Synthesis and cellular characterization of various nano-assemblies of cell penetrating peptide-epirubicin-polyglutamate conjugates for the enhancement of antitumor activity.

A new class of cell penetrating peptides (CPPs) named peptide amphiphile was designed to improve the intracellular uptake and the antitumor activity of epirubicin (EPR). Various amphiphilic CPPs were synthesized by solid phase peptide synthesis method and were chemically conjugated to EPR. Their corresponding nanoparticles (CPPs-E4 and CPPs-E8) were prepared via non-covalent binding of the peptides and polyanions. Cytotoxicity and anti-proliferative activity were evaluated by MTT assay. Cellular uptake was examined by flow cytometry and fluorescence microscopy. The CPPs exhibited slight cytotoxicity. Binding of polyglutamate to CPPs (CPPs-E4 and CPPs-E8 nanoparticles) decreased their cytotoxicity. CPPs-E8 nanoparticles showed lower cytotoxicity than CPPs-E4 nanoparticles. Cellular uptake of K3W4K3-E8, K2W4K2-E8 and W3K4W3-E8 reached 100% with no difference between each of the mentioned CPPs and its nanoparticles at 50 µM. The anti-proliferative activity of EPR was enhanced following conjugation to peptides and nanoparticles at 25 µM. CPPs-EPR-E4 and CPPs-E8-EPR nanoparticles displayed higher anti-proliferative activity than CPPs-EPR at 25 µM. CPPs-E8-EPR nanoparticles showed higher anti-proliferative activity than CPPs-E4-EPR. K3W4K3-E8-EPR nanoparticles exhibited the highest anti-proliferative activity at 25 µM. The synthesized peptide nanoparticles are proposed as suitable carriers for improving the intracellular delivery of EPR into tumor cells with low cytotoxicity and high antitumor activity.

Experiments and synthesis of bone-targeting epirubicin with the water-soluble macromolecular drug delivery systems of oxidized-dextran.

Epirubicin (EPI) is a broad spectrum antineoplastic drug, commonly used as a chemotherapy method to treat osteosarcoma. However, its application has been limited by many side-effects. Therefore, targeted drug delivery to bone has been the aim of current anti-bone-tumor drug studies. Due to the exceptional affinity of Bisphosphonates (BP) to bone, 1-amino-ethylene-1, 1-dephosphate acid (AEDP) was chosen as the bone targeting moiety for water-soluble macromolecular drug delivery systems of oxidized-dextran (OXD) to transport EPI to bone in this article. The bone targeting drug of AEDP-OXD-EPI was designed for the treatment of malignant bone tumors. The successful conjugation of AEDP-OXD-EPI was confirmed by analysis of FTIR and (1)H-NMR spectra. To study the bone-seeking potential of AEDP-OXD-EPI, an in vitro hydroxyapatite (HAp) binding assay and an in vivo experiment of bone-targeting capacity were established. The effectiveness of AEDP-OXD-EPI was demonstrated by inducing apoptosis and necrosis of MG-63 tumor cell line. The obtained experimental data indicated that AEDP-OXD-EPI is an ideal bone-targeting anti-tumor drug.

[Preparation and in vivo-in vitro evaluation of compound nanoparticles loaded with epirubicin hydrochloride and gadopentetate meglumine].

OBJECTIVE: To prepare and characterize the compound Epirubicin hydrochloride and Gadopentetate meglumine (Gd-DTPA) nanoparticles, and evaluate its properties from rabbits in vivo and in vitro. METHODS: The compound Epirubicin hydrochloride and Gd-DTPA nanoparticles were prepared by double emulsion-solvent evaporation method. The main effective factors were orthogonal designed. The characteristics such as drug entrapment efficiency, drug loading, and drug utilization were assayed in vitro. MR imaging effect of the VX2 rabbit hepatoma model were observed after injecting the drug-loaded nanoparticles through the hepatic artery intubation in vivo. RESULTS: The drug encapsulation efficiency of the nanoparticles, drug loading and drug utilization were 33.8% ± 3.4%, 0.225% ± 0.052%, and 69.6% ± 4.3% under the optimized prescription, respectively. The mean size of the nanoparticles was 180.6 nm, the drug release continued in 48 h with good MR imaging effect. CONCLUSIONS: Compound Epirubicin hydrochloride and Gd-DTPA Nanoparticles were in simply preparation and showed sustained drug release properties. These novel nanoparticles with detecting function could develop of epirubicin hydrochloride targeted therapy of liver cancer.

Synthesis of a covalent epirubicin-(C(3)-amide)-anti-HER2/neu immunochemotherapeutic utilizing a UV-photoactivated anthracycline intermediate.

