PEG-doxorubicin conjugates: influence of polymer structure on drug release, in vitro cytotoxicity, biodistribution, and antitumor activity

Polymer-drug conjugates (polymer therapeutics) are finding increasing use as novel anticancer agents. Here a series of poly(ethylene glycol) PEG-doxorubicin (Dox) conjugates were synthesized using polymers of linear or branched architecture (molecular weight 5000-20000 g/mol) and with different peptidyl linkers (GFLG, GLFG, GLG, GGRR, and RGLG). The resultant conjugates had a drug loading of 2.7-8.0 wt % Dox and contained <2.0% free drug (% total drug). All conjugates containing a GFLG linker showed approximately 30% release of Dox at 5 h irrespective of PEG molecular weight or architecture. The GLFG linker was degraded more quickly (approximately 57% Dox release at 5 h), and the other linkers more slowly (<16% release at 5 h), by lysosomal enzymes in vitro. In vitro there was no clear relationship between cytotoxicity toward B16F10 cells and the observed Dox release rate. All PEG conjugates were more than 10-fold less toxic (IC50 values > 2 microg/mL) than free Dox (IC50 value = 0.24 microg/mL). Biodistribution in mice bearing sc B16F10 tumors was assessed after administration of PEGs (5000, 10000, or 20000 g/mol) radioiodinated using the Bolton and Hunter reagent or PEG-Dox conjugates by HPLC. The 125I-labeled PEGs showed a clear relationship between Mw and blood clearance and tumor accumulation. The highest Mw PEG had the longest plasma residence time and consequently the greatest tumor targeting. The PEG-Dox conjugates showed a markedly prolonged plasma clearance and greater tumor targeting compared to free Dox, but there was no clear molecular weight-dependence on biodistribution. This was consistent with the observation that the PEG-Dox conjugates formed micelles in aqueous solution comprising 2-20 PEG-Dox molecules depending on polymer Mw and architecture. Although PEG-Dox showed greater tumor targeting than free Dox, PEG conjugation led to significantly lower anthracycline levels in heart. Preliminary experiments to assess antitumor activity against sc B16F10 in vivo showed the PEG5000linear (L)-GFLG-Dox and PEG10000branched (B)-GLFG-Dox (both 5 mg/kg Dox-equiv) to be the most active with T/C values of 146 and 143%, respectively. Free Dox did not show significant activity in this model (T/C = 121%). Dose escalation of PEG5000(L)-GFLG-Dox to 10 mg/kg Dox-equiv prolonged further animal survival (T/C = 161%). Using the Dox-sensitive model ip L1210 (where Dox displayed a T/C = 150% after single ip dose), the PEG5000(L)-GFLG-Dox displayed a maximum T/C of 141% (10 mg/kg Dox-equiv) using a once a day (x3) schedule. Further studies are warranted with PEG5000(L)-GFLG-Dox to determine its spectrum of antitumor activity and also the optimum dosing schedule before clinical testing.     

Analysis of lymphocyte-target conjugates by flow cytometry. I. Discrimination between killer and non-killer lymphocytes bound to targets and sorting of conjugates containing one or multiple lymphocytes

The use of flow cytometric analysis and sorting techniques for the enumeration and purification of lymphocyte-target conjugates was investigated. Murine cytotoxic T-lymphocytes (CTL) with killer effector function were identified and quantitated during a 3-hour cell-mediated cytotoxicity reaction using multiparameter analysis. Resolution of conjugates containing single and multiple lymphocytes was achieved by two-color fluorescence, and individual conjugate subpopulations were subsequently sorted for further analysis. To measure total and cytotoxic conjugate frequencies, CTL were labelled with FITC-conjugated Thy 1.2 antibody and dead target cells were stained with propidium iodide (PI). Size difference between the CTL and P815 tumor target cells, as measured by Coulter volume and axial light loss, facilitated detection of conjugates which were identified as both large and Thy 1.2-positive. Conjugates containing dead target cells possessed red fluorescence due to PI uptake. The frequency of conjugates containing cytotoxic activity increased with time during the cytotoxicity period and correlated with frequencies obtained in single-cell assays. Analysis of the distribution of single and multiple lymphocyte-bound conjugates was done by co-centrifugation of Hoechst-stained CTL and FITC-labeled P815 target cells. Analysis by two-color fluorescence effectively resolved conjugate populations containing different numbers of CTL and allowed their purification by cell sorting. The purity of the separate populations was confirmed by fluorescence microscopic inspection. The results of these studies demonstrate that flow cytometry can resolve target-bound and free CTL, measure cytolytic efficiency and specifically sort out cytometrically defined subgroups within the effector cell population.     

