Novel folated and non-folated pullulan bioconjugates for anticancer drug delivery.

Two new anticancer polymer therapeutics were designed for tumour cell targeting. The bioconjugates were synthesised by pullulan derivatisation with either doxorubicin or doxorubicin and folic acid. Pullulan was activated by periodate oxidation and functionalised by reductive conjugation with cysteamine and 1.9 kDa PEG(NH(2))(2). The cysteamine thiol groups were conjugated to doxorubicin through a pH-sensitive hydrazone spacer while the pending PEG-NH(2) functions of one derivatised pullulan batch were conjugated to folic acid to obtain one of the two polymer therapeutics. The reaction intermediates and the final products were characterised by mass spectrometry, UV-vis analysis and reverse phase and gel permeation chromatography. The folic acid-free derivative [(NH(2) PEG)-Pull-(Cyst-Dox)] contained 6.3% (w/w) doxorubicin while the folic acid-doxorubicin-coupled derivative [(FA-PEG)-Pull-(Cyst-Dox)] contained 6% (w/w) doxorubicin and 4.3% (w/w) folic acid. Photon correlation spectroscopy showed that (NH(2) PEG)-Pull-(Cyst-Dox) and (FA-PEG)-Pull-(Cyst-Dox) assembled into particles of about 150 and 100 nm diameter, respectively. The two bioconjugates displayed similar drug release profiles either at pH 7.4 buffer or in plasma, where less than 20% of doxorubicin was released within three days. At pH 5.5, both conjugates underwent complete drug release in about 40 h. In vitro studies carried out with KB tumour cells over-expressing folic acid receptor showed that both free doxorubicin and (FA-PEG)-Pull-(Cyst-Dox) were rapidly taken up by the cells, while the internalisation of the non-folated derivative was significantly slower. Cell viability studies did not show relevant difference between the two bioconjugates. After 72 h of incubation with folic acid receptor non-expressing MCF7 cells, the IC(50) values of doxorubicin, (NH(2)PEG)-Pull-(Cyst-Dox) and (FA-PEG)-Pull-(Cyst-Dox) were 0.3 μM, 1.2 μM and 3.1 μM, respectively. After incubation with KB cells over-expressing folic acid receptor, the IC(50) values were 0.4 μM, 1.8 μM and 1.1 μM, respectively. Pharmacokinetic studies showed that 4 h after intravenous administration of the conjugates to Balb/c mice about 40% of the administered drug equivalent dose was present in the bloodstream while in the case of unconjugated doxorubicin, 80% of the drug was cleared within 30 min. These findings suggest that the novel doxorubicin-pullulan bioconjugates possess suitable properties for passive tumour targeting. On the other hand, folic acid conjugation has been found to have limited effect on selective cell up-take.

Scomparin A ，Salmaso S ，Bersani S ，Satchi-Fainaro R ，Caliceti P ... -  《-》

Folate-decorated maleilated pullulan-doxorubicin conjugate for active tumor-targeted drug delivery.

A novel folate-decorated maleilated pullulan-doxorubicin conjugate (abbreviated as FA-MP-DOX) for active tumor targeting was set up. The structure of this conjugate was confirmed by (1)H NMR analysis. Furthermore, the conjugation efficiency, drug release property and stability of the conjugate were determined. The cellular uptake and cytotoxicity were assessed by using ovarian carcinoma A2780 cells as in vitro cell model. In vitro DOX release from FA-MP-DOX conjugate occurred at a faster rate at acidic pH compared to neutral pH (7.4). After 30 h of incubation at pH 2.5, 5.0 and 7.4 the released free DOX was about 68.71%, 50.08% and 26%, respectively. Based on the IC(50) values, the conjugate was found more effective with ovarian carcinoma A2780 cells than the parent drug after 48 h culture. These results suggested that FA-MP-DOX conjugate could be a promising doxorubicin carrier for its targeted and intracellular delivery.

Zhang H ，Li F ，Yi J ，Gu C ，Fan L ，Qiao Y ，Tao Y ，Cheng C ，Wu H ... -  《-》

Folate-mediated poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) nanoparticles for targeting drug delivery.