The C(3)-monoamine on the carbohydrate moiety (daunosamine -NH(2)-3') of epirubicin was reacted under anhydrous conditions with succinimidyl 4,4-azipentanoate to create a covalent UV-photoactivated epirubicin-(C(3)-amide) intermediate with primary amine-reactive properties. A synthetic covalent bond between the UV-photoactivated epirubicin-(C(3)-amide) intermediate and the ɛ-amine of lysine residues within the amino acid sequence of anti-HER2/neu monoclonal immunoglobulin was subsequently created by exposure to UV light (354 nm) for 15 minutes. Size-separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with immunodetection analysis and chemiluminescent autoradiographic imaging revealed a lack of IgG-IgG polymerization or degradative protein fragmentation of the covalent epirubicin-(C(3)-amide)-[anti-HER2/neu] immunochemotherapeutic. Retained binding-avidity of epirubicin-(C(3)-amide)-[anti-HER2/neu] was validated by cell-ELISA utilizing monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma SKBr-3 which highly overexpress membrane-associated HER2/neu complexes. Between epirubicin-equivalent concentrations of 10(-10) to 10(-6) M the covalent epirubicin-(C(3)-amide)-[anti-HER2/neu] immunochemotherapeutic consistently evoked levels of cytotoxic anti-neoplastic potency that were highly analogous to chemotherapeutic-equivalent concentrations of epirubicin. Cytotoxic anti-neoplastic potency of epirubicin-(C(3)-amide)-[anti-HER2/neu] against chemotherapeutic-resistant mammary adenocarcinoma SKBr-3 challenged with epirubicin-(C(3)-amide)-[anti-HER2/neu] at an epirubicin-equivalent concentration of 10(-6) M was 88.5% (e.g., 11.5% residual survival). Between final epirubicin-equivalent concentrations of 10(-8) and 10(-7) M there was a marked threshold increase in the mean cytotoxic anti-neoplastic activity for epirubicin-(C(3)-amide)-[anti-HER2/neu] from 9.9% to 66.9% (90.2% to 33.1% residual survival).

Effect of phospholipid composition on pharmacokinetics and biodistribution of epirubicin liposomes.

The effects of phospholipid composition on the pharmacokinetics (PK) and biodistribution of epirubicin (EPI) liposomes, as well as the in vitro macrophage uptake of various liposome formulations, were investigated. Three liposome formulations were investigated: HSPC:Chol (L-EPI; 5:4 molar ratio), HSPC:Chol:DSPG (D-EPI; 5:4:1 molar ratio), and HSPC:Chol:DSPG:DSPE-mPEG(2000) (S-EPI; 5:4:1:0.3 molar ratio). Small unilamellar liposomes were prepared by the modified thin-film hydration method with extrusion through polycarbonate filters, and EPI was remote loaded into liposomes by the transmembrane ammonium sulfate gradient method. Macrophages were used to evaluate in vitro the cellular uptake of EPI-loaded liposomes. The following decreasing order of uptake amount was observed: L-EPI>D-EPI>S-EPI. D-EPI showed a relatively low level of uptake, probably because of the steric hindrance provided by the glycerol head group on DSPG, protecting it from the direct recognization by cell-membrane receptors. With the presence of serum, uptake values for all liposome formulations were increased for the activation of the complement system. In the PK study, S-EPI showed significantly prolonged circulating time and reduced clearance. The following increasing order of area under the concentration versus time curve was observed among the various liposome formulations: L-EPI

[Preparation and quality control of compound epirubicin hydrochloride-loaded polymeric nanoparticles of L-lactic-co-glycolic acid].

OBJECTIVE: To prepare compound epirubicin hydrochloride-loaded polymeric nanoparticles of L-lactic-co-glycolic acid and establish their quality control. METHODS: The emulsion-solvent evaporation method was employed to prepare and freeze-dry the compound epirubicin hydrochloride-loaded polymeric nanoparticles of L-lactic-co-glycolic acid after the addition of lactose. The contents and cumulative release of epirubicin hydrochloride and dimeglumine gadopentetate were detected simultaneously by RP-HPLC (reverse phase-high performance liquid chromatography). RESULTS: The above nanoparticles were prepared and the quality standards for simultaneously determining the contents of epirubicin hydrochloride and dimeglumine gadopentetate established primarily. The contents of epirubicin hydrochloride and dimeglumine gadopentetate in compound preparation were 100.6% ± 1.6% and 99.1% ± 1.9% respectively. And two compositions could be completely released within 9 days. CONCLUSION: The preparation method of nanoparticles is simple and their quality control feasible.