Cytotoxicity, intracellular distribution and uptake of doxorubicin and doxorubicin coupled to cell-penetrating peptides in different cell lines: A comparative study

One of the major obstacles which are opposed to the success of anticancer treatment is the cell resistance that generally develops after administration of commonly used drugs. In this study, we try to overcome the tumour cell resistance of doxorubicin (Dox) by developing a cell-penetrating peptide (CPP)-anticancer drug conjugate in aim to enhance its intracellular delivery and that its therapeutic effects. For this purpose, two cell-penetrating peptides, penetratin (pene) and tat, derived from the HIV-1 TAT protein, were chemically conjugated to Dox. The cytotoxicity, intracellular distribution and uptake were accessed in CHO cells (Chinese Hamster Ovarian carcinoma cells), HUVEC (Human Umbilical Vein Endothelial Cells), differentiated NG108.15 neuronal cell and breast cancer cells MCF7drug-sensitive or MDA-MB 231 drug-resistant cell lines. The conjugates showed different cell killing activity and intracellular distribution pattern by comparison to Dox as assessed respectively by MTT-based colorimetric cellular cytotoxicity assay, confocal fluorescence microscopy and FACS analysis. After treatment with 3 μM with Dox-CPPs for 2 h, pene increase the Dox cytotoxicity by 7.19-fold in CHO cells, by 11.53-fold in HUVEC cells and by 4.87-fold in MDA-MB 231 cells. However, cytotoxicity was decreased in NG108.15 cells and MCF7. Our CPPs-Dox conjugate proves the validity of CPPs for the cytoplasmic delivery of therapeutically useful molecules and also a valuable strategy to overcome drug resistance.     

Poly(ethylene glycol)-poly(ester-carbonate) block copolymers carrying PEG-peptidyl-doxorubicin pendant side chains: synthesis and evaluation as anticancer conjugates

Water soluble polymer anticancer conjugates can improve the pharmacokinetics of covalently bound drugs by limiting cellular uptake to the endocytic route, thus prolonging plasma circulation time and consequently facilitating tumor targeting by the enhanced permeability and retention (EPR) effect. Many of the first generation antitumor polymer conjugates used nonbiodegradable polymeric carriers which limits the molecular weight that can be safely used to <40,000 g/mol. The aim of this ambitious study was to synthesize and evaluate a novel, prototype biodegradable polymeric system based on high molecular weight, water-soluble functionalized polyesters. The main polymeric platform was prepared from bis(4-hydroxy)butyl maleate (DBM) and poly(ethylene glycol) (PEG4000) blocks to give the polymer DBM2-PEG4000 containing biodegradable carbonate bonds and having a M(w) of 100,000-190,000 g/mol; M(n) of 37,000-53,000 g/mol, and M(w)/M(n) of 3.0-3.7. Using thioether linkages, this polymer was then grafted with HS-PEG3000-Gly-Phe-Lue-Gly doxorubicin (HS-PEG3000-GFLG-Dox) pendant side chains ( approximately 30 per DBM2-PEG chain). The final construct, DBM2-PEG4000-S-PEG3000-GFLG-Dox had a total Dox content of 3-4 wt % and a free Dox content of < or = 0.7% total Dox. During incubation with isolated lysosomal enzymes, the rate of Dox release from the polymer backbone was relatively slow (<5% release over 5 h) compared to that seen for PEG5000-GFLG-Dox alone (>20% over 5 h). The in vitro cytotoxicity was assessed using B16F10 murine melanoma (MTT assay). DBM2-PEG4000-S-PEG3000-GFLG-Dox was 10-20-fold less toxic than free Dox. In vivo antitumor activity of the DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates was assessed using a subcutaneous (s.c.) B16F10 murine melanoma model, and an intraperitoneal (i.p.) L1210 leukaemia model. The increased toxicity (attributed to poor solubility) and low antitumor activity of DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates compared to PEG5000-GFLG-Dox and HPMA copolymer-Dox conjugates was attributed to the slow rate of Dox release. The DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates were considered unfavorable as candidates for further development. However, the successful scale-up synthesis of DBM2-PEG4000-S-PEG3000 constructs suggest that they are worthy of further investigation as carriers for controlled release and targeting of less hydrophobic agents.     

