Synthesis of a New pH-Sensitive Folate–Doxorubicin Conjugate and its Antitumor Activity In Vitro

Folate–aminocaproic acid–doxorubicin (FA–AMA–DOX) was synthesized and characterized by H NMR spectroscopy and mass spectrometry. Cytotoxicity and cellular uptake experiments were performed in KB and HepG2 cells, which express folic acid receptor, and the cell line A549, which does not express folic acid receptor. Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cellular uptake was monitored using fluorescence microscopy. The amount of DOX released from FA–AMA–DOX was much greater at pH 5.0 than that at pH 6.5 or 7.4. The cytotoxicity of FA–AMA–DOX toward KB and HepG2 cells was greater than that of DOX or AMA–DOX at the same concentrations, and cytotoxicity could be attenuated by FA in a dose-dependent manner. On the contrary, the cytotoxicity of FA–AMA–DOX and AMA–DOX toward A549 cells was lower than that of DOX at the same concentration, and cytotoxicity could not be reduced by FA. Compared with FA–AMA, FA–AMA–DOX increased the intracellular accumulation of DOX in KB cells. These results suggested that FA–AMA–DOX have suitable attributes for the active targeting of folate-receptor-positive tumor cells and for releasing the chemotherapeutic agent, DOX, in situ; it therefore has potential as a novel cancer therapeutic. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:530–540, 2013     

Synthesis and evaluation of a novel ligand for folate-mediated targeting liposomes

Folate receptors (FRs) have been identified as cellular surface markers for cancer and leukemia. Liposomes containing lipophilic derivatives of folate have been shown to effectively target FR-expressing cells. Here, we report the synthesis of a novel lipophilic folate derivative, folate-polyethylene glycol-cholesterol hemisuccinate (F-PEG-CHEMS), and its evaluation as a targeting ligand for liposomal doxorubicin (L-DOX) in FR-expressing cells. Liposomes containing F-PEG-CHEMS, with a mean diameter of 120+/-20 nm, were synthesized by polycarbonate membrane extrusion and were shown to have excellent colloidal stability. The liposomes were taken up selectively by KB cells, which overexpress FR-alpha. Compared to folate-PEG-cholesterol (F-PEG-Chol), which contains a carbamate linkage, F-PEG-CHEMS better retained its FR-targeting activity during prolonged storage. In addition, F-PEG-CHEMS containing liposomes loaded with DOX (F-L-DOX) showed greater cytotoxicity (IC(50)=10.0muM) than non-targeted control L-DOX (IC(50)=57.5 microM) in KB cells. In ICR mice, both targeted and non-targeted liposomes exhibited long circulation properties, although F-L-DOX (t(1/2)=12.34 h) showed more rapid plasma clearance than L-DOX (t(1/2)=17.10h). These results suggest that F-PEG-CHEMS is effective as a novel ligand for the synthesis of FR-targeted liposomes.     

Novel,nano-sized,liposome-encapsulated polyamidoamine dendrimer derivatives facilitate tumour targeting by overcoming the polyethylene glycol dilemma and integrin saturation obstacle

Drug delivery systems (DDSs) commonly employ arginine–glycine–aspartic acid (RGD) peptides with polyethylene glycol (PEG)-dependent enhanced permeability and retention (EPR) effect to optimise tumour-targeting. However, the PEG dilemma and integrin saturation obstacle are major challenges. To address these issues, we constructed a novel, nano-sized DDS by encapsulating doxorubicin (DOX)-loaded folic acid derivatives of polyamidoamine dendrimer (PAMAM G5.0) in cyclic RGD-tyrosine-lysine pentapeptide (c[RGDyK])-modified liposomes (RGD-SL[FND/DOX]), prepared using thin-film hydration, film-dispersion and hydration-sonication. The liposomes were PEGylated, sterically stabilised and pH-sensitive. In vitro, RGD-SL[FND/DOX] showed pH-sensitive holistic FND/DOX release, and pH-dependent uptake and cytotoxicity in human cancer KB cells. At pH 7.4, RGD-SL[FND/DOX] demonstrated greater cellular uptake and cytotoxicity than relevant control formulations (except FND/DOX) did, although this advantage disappeared at pH 6.5. In vivo, RGD-SL[FND/DOX] inhibited S180 sarcoma xenografted tumour growth in Kunming mice more effectively than FND/DOX did. These findings demonstrate the feasibility of constructing double-stage tumour-targeting nano-sized DDSs such as RGD-SL[FND/DOX]. c[RGDyK] and the EPR effect, facilitated by the particle size (about 110?nm) and PEGylation, helped to target the DDS to the tumour tissue, while the subsequent pH-dependent release of FND/DOX and folic acid-mediated endocytosis specifically targeted the tumour cells, thereby overcoming the PEG dilemma and integrin saturation obstacle.     

