. Phenotypic expression analysis and functional colony forming units (CFU) showed significant ex vivo expansion of HSPC, particularly of CD45(+)CD34(+)CD38(-) population and granulocyte-macrophage (GM) colonies, in f-SWCNT-COOH augmented cultures supplemented with basal cytokines. In vivo data suggested that f-SWCNT-COOH expanded UCB-MNC could repopulate immunodeficient mice models with minimal acute or sub-acute symptoms of graft-versus-host disease (GVHD) and f-SWCNT-COOH dependent toxicity. FROM THE CLINICAL EDITOR: In this paper a novel method is presented by using single wall functionalized carbon nanotubes to enhance viability and ex vivo expansion of freeze-thawed, non-enriched hematopoietic stem and progenitor cells in human umbilical cord blood -mononucleated cells. Detailed data is presented about enhanced viability, including improved repopulation of immunodeficient mice models with minimal acute or sub-acute symptoms of graft-versus-host disease.

Role of oxidative stress, endoplasmic reticulum stress and ERK activation in triptolide-induced apoptosis.

Since its isolation from Tripterygium wilfordii in 1972, triptolide has been shown to possess potent anticancer activity against a variety of cancers, and has entered phase I clinical trial. It is a diterpenoid triepoxide that acts through multiple molecular targets and signaling pathways. The mitogen-activated protein kinases are well known for their modulation of cell survival and proliferation. In particular, the ERK pathway has a dual role in cell proliferation and cell death. Thus far, data on the effect of triptolide on ERK signaling remain limited. In our current study, we have shown for the first time that ERK activation rather than inhibition occurred in a dose- and time-dependent manner following triptolide treatment in MDA-MB-231 breast cancer cells. ERK activation was crucial in mediating triptolide-induced caspase-dependent apoptosis. Tritpolide-induced ERK activation modulated the expression of the Bcl-2 protein family member Bax but was not involved in the downregulation of Bcl-xL expression. Signals acted upstream of ERK activation included generation of reactive oxygen species (ROS) and endoplasmic reticulum stress predominantly via the PERK­eIF2α pathway, as the MEK inhibitor U0126 did not inhibit the phosphorylation of PERK and eIF2α or the generation of ROS.

Liposome co-encapsulation of synergistic combination of irinotecan and doxorubicin for the treatment of intraperitoneally grown ovarian tumor xenograft.

Liposome co-encapsulation of synergistic anti-cancer drug combination is an emerging area that has demonstrated therapeutic benefit in clinical trials. Remote loading of two or more drugs into a single liposome constitutes a new challenge that calls for a re-examination of drug loading strategies to allow the loading of the drug combination efficiently and with high drug content. In this study, the Mn(2+) gradient coupled with A23187 ionophore was applied in the sequential co-encapsulation of doxorubicin and irinotecan, as this drug loading method is capable of remotely loading drugs by apparently two different mechanisms, namely, coordination complexation and pH gradient. Doxorubicin and irinotecan could be co-encapsulated into liposomes in a wide range of drug-to-drug ratios, with encapsulation efficiencies of > 80%. The total encapsulated drug content was non-linearly correlated with increases in the intraliposomal Mn(2+) concentration, with a maximum total drug-to-lipid molar ratio of 0.8:1 achieved with 600 mM Mn(2+). This high encapsulated drug content did not affect the stability of the co-encapsulated liposomes upon storage for six months. Regardless of the encapsulated drug amount, the liposomes did not exhibit the fiber bundle precipitate morphology but rather an undefined structural organization in the aqueous core. The co-encapsulated liposome formulation was further tested in an intraperitoneally grown, human ovarian tumor xenograft model, and was shown to significantly improve the survival of the tumor-bearing animals. The improvement in therapeutic efficacy was possibly due to the increase in systemic drug exposure, with the maintenance of the synergistic molar drug ratio of 1:1 in circulation.