Solution studies on the complex of 4'-epiadriamycin-d-(CGATCG)2 followed by time-resolved fluorescence measurement, diffusion ordered spectroscopy and restrained molecular dynamics simulations.

4'-Epiadriamycin is a better-tolerated anthracycline drug, due to lesser cardiotoxicity. We report here a study of the 2:1 complex of 4'-epiadriamycin-d-(CGATCG)(2) by proton Nuclear Magnetic Resonance Spectroscopy which show the absence of sequential connectivities between C1pG2 and C5pG6 base pair steps and presence of intermolecular cross peaks of the drug and DNA. Our studies establish the role of 9OH, NH3+, 7O, 4OCH(3) groups in binding to DNA. Time-resolved fluorescence measurement and diffusion ordered spectroscopic studies reveal the formation of complex. The nonspecific interactions as well as those essential for biological activity are discussed along with its medicinal importance.

Development of dextran derivatives with cytotoxic effects in human urinary bladder cancer cell lines.

BACKGROUND: In a previous study we reported on a new approach describing intravesical instillation of charged dextran in patients with superficial bladder carcinoma. The cationic derivative showed a strong tumor-selective accumulation. To develop this approach, the present study investigates the cytotoxic effect of cationic dextran derivatives on two urinary bladder cancer cell lines (J82 and 5637). METHODS: The dextran conjugates were prepared by periodate activation and subsequent coupling by reductive amination. A fluorimetric cytotoxicity assay (FMCA) was used for the cytotoxicity assay. The tumor cells were seeded into 96-well microtiter plates and different cationic dextran derivatives were added and incubated for 72 hours. RESULTS: The results showed that cationic epirubicin-dextran had a clear inhibitory effect on the growth in both cell lines (40-95% growth inhibition). The corresponding values for epirubicin (the reference) was 90-100% inhibition. Interestingly, cationic dextran had, by itself, a growth inhibitory effect. This cytotoxic effect could be strongly enhanced to be almost equal to the reference by changing the cationic sidegroup to aminohexane. Dextran alone showed no effect. CONCLUSION: The finding that cationic dextran by itself can be made cytotoxic, together with its capacity to accumulate in superficial bladder cancer, suggests possibilities for new therapeutic constructs. Cationic dextran with different cationic side-groups and in combination with cytotoxic drugs will be studied further. The cytotoxic mechanism needs to be elucidated.

Epidoxoform: a hydrolytically more stable anthracycline-formaldehyde conjugate toxic to resistant tumor cells.

The recent discovery that the formaldehyde conjugates of doxorubicin and daunorubicin, Doxoform and Daunoform, are cytotoxic to resistant human breast cancer cells prompted the search for hydrolytically more stable anthracycline-formaldehyde conjugates. Doxoform and Daunoform consist of two molecules of the parent drug bound together with three methylene groups, two forming oxazolidine rings and one binding the oxazolidines together at their 3'-amino nitrogens. The 4'-epimer of doxorubicin, epidoxorubicin, reacts with formaldehyde at its amino alcohol functionality to produce a conjugate, Epidoxoform, in 59% yield whose structure consists of two molecules of epidoxorubicin bound together with three methylene groups in a 1, 6-diaza-4,9-dioxabicyclo[4.4.1]undecane ring system. The structure was established from spectroscopic data and is consistent with products from reaction of simpler vicinal trans-amino alcohols with formaldehyde. Epidoxoform hydrolyzes at pH 7.3 to an equilibrium mixture with dimeric and monomeric epidoxorubicin-formaldehyde conjugates without release of formaldehyde or epidoxorubicin. The hydrolysis follows the rate law (A if B) if C + D where A (Epidoxoform) is in rapid equilibrium with B, and B is in slow equilibrium with C and D. The forward rate constant for A/B going to C+D gives a half-life of approximately 2 h at 37 degrees C. At equilibrium the mixture is stable for at least 2 days. At pH 6.0, hydrolysis proceeds with first-order kinetics to epidoxorubicin and formaldehyde with a half-life of 15 min at 37 degrees C. Epidoxoform and epidoxorubicin plus formaldehyde react with the self-complementary DNA octamer (GC)4 to yield five drug-DNA adducts which have structures analogous to the doxorubicin-DNA adducts from reaction of Doxoform with (GC)4. Epidoxoform is 3-fold more toxic to MCF-7 human breast cancer cells and greater than 120-fold more toxic to MCF-7/ADR resistant cells than epidoxorubicin. Epidoxoform in equilibrium with its hydrolysis products is greater than 25-fold more toxic to resistant cells with respect to epidoxorubicin.