Targeting of folate-conjugated liposomes with co-entrapped drugs to prostate cancer cells via prostate-specific membrane antigen (PSMA)

Folate-targeted liposomes (FTL) were tested as drug delivery vehicles to PSMA-positive cancer cells. We used FL with co-entrapped mitomycin C lipophilic prodrug (MLP) and doxorubicin (DOX), and the LNCaP prostate cancer cell line which expresses PSMA but is negative for folate receptor. A major increase in cell drug levels was observed when LNCaP cells were incubated with FTL as compared to non-targeted liposomes (NTL). MLP was activated to mitomycin C, and intracellular and nuclear fluorescence of DOX was detected, indicating FTL processing and drug bioavailability. PMPA (2-(phosphonomethyl)-pentanedioic acid), a specific inhibitor of PSMA, blocked the uptake of FTL into LNCaP cells, but did not affect the uptake of FTL into PSMA-deficient and folate receptor-positive KB cells. The cytotoxic activity of drug-loaded FTL was found significantly enhanced when compared to NTL in LNCaP cells. FTL may provide a new tool for targeted therapy of cancers that over-express the PSMA receptor.     

Design and development of folate appended liposomes for enhanced delivery of 5-FU to tumor cells

Folate appended sterically-stabilized liposomes (FA-SL) were investigated for tumor targeting. Liposomes were prepared using HSPC, cholesterol and FA-polyethylene glycol (PEG)-SA. The liposomes with polyethylene glycol (PEG) without folic acid which has similar lipid composition were used for comparison. Liposomal preparations were characterized for shape, size and percent entrapment. The average size of liposomes was found to be in range 124-163 nm and maximum drug entrapment was found to be 34.2-40.3%. In vitro drug release from the formulations is obeying fickian release kinetics. Cellular uptake and IC(50) values of the FR-targeted formulation were determined in vitro in FR (+) B16F10 melanoma cells. In vitro cell binding of FA-SL exhibits 11-folds higher binding to B16F10 melanoma cells in comparison to SL. In vivo cytotoxicy assay on FR targeted liposomes gave IC(50) of 1.87 microM and non-targeted liposomes gave IC(50) of 4.02 microM. In therapeutic experiments 5-fluorouracil (5-FU), SL and FA-SL were administered at the dose of 10 mg 5-FU/kg body weight to B16F10 tumor bearing Balb/c mice. Administration of FA-SL formulation results in effective reduction in tumor growth as compared with free 5-FU and SL. Results indicate that folic acid appended SL bearing 5-FU are significantly (P < 0.01) active against primary tumor and metastasis than non-targeted sterically-SL. Thus, it could be concluded that folate coupled liposomal formulations enhanced drug uptake by tumor cells.     

Estrogen-anchored pH-sensitive liposomes as nanomodule designed for site-specific delivery of doxorubicin in breast cancer therapy

The present investigation reports the development of nanoengineered estrogen receptor (ER) targeted pH-sensitive liposome for the site-specific intracellular delivery of doxorubicin (DOX) for breast cancer therapy. Estrone, a bioligand, was anchored on the surface of pH-sensitive liposome for drug targeting to ERs. The estrone-anchored pH-sensitive liposomes (ES-pH-sensitive-SL) showed fusogenic potential at acidic pH (5.5). In vitro cytotoxicity studies carried out on ER-positive MCF-7 breast carcinoma cells revealed that ES-pH-sensitive-SL formulation was more cytotoxic than non-pH-sensitive targeted liposomes (ES-SL). The flow cytometry analysis confirmed significant enhanced uptake (p < 0.05) of ES-pH-sensitive-SL by MCF-7 cells. Intracellular delivery and nuclear localization of the DOX was confirmed by fluorescence microscopy. The mechanism for higher cytotoxicity shown by estrone-anchored pH-sensitive liposomal-DOX was elucidated using reactive oxygen species (ROS) determination. The in vivo biodistribution studies and antitumor activities of formulations were evaluated on tumor bearing female Balb/c mice followed by intravenous administration. The ES-pH-sensitive-SL efficiently suppressed the breast tumor growth in comparison to both ES-SL and free DOX. Serum enzyme activities such as LDH and CPK levels were assayed for the evaluation of DOX induced cardiotoxicity. The ES-pH-sensitive-SL accelerated the intracellular trafficking of encapsulated DOX, thus increasing the therapeutic efficacy. The findings support that estrone-anchored pH-sensitive liposomes could be one of the promising nanocarriers for the targeted intracellular delivery of anticancer agents to breast cancer with reduced systemic side effects.     