Intercellular cytosolic transfer correlates with mesenchymal stromal cell rescue of umbilical cord blood cell viability during ex vivo expansion.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have been observed to participate in tissue repair and to have growth-promoting effects on ex vivo co-culture with other stem cells. METHODS: In order to evaluate the mechanism of MSC support on ex vivo cultures, we performed co-culture of MSC with umbilical cord blood (UCB) mononuclear cells (MNC) (UCB-MNC). RESULTS: Significant enhancement in cell growth correlating with cell viability was noted with MSC co-culture (defined by double-negative staining for Annexin-V and 7-AAD; P < 0.01). This was associated with significant enhancement of mitochondrial membrane potential (P < 0.01). We postulated that intercellular transfer of cytosolic substances between MSC and UCB-MNC could be one mechanism mediating the support. Using MSC endogenously expressing green fluorescent protein (GFP) or labeled with quantum dots (QD), we performed co-culture of UCB-MNC with these MSC. Transfer of these GFP and QD was observed from MSC to UCB-MNC as early as 24 h post co-culture. Transwell experiments revealed that direct contact between MSC and UCB-MNC was necessary for both transfer and viability support. UCB-MNC tightly adherent to the MSC layer exhibited the most optimal transfer and rescue of cell viability. DNA analysis of the viable, GFP transfer-positive UCB-MNC ruled out MSC transdifferentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB-MNC viability (P < 0.0001). CONCLUSIONS: Collectively, the data suggest that intercellular transfer of cytosolic materials could be one novel mechanism for preventing UCB cell death in MSC co-culture.

Lyophilization of cholesterol-free PEGylated liposomes and its impact on drug loading by passive equilibration.

The obstacles in translating liposome formulations into marketable products could be attributed to their physical instabilities upon long-term storage as aqueous dispersions. Lyophilization is the most commonly used technique to improve physical stability of liposomes. The development of stable, lyophilized liposomes is focused primarily on the cholesterol-containing liposomes or pure phosphatidylcholine-based liposomes, with minimal studies on cholesterol-free, pegylated (CF-PEG) liposomes which have emerged as an important class of liposome drug carriers. Hence, it is our interest to investigate the effect of lyophilization on CF-PEG liposomes, and specifically, on drug loading via the passive equilibration method. Three different sugar cryoprotectants were used at two different sugar-to-lipid molar ratios (S/L). Our results demonstrated that CF-PEG liposomes lyophilized with sucrose at S/L=5:1 yielded the best cryoprotective effect, as characterized by size, polydispersity indices, and microscopic examination upon liposome reconstitution. The lyophilized liposomes had low water content of 2.59 ± 0.18%. Of note, lyophilized CF-PEG liposomes exhibited two-fold increase in drug content when carboplatin was loaded via the passive equilibration method, and the in vitro drug release profile of these liposomes were not different from that of the non-lyophilized counterparts. Taken together, we envisioned that a stable, lyophilized empty CF-PEG liposome system could be coupled to hydrophilic drug loading via the passive equilibration method to produce a liposomal drug kit product.

Potent therapeutic activity of folate receptor-targeted liposomal carboplatin in the localized treatment of intraperitoneally grown human ovarian tumor xenograft.

Intraperitoneal (IP) therapy with platinum (Pt)-based drugs has shown promising results clinically; however, high locoregional concentration of the drug could lead to adverse side effects. In this study, IP administration was coupled with a folate receptor-targeted (FRT) liposomal system, in an attempt to achieve intracellular delivery of the Pt-based drug carboplatin in order to increase therapeutic efficacy and to minimize toxicity. In vitro and in vivo activity of FRT carboplatin liposomes was compared with the activity of free drug and nontargeted (NT) carboplatin liposomes using FR-overexpressing IGROV-1 ovarian cancer cells as the model. Significant reduction in cell viability was observed with FRT liposomes, which, compared with the free drug, provided an approximately twofold increase in carboplatin potency. The increase in drug potency was correlated with significantly higher cellular accumulation of Pt resulting from FRT liposomal delivery. Further evaluation was conducted in mice bearing intraperitoneally inoculated IGROV-1 ovarian tumor xenografts. A superior survival rate (five out of six animals) was achieved in animals treated with FRT carboplatin liposomes, injected intraperitoneally with a dose of 15 mg/kg and following a schedule of twice-weekly administration for 3 weeks. In contrast, no survivors were observed in the free drug or NT carboplatin liposome groups. The presence of cancer cells in lung and liver tissues was observed in the saline, free carboplatin, and NT carboplatin liposome groups. However, there was no sign of cancer cells or drug-related toxicity detected in tissues from the animals treated with FRT carboplatin liposomes. The results of this study have demonstrated for the first time that the approach of coupling IP administration with FRT liposomal delivery could provide significantly improved therapeutic efficacy of carboplatin in the treatment of metastatic ovarian cancer.