Synthesis and cytotoxic activities of beta-carboline amino acid ester conjugates

Beta-carboline represents a class of compounds with potent anti-tumor activity by intercalating with DNA. To further enhance the cytotoxic potency and bioavailability of beta-carboline, a series of novel beta-carboline amino acid ester conjugates were designed and synthesized, and the cytotoxic activities of these compounds were tested using a panel of human tumor cell lines. In addition, the membrane permeability of these compounds was evaluated in vitro using a Caco-2 cell monolayer model. The beta-carboline amino acid ester conjugates demonstrated improved cytotoxic activity compared to the parental beta-carbolines. In particular, the Lys/Arg conjugates were the most potent analogs with an IC(50) value of 4 and 1 microM against human cervical carcinoma cells. The low interaction energy of Arg conjugate based on molecular modeling may contribute to its enhanced cytotoxicity. Taken together, this study provided new insights into structure-activity relationships in the beta-carboline amino acid ester conjugates and identified the beta-carboline Lys/Arg conjugates as promising lead compounds for further in vivo biological and molecular evaluation.     

Synthesis,in vitro stability,and antiproliferative effect of d‐cysteine modified GnRH‐doxorubicin conjugates

Overexpression of gonadotropin‐releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH, [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH, and [d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH were conjugated with doxorubicin (Dox), respectively, through N‐succinimidyl‐3‐maleimidopropionate as a linker to afford three new GnRH‐Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP‐HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor‐positive MCF‐7 human breast cancer cell line by MTT assay. The three GnRH‐Dox conjugates showed improved metabolic stability in human serum in comparison with AN‐152. The antiproliferative effect of conjugate II ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH‐Dox) on MCF‐7 cells was higher than that of conjugate I ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH‐Dox) and conjugate III ([d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH‐Dox), and the cytotoxicity of conjugate II against GnRH receptor‐negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.     

Non-Polar Chemical Constituents of Atemoya and Evaluation of the Cytotoxic and Antimicrobial Activity

This study aimed to identify the chemical composition of essential oil from fruits (EOAF) and the hexanic crude extract from aerial parts (At-Hex) of atemoya (Annona cherimola x Annona squamosa), a hybrid belonging to the Annonaceae family. Cytotoxic and antimicrobial activity was also evaluated. OEAF was obtained by hydrodistillation using a Clevenger apparatus, and their composition was analyzed by gas chromatography-mass spectrometry (GC-MS) analyses. Cytotoxicity was tested against human tumor cell lines HCT-116 (colon carcinoma), SF-295 (glioblastoma), OVCAR-8 (ovarian carcinoma) and HL60 (leukemia) using 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide (MTT) assay, while antimicrobial activity was conducted by bioauthography method against eleven microorganisms strains. Twenty-four compounds were identified in the EOAF and twenty-nine in At-Hex. The monoterpenes linalool (25.70%), α-pinene (10.38%), β-pinene (9.12%), transocimene (7.43%), and the sesquiterpene bicyclogermacrene (12.58%) were the major constituents of EOAF, whereas the sesquiterpene spathulenol (13.91%) was the main compound of At-Hex. At-Hex showed a high cytotoxicity against SF-295 (glioblastoma). These findings show an important chemotaxonomic contribution for the Annonaceae family, mainly for the Annona genus. Atemoya proved to be a promising source of substances with potential cytotoxic activity.     

Hexose keto-C-glycoside conjugates: design, synthesis, cytotoxicity, and evaluation of their affinity for the glucose transporter glut-1

The design, synthesis, cytotoxicity, and biological evaluation of carbohydrate/C-glycoside conjugates are described. The design concept is predicted on the idea that physiological barriers like the blood brain barrier could be crossed selectively by using glucose or glucose derivative/drug conjugates. The study demonstrates that, (1) carbohydrates and C-glycosides can be bonded at nonanomeric positions by the reaction of carbohydrate triflates with C-glycoside alkoxydes in the presence of DMPU; (2) there is a structure-activity relationship between the cytotoxicity of the conjugate and the nature of the carbohydrate residue; and (3) peracetylated hexose keto-C-glycoside conjugates are the most cytotoxic keto-C-glycosides.     