Synthesis of transferrin (Tf) conjugated liposomes via Staudinger ligation

Staudinger ligation was evaluated as a strategy for synthesizing receptor targeted liposomes. First, an activated lipid derivative was synthesized by reacting dioleoyl phosphatidylethanolamine (DOPE) and 2-(diphenylphosphino) terephthalic acid 1-methyl 4-penta-fluorophenyldiester. Second, transferrin (Tf) was activated with p-azidophenyl isothiocyanate. Third, liposomes containing the activated lipid were prepared and then coupled to the activated Tf via the Staudinger reaction. These liposomes were evaluated in KB cells for cellular uptake and cytotoxicity, and in mice for pharmacokinetic properties. Tf-derivatized liposomes encapsulating calcein prepared by this conjugation method effectively targeted Tf receptor expressing KB cells. In addition, the Tf-targeted liposomes entrapping doxorubicin showed greatly enhanced in vitro cytotoxicity relative to non-targeted control liposomes. Pharmacokinetic parameters indicated that these liposomes retained long circulating properties relative to the free drug. In summary, Staudinger ligation is an effective method for the synthesis of receptor targeted liposomes.     

Reversal of multidrug resistance by transferrin-conjugated liposomes co-encapsulating doxorubicin and verapamil.

PURPOSE: Liposomes co-encapsulating doxorubicin (DOX) and verapamil (VER), and conjugated to transferrin (Tf-L-DOX/VER) were synthesized and evaluated in K562 leukemia cells. The design of this formulation was aimed at selective targeting of tumor cells, reducing cardiotoxicity of DOX and VER, as well as overcoming P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) phenotype. METHODS: The liposomes were prepared by polycarbonate membrane extrusion, followed by pH-gradient driven remote loading and Tf conjugation. Kinetics of in vitro release of DOX and VER from liposomes was determined by measuring changes in the concentration of encapsulated drugs. Uptake of Tf-conjugated liposomes by K562 cells was evaluated by fluorescence microscopy and by fluorometry. Cytotoxicities of various formulations of DOX were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide (MTT) assay. RESULTS: Efficiencies for liposomal loading of DOX and VER were 95% and 70%, respectively. The mean particle diameter for the liposomes was approximately 110nm. Rates of release for DOX and VER were similar in singly-loaded and co-loaded liposomes. Tf-L-DOX/VER showed efficient uptake by the TfR+ K562 cells. In DOX-resistant K562 cells (K562/DOX), Tf-L-DOX/VER showed 5.2 and 2.8 times greater cytotoxicity (IC50 = 4.18 muM) than non-targeted liposomes (L-DOX/VER) (IC50 = 21.7 muM) and Tf-targeted liposomes loaded with DOX alone (Tf-L-DOX) (IC50 = 11.5 muM), respectively. CONCLUSIONS: The combination of TfR targeting and co-encapsulation of DOX and VER was highly effective in overcoming drug resistance in K562 leukemia cells.     