Perorally active nanomicellar formulation of quercetin in the treatment of lung cancer.

BACKGROUND: Realizing the therapeutic benefits of quercetin is mostly hampered by its low water solubility and poor absorption. In light of the advantages of nanovehicles in the delivery of flavanoids, we aimed to deliver quercetin perorally with nanomicelles made from the diblock copolymer, polyethylene glycol (PEG)-derivatized phosphatidylethanolamine (PE). METHODS: Quercetin-loaded nanomicelles were prepared by using the film casting method, and were evaluated in terms of drug incorporation efficiency, micelle size, interaction with Caco-2 cells, and anticancer activity in the A549 lung cancer cell line and murine xenograft model. RESULTS: The incorporation efficiency into the nanomicelles was ≥88.9% when the content of quercetin was up to 4% w/w, with sizes of 15.4-18.5 nm and polydispersity indices of <0.250. Solubilization of quercetin by the nanomicelles increased its aqueous concentration by 110-fold. The quercetin nanomicelles were stable when tested in simulated gastric (pH 1.2) and intestinal (pH 7.4) fluids, and were non-toxic to the Caco-2 cells as reflected by reversible reduction in transepithelial electrical resistance and ≤25% lactose dehydrogenase release. The anticancer activity of quercetin could be significantly improved over the free drug through the nanomicellar formulation when tested using the A549 cancer cell line and murine xenograft model. The nanomicellar quercetin formulation was well tolerated by the tumor-bearing animals, with no significant weight loss observed at the end of the 10-week study period. CONCLUSION: A stable PEG-PE nanomicellar formulation of quercetin was developed with enhanced peroral anticancer activity and no apparent toxicity to the intestinal epithelium.

Increased ERK activation and cellular drug accumulation in the enhanced cytotoxicity of folate receptor-targeted liposomal carboplatin.

Folate receptor-targeted (FRT) liposomes for carboplatin were developed and evaluated in FR-positive and FR-negative cell lines, KB and A549, respectively, for their cytotoxic effects. Significant enhancement in carboplatin potency and intracellular drug accumulation was observed in KB cells when treated with FRT liposomes, compared to free drug and non-targeted liposomes. No enhancement was observed in the FR-negative A549 cells. The increase in carboplatin potency was hypothesized to be associated with an increase in the formation of DNA-platinum adducts resulted from an increase in cellular accumulation of the drug. Surprisingly, FRT carboplatin liposomes showed significantly lower levels of DNA-platinum adducts in comparison to free drug. To elucidate this discrepancy, activation of extracellular signal-regulated protein kinase (ERK) was probed, which has been suggested as an alternative mechanism of carboplatin action. FRT liposomes loaded with carboplatin exhibited the highest level of ERK phosphorylation, and the cytotoxic effect of FRT carboplatin liposomes could be reversed by the MEK/ERK inhibitors, U0126 and PD98059. Importantly, empty FRT liposomes could significantly increase ERK phosphorylation in a concentration-dependent manner without causing toxicity to cells. For the first time, increased potency of carboplatin delivered by FRT liposomes was found to be associated with other molecular targets in addition to DNA-platinum adduct formation. Collectively, the current study suggests a novel mechanism by which FRT liposomes could sensitize cancer cells to drug treatment via modulation of ERK-related cell survival signals.