A novel targeting drug delivery system (TDDS) has been developed. Such a TDDS was prepared by W(1)/O/W(2) solvent extraction/evaporation method, adopting poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] as the drug carrier, folic acid (FA) as the targeting ligand, and doxorubicin (DOX) as the model anticancer drug. The average size, drug loading capacity and encapsulation efficiency of the prepared DOX-loaded, folate-mediated P(HB-HO) nanoparticles (DOX/FA-PEG-P(HB-HO) NPs) were found to be around 240 nm, 29.6% and 83.5%. The in vitro release profile displayed that nearly 50% DOX was released in the first 5 days. The intracellular uptake tests of the nanoparticles (NPs) in vitro showed that the DOX/FA-PEG-P(HB-HO) NPs were more efficiently taken up by HeLa cells compared to non-folate-mediated P(HB-HO) NPs. In addition, DOX/FA-PEG-P(HB-HO) NPs (IC(50)=0.87 microM) showed greater cytotoxicity to HeLa cells than other treated groups. In vivo anti-tumor activity of the DOX/FA-PEG-P(HB-HO) NPs showed a much better therapeutic efficacy in inhibiting tumor growth, and the final mean tumor volume was 178.91+/-17.43 mm(3), significantly smaller than normal saline control group (542.58+/-45.19 mm(3)). All these results have illustrated that our techniques for the preparing of DOX/FA-PEG-P(HB-HO) NPs developed in present work are feasible and these NPs are effective in selective delivery of anticancer drug to the folate receptor-overexpressed cancer cells. The new TDDS may be a competent candidate in application in targeting treatment of cancers.

Zhang C ，Zhao L ，Dong Y ，Zhang X ，Lin J ，Chen Z ... -  《-》

A comparative study of folate receptor-targeted doxorubicin delivery systems: dosing regimens and therapeutic index.

Ligand-receptor mediated targeting may affect differently the performance of supramolecular drug carriers depending on the nature of the nanocarrier. In this study, we compare the selectivity, safety and activity of doxorubicin (Dox) entrapped in liposomes versus Dox conjugated to polymeric nanocarriers in the presence or absence of a folic acid (FA)-targeting ligand to cancer cells that overexpress the folate receptor (FR). Two pullulan (Pull)-based conjugates of Dox were synthesized, (FA-PEG)-Pull-(Cyst-Dox) and (NH2-PEG)-Pull-(Cyst-Dox). The other delivery systems are Dox loaded PEGylated liposomes (PLD, Doxil®) and the FR-targeted version (PLD-FA) obtained by ligand post-insertion into the commercial formulation. Both receptor-targeted drug delivery systems (DDS) were shown to interact in vitro specifically with cells via the folate ligand. Treatment of FR-overexpressing human cervical carcinoma KB tumor-bearing mice with three-weekly injections resulted in slightly enhanced anticancer activity of PLD-FA compared to PLD and no activity for both pullulan-based conjugates. When the DDS were administered intravenously every other day, the folated-Pull conjugate and the non-folated-Pull conjugate displayed similar and low antitumor activity as free Dox. At this dosing regimen, the liposome-based formulations displayed enhanced antitumor activity with an advantage to the non-folated liposome. However, both liposomal formulations suffered from toxicity that was reversible following treatment discontinuation. Using a daily dosing schedule, with higher cumulative dose, the folated-Pull conjugate strongly inhibited tumor growth while free Dox was toxic at this regimen. For polymeric constructs, increasing dose intensity and cumulative dose strongly affects the therapeutic index and reveals a major therapeutic advantage for the FR-targeted formulation. All DDS were able to abrogate doxorubicin-induced cardiotoxicity. This study constitutes the first side-by-side comparison of two receptor-targeted ligand-bearing systems, polymer therapeutics versus nanoparticulate systems, evaluated in the same mouse tumor model at several dosing regimens.

Scomparin A ，Salmaso S ，Eldar-Boock A ，Ben-Shushan D ，Ferber S ，Tiram G ，Shmeeda H ，Landa-Rouben N ，Leor J ，Caliceti P ，Gabizon A ，Satchi-Fainaro R ... -  《-》

Amphiphilic multi-arm-block copolymer conjugated with doxorubicin via pH-sensitive hydrazone bond for tumor-targeted drug delivery.

Prabaharan M ，Grailer JJ ，Pilla S ，Steeber DA ，Gong S ... -  《-》