Enhanced tumor-targeted delivery of anticancer drugs by folic acid-conjugated pH-sensitive polymeric micelles

In order to enhance the targeted delivery of anticancer drugs by polymeric micelles, folic acid (FA), the ligand of folate receptor (FR) over-expressed in the most cancer cells, modified pH-sensitive polymeric micelles were designed and fabricated to encapsulate doxorubicin (DOX) by combination of pH-sensitive amphiphilic polymer poly(2-ethyl-2-oxazoline)-poly(D,L-lactide) with FA-conjugated poly(2-ethyl-2-oxazoline)-poly(D,L-lactide). The prepared micelles were characterized to have about 36 nm in diameter with narrow distribution, well-defined spherical shape observed under TEM and pH-responsive drug release behavior. Moreover, the tumor targeting ability of the FA-modified pH-sensitive polymeric micelles was demonstrated by the cellular uptake, in vitro cytotoxicity to FR-positive KB cells and in vivo real time near-infrared fluorescence imaging in KB tumor-bearing nude mice. The efficient drug delivery by the micelles was ascribed to the synergistic effects of FR-mediated targeting and pH-triggered drug release. In conclusion, the designed FR-targeted pH-sensitive polymeric micelles might be of great potential in tumor targeted delivery of water-insoluble anticancer drugs.     

Cytotoxicity of doxorubicin‐loaded Con A‐liposomes

The present study investigated the potential of Concanavalin A lectin (Con A) conjugated to liposomes (Con A‐liposomes) for targeting doxorubicin (DOX) to cells. The physicochemical properties and the cytotoxicity of DOX‐loaded Con A‐liposomes were evaluated. DOX‐loaded Con A‐liposomes were prepared by incubation of DOX‐loaded liposomes with a Con A‐SATA derivative. Lectin biological activity was monitored before and after conjugation by a hemagglutinating assay. The cytotoxicity of DOX‐loaded Con A‐liposomes was evaluated in terms of the inhibition of NCI‐H299 and HEp‐2 cell proliferation using the MTT method. The affinity of lectinized liposomes with these cells was thus assessed by evaluating the cytotoxic effect of the DOX released into cells. Stable DOX‐loaded Con A‐liposomes were obtained and their high affinity for cells was corroborated. The encapsulation of DOX into Con A‐liposomes produced an inhibition of roughly 70% of Hep‐2 cell proliferation and 50% of cell inhibition was verified on HCI‐H292. DOX in solution was able to inhibit only 20% of cell proliferation for both cell lines. Unloaded Con A‐liposomes were not cytotoxic. The encapsulation of DOX into Con A‐liposomes improves drug penetration into cells, thereby enhancing its cytotoxicity, especially in Hep‐2 cells. Drug Dev. Res. 67:430–437, 2006. © 2006 Wiley‐Liss, Inc.     

Selectivity of folate conjugated polymer micelles against different tumor cells

Folate or folic acid has been employed as a targeting moiety of various anticancer agents to increase their cellular uptake within target cells since folate receptors are vastly overexpressed in several human tumors. In this study, a biodegradable polymer poly(d,l-lactide-co-glycolide)-poly(ethylene glycol)-folate (PLGA-PEG-FOL) was used to form micelles for encapsulating anticancer drug doxorubicin (DOX). The drug loading content, encapsulation efficiency and in vitro release were characterized. To evaluate the targeting ability of the folate conjugated micelles, the cytotoxicity and cellular uptake of DOX-loaded micelles on three cancer cell lines with different amount of folate receptors (KB, MATB III, C6) and normal fibroblast cells (CCL-110) were compared. The cytotoxicity of PLGA-PEG-FOL micelles to cancer cells was found to be much higher than that of normal fibroblast cells, demonstrating that the folate conjugated micelles has the ability to selectively target to cancer cells. For normal cells, the cellular uptake of PLGA-PEG-FOL micelles was similar to PLGA-PEG micelles without folate conjugation, and was substantially lower than that of cancer cells. In addition, the cell cycle analysis showed that the apoptotic percentage of normal fibroblasts was substantially lower compared with the cancer cells after exposing to DOX-loaded PLGA-PEG-FOL micelles. An optimal folate amount of approximately 40-65% on the micelles was found to be able to kill cancer cells but, at the same time, to have very low effect to normal cells.     