Adaptability of carp neutrophilic granulocytes to environmental temperatures: spontaneous cytotoxic activity and cell-adherent activity

This study observed the adaptability of carp neutrophilic granulocytes possessing spontaneous cytotoxic activity to different environmental temperatures. To study the adaptability of neutrophilic granulocytes, two different temperatures (25° C and 10° C) were selected, both for rearing and forin vitroassays, in which the cytotoxicity and the adherent rate against K562 target cells were measured. The cytotoxicity and adherent rate of neutrophilic granulocytes from carp kept at 25° C for 30 days were higher when assayed at 25° C than when assayed at 10° C. On the other hand, in carp acclimated from 25° C to 10° C, the cytotoxicities and adherent rates, when assayed at 25° C, decreased with increasing acclimation times, eventually becoming smaller than the values obtained when assayed at 10° C. After the fish kept at 10° C for a long period were re-acclimated to 25° C, these activities assayed at 25° C again became higher than the activities assayed at 10° C. These results indicated that carp neutrophilic granulocytes adapt their cytotoxic activity and adherent activity to different environmental temperatures. A change in cellular composition in the head kidney was also observed in carp kept at different environmental temperatures. The percentage of neutrophilic granulocytes became higher and lymphocytes became lower in carp that were kept at 10° C for a long period compared with carp that were kept at 25° C for a long period.     

Density gradient fractionation of effector cells in human natural cell-mediated cytotoxicity

In order to separate and characterize cytotoxic effector cells in natural cell-mediated cytotoxicity (NCMC), human lymphocytes were fractionated by Percoll continuous density gradient centrifugation (Pharmacia, Piscataway, N.J.). Lymphocytes from normal donors were fractionated through a 35-ml gradient and 2- or 3-ml aliquots were collected, counted, and grouped into three or more fractions in order to obtain sufficient cells for testing. Fractions of cells were tested for cytotoxicity in a 4-hr chromium release test and/or a 40-hr [³H]proline assay. Cell markers were assessed by testing for cells forming E rosettes, EA rosettes, and for cells with surface membrane immunoglobulin (SMIg). The lightest fraction contained larger cells and also usually contained the highest concentrations of cells with receptors for the Fc portion of IgG (FcR + cells), although slight variations were seen among individual donors. Results of cytotoxicity tests showed that cells from the top portions of the Percoll gradient had consistently greater cytotoxic activity on a per cell basis than the denser cells sedimenting lower. Estimation of cytotoxic activity in lytic units showed that 54–75% of the activity was recovered in the top 26–29% of the cells. This approach to investigating cell-mediated cytotoxicity should yield useful information regarding cellular interaction in, and regulation of, cytotoxic activities.     

The synthesis of doxorubicin-conjugated magnetite nanoparticles and their magnetic resonance and cytotoxic properties

The possibility of increasing the effectiveness of antitumor drugs such as doxorubicin by preparing its complex with ultrafine magnetic iron oxide nanoparticles is considered. A method for binding doxorubicin molecules to magnetic nanoparticles via citric acid is proposed. The main magnetic properties of the obtained conjugates were studied by proton relaxometry and Mössbauer spectroscopy, while their cytotoxic activity was evaluated via spectrophotometric MTT assay in HeLa cells. It was shown that the conjugates of magnetite nanoparticles with doxorubicin are characterized by a high level of contrast in magnetic resonance imaging. The magnetic properties of doxorubicin-free and bound magnetite nanoparticles are mainly determined by the average size of nanoobjects and the phase composition and slightly depend on the composition of the stabilizing shell. The cytotoxic effect of the synthesized conjugates of magnetite nanoparticles with doxorubicin is higher than that of unbound doxorubicin. This makes it possible to increase the antitumor effect of doxorubicin and control the dynamics of its delivery in the form of a conjugate into the disease focus due to the magnetic contrast properties of nanoparticles.     

Co-Encapsulation of Doxorubicin With Galactoxyloglucan Nanoparticles for Intracellular Tumor-Targeted Delivery in Murine Ascites and Solid Tumors

Doxorubicin (Dox) treatment is limited by severe toxicity and frequent episodes of treatment failure. To minimize adverse events and improve drug delivery efficiently and specifically in cancer cells, encapsulation of Dox with naturally obtained galactoxyloglucan polysaccharide (PST001), isolated from Tamarindus indica was attempted. Thus formed PST-Dox nanoparticles induced apoptosis and exhibited significant cytotoxicity in murine ascites cell lines, Dalton’s lymphoma ascites and Ehrlich’s ascites carcinoma. The mechanism contributing to the augmented cytotoxicity of nanoconjugates at lower doses was validated by measuring the Dox intracellular uptake in human colon, leukemic and breast cancer cell lines. PST-Dox nanoparticles showed rapid internalization of Dox into cancer cells within a short period of incubation. Further, in vivo efficacy was tested in comparison to the parent counterparts - PST001 and Dox, in ascites and solid tumor syngraft mice models. Treatment of ascites tumors with PST-Dox nanoparticles significantly reduced the tumor volume, viable tumor cell count, and increased survival and percentage life span in the early, established and prophylactic phases of the disease. Administration of nanoparticles through intratumoral route delivered more robust antitumor response than the intraperitoneal route in solid malignancies. Thus, the results indicate that PST-Dox nanoparticles have greater potential compared to the Dox as targeted drug delivery nanocarriers for loco regional cancer chemotherapy applications.     

