Nanoconjugate based on polymalic acid for tumor targeting

Abstract

A new prototype of polymer-derived drug delivery system, the nanoconjugate Polycefin, was tested for its ability to accumulate in tumors based on enhanced permeability and retention (EPR) effect and receptor mediated endocytosis. Polycefin was synthesized for targeted delivery of Morpholino antisense oligonucleotides into certain tumors. It consists of units that are covalently conjugated with poly(beta-l-malic acid) (M(w) 50,000, M(w)/M(n) 1.3) highly purified from cultures of myxomycete Physarum polycephalum. The units are active in endosomal uptake, disruption of endosomal membranes, oligonucleotide release in the cytoplasm, and protection against enzymatic degradation in the vascular system. The polymer is biodegradable, non-immunogenic and non-toxic. Polycefin was also coupled with AlexaFluor 680 C2-maleimide dye for in vivo detection. Nude mice received subcutaneous injections of MDA-MB 468 human breast cancer cells into the left posterior mid-dorsum or intracranial injections of human glioma cell line U87MG. Polycefin at concentration of 2.5mg/kg was injected via the tail vein. In vivo fluorescence tumor imaging was performed at different time points, 0-180 min up to 24h after the drug injection. The custom-made macro-illumination imaging MISTI system was used to examine the in vivo drug accumulation in animals bearing human breast and brain tumors. In breast tumors the fluorescence signal in large blood vessels and in the tumor increased rapidly until 60 min and remained in the tumor at a level 6 times higher than in non-tumor tissue (180 min) (p<0.003). In brain tumors drug accumulated selectively in 24h without any detectable signal in non-tumor areas. The results of live imaging were corroborated histologically by fluorescence microscopic examination of various organs. In addition to tumors, only kidney and liver showed some fluorescent signal.

Fig. 1

Poly(malic acid) from the slime mold Physarum polycephalum. β-Poly(L-malate) is synthesized and secreted by the plasmodium. The polymer is harvested from the broth of shaken cultures and isolated by ion exchange DEAE-chromatography and ethanol precipitation. From the highly purified β-poly(L-malic acid) Polycefin is synthesized by chemical conjugation of functional groups as described [2].

Fig. 2

Schematic view of Polycefin. Panel a, schematic formula showing the composition of the nanoconjugate. The functional units (modules) have been chemically conjugated to the carboxyl groups of the PMLA platform [2]. Per cent values refer to total pendant carboxyl residues (100%). AON1 and AON2 are the Morpholino antisense oligonucleotides that target the mRNAs of laminin-8 α4 and β1 chains. The drug releasing unit is the disulfide group. Mouse anti-human TfR mAb targets tumor cells at their cell surface. PEG protects against enzymatic degradation. The fluorescent reporter group, AexaFuor 680, serves in vivo fluorescence imaging. L-Leucine ethylester moieties function in endosomal escape. The groups of 7.27% is the product of sulfhydryl blocking at the end of Polycefin synthesis and has no assigned function. Panel b shows a cartoon of Polycefin with the functional modules described in panel a.

Fig. 3

In vivo fluorescence imaging (MISTI) of mouse brain with xenografted human U87MG glioma 24 h after I.V. injection of Polycefin. The tumor area shows distinct fluorescent signal.

Fig. 4

In vivo fluorescence images of mice with xenografted human MDA-MB 468 breast tumor before treatment and in 2, 10, and 60 min after intravenous injection of AlexaFluor 680-labeled Polycefin (a). Fluorescence intensity analysis was performed by selecting small areas within the tumor and normal regions as shown in the top panel. The time course fluorescence intensities in tumor area (solid line) and normal area (dotted line) are shown in graph (b). Polycefin circulated in blood vessels in early phase (2–10 min), but declined after 60 min (a and b). However, the drug kept retaining in tumor tissues even after 60 min (b).

Fig. 5

Sections of tissues including brain, lung, heart, liver, kidney, normal breast, and breast tumor were analyzed for the presence of Polycefin after I.V. injection into mice. Serial sections for each corresponding tissue were prepared to detect fluorescence of the conjugate by microscopy. (a) Hematoxylin-eosin staining. (b) Fluorescence of Polycefin. Tissue sections in (a) and (b) are not identical. Red, Polycefin-AlexaFluor 680 fluorescence; green, non-specific autofluorescence. The positive signal was seen 3 h after I.V. administration in implanted breast tumor, normal liver and kidney, whereas it was not detected in other organs, such as heart, lung, brain and normal breast. Accumulation in liver and kidney was expected, because they participate in drug metabolism and elimination.