Vascular targeting of doxorubicin using cationic liposomes

Tumor vessel has been recognized as an important target for anticancer therapy. Cationic liposomes have been shown to selectively target tumor endothelial cells, thus can potentially be used as a carrier for chemotherapy agents. In this study, cationic liposomes containing 20 mol% cationic lipid dimethyl dioctadecyl ammonium bromide (DDAB) and loaded with doxorubicin (DOX) were prepared and characterized. The cationic liposomal DOX showed 10.8 and 9.1 times greater cytotoxicity than control PEGylated liposomal DOX in KB oral carcinoma and L1210 murine lymphocytic leukemia cells, and 7.7- and 6.8-fold greater cytotoxicity compared to control neutral non-PEGylated liposomal DOX, repectively, in these two cell lines. Although cationic liposomal DOX had higher tumor accumulation at 30 min after intravenous administration compared to control liposomes (p<0.05), DOX uptake of these liposomes at 24h post-injection was similar to that of PEGylated liposomal DOX (p>0.05) and approximately twice the levels of the free drug and non-PEGylated liposomes. In a murine tumor model generated using L1210 cells, increased survival rate was obtained with cationic liposomal DOX treatment compared to free DOX (p<0.01), neutral liposome control (p<0.01), as well as PEGylated liposomes (p<0.05). In conclusion, the cationic liposomal DOX formulation produced superior in vitro cytotoxicity and in vivo antitumor activity, and warrants further investigation.     

Engineering a folic acid-decorated ultrasmall gemcitabine nanocarrier for breast cancer therapy: Dual targeting of tumor cells and tumor-associated macrophages

Combination of passive targeting with active targeting is a promising approach to improve the therapeutic efficacy of nanotherapy. However, most reported polymeric systems have sizes above 100 nm, which limits effective extravasation into tumors that are poorly vascularized and have dense stroma. This will, in turn, limit the overall effectiveness of the subsequent uptake by tumor cells via active targeting. In this study, we combined the passive targeting via ultra-small-sized gemcitabine (GEM)-based nanoparticles (NPs) with the active targeting provided by folic acid (FA) conjugation for enhanced dual targeted delivery to tumor cells and tumor-associated macrophages (TAMs). We developed an FA-modified prodrug carrier based on GEM (PGEM) to load doxorubicin (DOX), for co-delivery of GEM and DOX to tumors. The co-delivery system showed small particle size of ～10 nm in diameter. The ligand-free and FA-targeted micelles showed comparable drug loading efficiency and a sustained DOX release profile. The FA-conjugated micelles effectively increased DOX uptake in cultured KB cancer cells that express a high level of folate receptor (FR), but no obvious increase was observed in 4T1.2 breast cancer cells that have a low-level expression of FR. Interestingly, in vivo, systemic delivery of FA-PGEM/DOX led to enhanced accumulation of the NPs in tumor and drastic reduction of tumor growth in a murine 4T1.2 breast cancer model. Mechanistic study showed that 4T1.2 tumor grown in mice expressed a significantly higher level of FOLR2, which was selectively expressed on TAMs. Thus, targeting of TAM may also contribute to the improved in vivo targeted delivery and therapeutic efficacy.     

Targeted Sialic Acid–Doxorubicin Prodrugs for Intracellular Delivery and Cancer Treatment

Purpose To evaluate a novel targeted anticancer prodrug consisting of several copies of sialic acid (SA, targeting moiety), doxorubicin (DOX), citric acid (multifunctional spacer) and poly(ethylene glycol) (PEG, carrier). Methods α, ω bis carboxyl PEG was covalently conjugated with multiple copies of SA and DOX through a citric acid spacer and characterized by proton nuclear magnetic resonance (¹HNMR), matrix-assisted laser desorption/ionization-time of flight (MALDI/TOF), and high-performance liquid chromatography (HPLC). The molecular models of conjugates were established using ChemDraw software. Stability, spontaneous and esterase-stimulated drug release was analyzed by HPLC. Cellular internalization (fluorescence microscopy) and cytotoxicity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay] of free DOX and prodrugs were evaluated. Results ¹HNMR, MALDI/TOF, and HPLC showed the formation of the PEG prodrug conjugates. More than 40% of the drug was released from its conjugate in the presence of esterase enzyme, whereas the conjugate was stable at pH 7.4 in the absence of enzyme. Molecular modeling studies showed stable conformations of conjugates. The targeted prodrug conjugates with two copies of SA and DOX showed enhanced cytotoxicity when compared with non-targeted prodrugs and free DOX. Conclusions Targeting of the conjugate to cancer cells by SA with increased copies of targeting moiety and anticancer drug enhanced prodrug uptake by cancer cells and cytotoxicity of the prodrug.     