The order of hepatic cytotoxicity of bile salts in vitro does not agree with that examined in vivo in rats

Using an enzyme release from isolated rat hepatocytes incubated with a bile salt as a marker, the cytotoxic order of bile salts was found to be taurochenodeoxycholate, glycochenodeoxycholate greater than tauroursodeoxycholate, glycoursodeoxycholate, cholate greater than taurocholate, glycocholate. Thus, the cytotoxicity of conjugates of ursodeoxycholate was greater than that of conjugates of cholate. However, these data do not agree with the order of cytotoxicity of these bile salts previously studied in vivo by the authors which demonstrated the least cytotoxic nature of conjugates of ursodeoxycholate.     

Doxorubicin coupled to penetratin promotes apoptosis in CHO cells by a mechanism involving c-Jun NH2-terminal kinase

Doxorubicin (Dox) has demonstrated potent activity in treating malignant lymphomas but its therapeutic efficacy is hampered by induction of cardiotoxicity. This side effect is related to the ability of the drug to generate reactive oxygen species in cells. Previously, we demonstrated that coupling Dox to penetratin (Pen), a cell penetrating peptide, represent a valuable strategy to overcome drug resistance in CHO cells. In the present study, we evaluated the consequences of the conjugation of Dox to Pen in term of apoptosis induction. When tested on CHO cells, Dox-Pen generated a typical apoptotic phenotype but at lower dose that needed for unconjugated Dox. Cell death induction was associated with chromatin condensation, caspase activation, Bax oligomerisation and release of cytochrome c. By using reactive oxygen species and c-jun NH2-terminal kinase (JNK) inhibitors, we prevented Dox- and Dox-Pen-induced CHO cell death. The chimeric soluble DR5 receptor that inhibits TRAIL induced cell death does not prevent Dox or Dox-Pen-induced cytotoxicity. These observations indicate that conjugation of Dox to cell penetrating peptide does not impair the ability of the drug to trigger cell death through activation of the intrinsic pathway involving c-Jun NH2-terminal kinase but could exhibit less toxic side effects and could warrant its use in clinic.     

The synthesis of a novel Crizotinib heptamethine cyanine dye conjugate that potentiates the cytostatic and cytotoxic effects of Crizotinib in patient-derived glioblastoma cell lines

We describe the synthesis of drug-dye conjugate 1 between anaplastic lymphoma kinase inhibitor Crizotinib and heptamethine cyanine dye IR-786. The drug-dye conjugate 1 was evaluated in three different patient-derived glioblastoma cell lines and showed potent cytotoxic activity with nanomolar potency (EC₅₀: 50.9 nM). We also demonstrate evidence for antiproliferative activity of 1 with single digit nanomolar potency (IC₅₀: 4.7 nM). Furthermore, the cytotoxic effects conveyed a dramatic, 110-fold improvement over Crizotinib. This improvement was even more pronounced (492-fold) when 1 was combined with Temozolomide, the standard drug for treatment for glioblastoma. This work lays the foundation for future exploration of similar tyrosine kinase inhibitor drug-dye conjugates for the treatment of glioblastoma.     

Progressive changes in cytotoxic potential during mixed lymphocyte culture.

In a previous study it was shown that at least one round of DNA synthesis is required for initial expression of cytotoxic function in mouse lymphocytes responding to alloantigen in vitro. In the experiments reported here we ask whether subsequent rounds of cell division are required simply for clonal expansion of this initial level of cytotoxic function within the population, or whether the amount of cytotoxicity per cytotoxic cell is altered during subsequent rounds of cell division. The amount of cytotoxicity per unit number of cells at various stages of culture was compared with the frequency of cytotoxic cells as estimated principally by effector-target cell conjugates. Our results strongly suggest that the amount of cytotoxicity per cell (cytotoxic potential) is not a static property of cytotoxic cells, but can be modulated up or down during the course of a reaction.     

