Design of a novel nanoconjugate based on poly(β-l-malic acid) for intracellular delivery of anti-tumor agents

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Introduction:
 Naturally occurring poly(β-L-malic acid) (PMLA) was developed as a promising carrier for delivery of anti-tumor agents and cancer treatment (Fujita et al. Angiogenesis, 2006; Fujita et al. J Control Release, 2007). Laminin-411 is overexpressed in gliomas and deposited in newly formed tumor blood vessel basement membranes. Laminin-411 is associated with glioma recurrence and shorter patient survival time. The new nanobioconjugate Polycefin based on PMLA scaffold was evaluated in vitro and in vivo for tumor growth suppression.
 Material and methods:
 The new version of Polycefin (Polycefin-LLL) designed to pass through blood brain barrier (BBB) contains three key components: antisense Morpholino oligos against laminin-411 α4 and β1 chains, tandem targeting anti-transferrin receptor (TfR) antibodies, and new pH-sensitive endosome escape unit, L-leucylleucylleucine (trileucine, LLL). Endosomal escape capability of Polycefin-LLL conjugates with 10%, 25%, or 40% LLL was evaluated in vitro using liposome leakage assay. Conjugation of tandem anti-TfR antibodies on Polycefin was validated by HPLC and functional activity, by ELISA. Western blot was used to examine the inhibition of laminin-411 synthesis by human glioma cells U87MG in the presence of Polycefin-LLL. U87MG human glioma cells were inoculated intracranially into nude mice. Polycefin variants were injected intravenously in each group of animals (n=8 per group). Imaging analysis of Polycefin variant accumulation in vivo was performed using Xenogen IVIS 200 imager.
 Results:
 In vitro. Polycefin-LLL (40%) demonstrated liposome leakage at pH 5.0. However, conjugates with dileucine and tetraleucine were not effective at pH 5.0 and 7.5. Polycefin-leucine ethyl ester 40% induced liposome leakage in a pH-independent manner. Therefore, Polycefin-LLL has the most physiological potential to escape endosome and release the drug intracellularly. In cell viability study, Polycefin-LLL was non-toxic up to 500 μg/ml for U87MG cells. By Western blot analysis Polycefin-LLL completely inhibited secretion of α4 and β1 protein chains of laminin-411 by U87MG cells, suggesting that this pH-sensitive Polycefin was able to successfully deliver and liberate the antisense oligos into the cytoplasm. In vivo. Mice in all Polycefin- and PBS-treated groups were sacrificed on day 48 after tumor cell inoculation and tumor size was measured. After the treatment with Polycefin-LLL (40%) the average tumor size was 4 mm³, compared to 38 mm³ in the group treated with Polycefin-leucine ethyl ester 40% (the original Polycefin), (p<0.05), and 47 mm³ in the group treated with PBS (p<0.01).
 Conclusion:
 The newly designed Polycefin-LLL nanoconjugate, which is pH-sensitive, non-toxic, and biodegradable, proves to be the most effective for intracellular delivery of active anticancer agents.