Disaccharide-modified liposomes and their in vitro intracellular uptake

Sterically stabilized liposomes (SSL) were known to be accumulated passively in cancer due to the effect of enhanced permeability and retention (EPR). However, drug delivery via SSL to cancer seemed to show an insufficient improvement of chemotherapeutic efficacy. Herein, carbohydrate-binding proteins (lectins) of cell surface, which express on the plasmic membrane of many malignant cells, can be a good model of surface-modified liposomes. In this study, we investigated the in vitro characteristics of liposomes of which the surface was modified with a disaccharide molecule, sucrose or maltose. The disaccharide-modified lipids such as sucrose-modified lipid and maltose-modified lipid, in which the disaccharide was conjugated to the one end of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol)-2000 (DSPE-PEG2000), was synthesized. The disaccharide-modified liposomes were prepared by thin film-hydration method and then doxorubicin (DOX), an anticancer drug, was loaded to the prepared liposomes by the remote loading method with ammonium ion gradient. Flow cytometry and confocal microscopy analyses showed that the disaccharide-modified liposomes enhanced the intracellular uptake of liposomes into various cancer cell lines via lectin-mediated endocytosis. The disaccharide-modified liposomes in which DOX was loaded inside of liposomes exhibited higher cytotoxicity against various cancer cells than DOX-loaded SSL did. These results suggest that disaccharide-modified liposomes may be promising cancer targeting carriers which can enhance intracellular uptake and cytotoxicity of the drug-loaded liposomes via lectin-mediated endocytosis.     

Peptide-directed HPMA copolymer-doxorubicin conjugates as targeted therapeutics for colorectal cancer

Synthetic oligopeptides have emerged as a promising class of targeting ligands, providing a variety of choices for the construction of conjugates for desired ligand functionality. To explore the potential of short peptides as ligands for targeted delivery of macromolecular therapeutics for colorectal cancer (CRC), fluorescently labelled HPMA copolymers—bearing either G3-C12 or GE11 for targeting galectin-3 and epidermal growth factor receptor (EGFR), respectively—were synthesised and the mechanisms of their internalisation and subcellular fate in CRC cells were studied. The targetability of the G3-C12 bearing copolymers towards galectin-3 was further compared to that of galactose-containing copolymers. The resulting G3-C12-bearing conjugate actively and selectively targets CRC tumour cells over-expressing galectin-3 and exhibits superior targetability to galectin-3 when compared to the galactose-bearing copolymer. GE11 copolymer conjugate binds specifically and efficiently to EGFR over-expressing cells, thus mediating internalisation to a significantly higher extent relative the copolymer conjugated to a scrambled sequence peptide. We further incorporated doxorubicin (DOX) into GE11 bearing copolymer via an acid-labile hydrazone bond. The GE11-DOX copolymer conjugate demonstrated higher cytotoxicity toward EGFR over-expressing cells relative to the control non-targeted DOX conjugate. Altogether, our results show a proof of principle for the selective delivery of DOX to the target CRC cells.     

Development,characterization and cancer targeting potential of surface engineered carbon nanotubes

Abstract

The aim of the present study was to assess the in vitro and in vivo potential of doxorubicin-loaded, folic acid appended engineered multi-walled carbon nanotubes (DOX/FA-PEG-MWCNTs) for efficient tumor targeting. The loading efficiency was determined to be 92.0?±?0.92 (DOX/FA-PEG-MWCNTs) in phosphate buffer solution (pH 7.4) ascribed to π–π stacking interaction. The developed nanoconjugates were evaluated for in vitro DOX release, erythrocytes toxicity, ex vivo cytotoxicity and cell uptake studies on MCF-7 (breast cancer cell line). The DOX/FA-PEG-MWCNTs nanoconjugate affords higher efficacy in tumor growth suppression due to its stealth nature and most preferentially taken up by the cultured MCF-7 through caveolae-mediated endocytosis as compared to free DOX. The in vivo studies were performed to determine the pharmacokinetics, biodistribution and antitumor efficacy on tumor bearing female Sprague Dawley rats and improved pharmacokinetics confirm the function of FA-PEG conjugated CNTs. The median survival time for tumor bearing rats treated with DOX/FA-PEG-MWCNTs (30?d) was extended very significantly as compared to free DOX (p?<?0.001). The results concluded that developed water-soluble nano-conjugates might emerge as “safe and effective” nano-medicine in cancer treatment by minimizing the side effects with and Generally Regarded as Safe prominence.     