Using small-angle neutron scattering to study the solution conformation of N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates

Our past research developed two N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates that became the first synthetic polymer-anticancer conjugates to be evaluated clinically. The first, FCE28068, contained Dox bound to the polymeric carrier via a tetrapeptidic linker (glycine-phenylalanine-leucine-glycine (GFLG)) (Mw approximately 30,000 g/mol; approximately 8 wt % drug), and the second, FCE28069, contained additionally galactosamine (Gal) (Mw approximately 30,000 g/mol; approximately 7.5 wt % Dox) again bound by a GFLG linker. Galactosamine was included to promote hepatocyte/hepatoma targeting via the asialoglycoprotein receptor. Both conjugates showed antitumor activity and were clinically less toxic than free Dox (2-5 fold). However, despite their similar chemical characteristics, the conjugates displayed a significantly different maximum-tolerated dose (MTD) in patients. The aim of this study, therefore, was to use small-angle neutron scattering (SANS) to explore the solution behavior of a small library of HPMA polymer conjugates including FCE28068, FCE28069, and their pharmaceutical formulations, plus as reference compounds HPMA copolymer-GFLG conjugates containing aminopropanol (Ap) or galactosamine (Gal) alone (i.e., without Dox). The SANS data obtained showed that HPMA copolymer-GFLG-Ap conjugates (containing 5 and 10 mol % side chains) showed evidence of polymer aggregation, however, no indication of aggregation was observed for FCE28068 and FCE28069 over the concentration range studied (2.5-50 mg/mL). Clear differences in the scattering behavior for the two conjugates were observed at equivalent concentration. Data were best fitted by a model for polydisperse Gaussian coils, and the HPMA copolymer-Dox conjugate with Gal (FCE28069) exhibited a larger radius of gyration (Rg) (by approximately 2.5 nm) compared to FCE28068. In conclusion, we have shown that SANS will be a valuable tool to elucidate conformation-performance relationships for polymer-drug conjugates.     

A non-crosslinking platinum-acridine hybrid agent shows enhanced cytotoxicity compared to clinical BCNU and cisplatin in glioblastoma cells

Using clonogenic survival assays, we demonstrated that a new platinum-acridine hybrid agent, PT-ACRAMTU, is cytotoxic in SNB19 and U87MG glioblastoma cells at low-micromolar concentrations. PT-ACRAMTU is more cytotoxic than ACRAMTU (the platinum-free acridine), acts in a time and dose dependent manner, and appears to generate an apoptotic response in both cell lines on the basis of increased caspase-3 activity.     

In situ gellable oxidized citrus pectin for localized delivery of anticancer drugs and prevention of homotypic cancer cell aggregation

The aim of this study was to develop in situ gellable hydrogels composed of periodate oxidized citrus pectin (OP) for localized anticancer drug delivery and evaluate the potential of OP to inhibit cancer metastasis. Doxorubicin (Dox) was coupled to OP by imine bonds. Adipic dihydrazide (ADH) was used for cross-linking of the Dox-OP conjugates. The Dox-OP conjugate solution gelled within 2 min after addition of ADH. The release rate of Dox from the hydrogels was controllable by an additive amount of ADH. The released Dox retained anticancer activity. OP was shown to have a potency to prevent homotypic cancer cell aggregation compared to unmodified citrus pectin, strongly suggesting that OP released from hydrogels in vivo will inhibit cancer metastasis. These results indicate that OP hydrogels have the potential to prevent progression of primary cancer by the released Dox and generation of metastatic cancer by the released OP.     

Luteolin as a glycolysis inhibitor offers superior efficacy and lesser toxicity of doxorubicin in breast cancer cells

Luteolin (Lu) exhibits a wide spectrum of anti-tumor activities, the present study was to observe whether Lu can sensitize breast cancer cells to doxorubicin (Dox) and to explain the basis underlying this phenomenon. In vitro, Lu at dose less than 100 μM had only slight effect on cells growth and cytotoxicity of Dox in 4T1 and MCF-7 cells under normoxia, but it could reverse tumor resistance to Dox and promote death of tumor cells under hypoxia. In vivo, Lu alone had also no effect on tumor growth delay, however, it could offer superior efficacy and lesser toxicity of Dox in 4T1 and MCF-7 bearing mice. Further study showed that Lu was able to suppress glycolytic flux but did not affect glucose uptake, the P-glycoprotein, anti-oxidative enzymes under hypoxia in vitro, and had not also effect on the intratumor Dox level in vivo. In addition, the activity of SOD and CAT was increased in serum and was decreased in tumor by Lu in vivo. These results suggest that luteolin as a glycolytic inhibitor might be a new adjuvant agent for chemotherapy.