Landscape phage fusion protein-mediated targeting of nanomedicines enhances their prostate tumor cell association and cytotoxic efficiency

Tumor-specific cytotoxicity of drugs can be enhanced by targeting them to tumor receptors using tumor-specific ligands. Phage display offers a high-throughput approach to screen for the targeting ligands. We have successfully isolated phage fusion peptides selective and specific for PC3 prostate cancer cells. Also, we have demonstrated a novel approach of targeting liposomes through tumor-specific phage fusion coat proteins, exploiting the intrinsic properties of the phage coat protein as an integral membrane protein. Here we describe the production of Rhodamine-labeled liposomes as well as doxorubicin-loaded long-circulating liposomes targeted to PC3 prostate tumor cells via PC-specific phage peptides, as an extension of our previous studies. Targeting of labeled liposomes was demonstrated using fluorescence microscopy as well as flow cytometry. Targeting of doxorubicin-loaded liposomes enhanced their cytotoxic effect against PC3 cells in vitro, indicating a possible therapeutic advantage. The simplicity of the approach for generating targeted liposomes coupled with the ability to rapidly obtain tumor-specific phage fusion proteins via phage display may contribute to a combinatorial system for the production of targeted liposomal therapeutics for advanced stages of prostate tumor.From the Clinical EditorThis paper demonstrates targeting cytotoxic agents to tumor receptors using tumor-specific ligands. The authors describe the production of Rhodamine-labeled liposomes as well as doxorubicin loaded long circulating liposomes targeted to PC3 prostate tumor cells via PC-specific phage peptides. This approach may be especially relevant for advanced prostate tumors.     

Capillary electrophoresis monitors enhancement in subcellular reactive oxygen species production upon treatment with doxorubicin

This study investigated the role of doxorubicin (DOX) accumulation in reactive oxygen species (ROS) production detected in individually electrophoresed organelles, including mitochondria, acidic organelles, and peroxisomes. While bulk measurements of ROS production in cells and organelles are not capable of discriminating between the effects of preparative procedures on measured ROS production, capillary electrophoresis with dual laser-induced detection of individual organelles demonstrated a difference in the measured ROS production as a result of various preparative procedures. Using this technique, the three different types of detected organelles (i) produce ROS and do not have detectable levels of DOX, (ii) contain detectable DOX but do not produce ROS, or (iii) produce ROS and accumulate DOX. The third type displays two subpopulations of organelles, one of which demonstrated a direct relationship between DOX uptake and subsequent ROS production, corresponding most likely to mitochondria, and a second one with low DOX uptake but large variation in ROS production, corresponding most likely to acidic organelles.     

Reversal activity of nanostructured lipid carriers loading cytotoxic drug in multi-drug resistant cancer cells

To overcome multi-drug resistance (MDR) of cancer cells, paclitaxel (PTX) and doxorubicin (DOX)-loaded nanostructured lipid carriers (NLC) were prepared by solvent diffusion method using monostearin as solid lipid and oleic acid as liquid lipid matrix. The cytotoxicities and reversal activity of drug-loaded NLC were tested against human breast cancer (MCF-7) cells, human ovarian cancer (SKOV3) cells and their multi-drug resistant (MCF-7/ADR and SKOV3-TR30) cells. The chemical conjugant of folic acid and stearic acid (FA-SA) was further synthesized to prepare folated NLC. Comparing with taxol and doxorubicin solution, the NLC loading PTX exhibited high cytotoxicities in MCF-7 and MCF-7/ADR cells, while the NLC loading DOX only indicated high cytotoxicity in MCF-7/ADR cells. The reversal powers of the NLC loading PTX and DOX were 34.3 and 6.4 folds, respectively. The NLC loading PTX and DOX showed the same trends of enhanced cytotoxicity against SKOV3 and SKOV3-TR30 cells. The reversal powers were 31.3 and 2.2 folds for the NLC loading PTX and DOX, respectively. The modification of NLC with FA-SA could further enhance the cytotoxicities of drug in drug sensitive and drug resistant cells.