Polycefin, a new prototype of a multifunctional nanoconjugate based on poly(beta-L-malic acid) for drug delivery

Abstract

A new prototype of nanoconjugate, Polycefin, was synthesized for targeted delivery of antisense oligonucleotides and monoclonal antibodies to brain tumors. The macromolecular carrier contains: 1. biodegradable, nonimmunogenic, nontoxic beta-poly(L-malic acid) of microbial origin; 2. Morpholino antisense oligonucleotides targeting laminin alpha4 and beta1 chains of laminin-8, which is specifically overexpressed in glial brain tumors; 3. monoclonal anti-transferrin receptor antibody for specific tissue targeting; 4. oligonucleotide releasing disulfide units; 5. L-valine containing, pH-sensitive membrane disrupting unit(s), 6. protective poly(ethylene glycol); 7. a fluorescent dye (optional). Highly purified modules were conjugated directly with N-hydroxysuccinimidyl ester-activated beta-poly(L-malic acid) at pendant carboxyl groups or at thiol containing spacers via thioether and disulfide bonds. Products were chemically validated by physical, chemical, and functional tests. In vitro experiments using two human glioma cell lines U87MG and T98G demonstrated that Polycefin was delivered into the tumor cells by a receptor-mediated endocytosis mechanism and was able to inhibit the synthesis of laminin-8 alpha4 and beta1 chains at the same time. Inhibition of laminin-8 expression was in agreement with the designed endosomal membrane disruption and drug releasing activity. In vivo imaging showed the accumulation of intravenously injected Polycefin in brain tumor tissue via the antibody-targeted transferrin receptor-mediated endosomal pathway in addition to a less efficient mechanism known for high molecular mass biopolymers as enhanced permeability and retention effect. Polycefin was nontoxic to normal and tumor astrocytes in a wide range of concentrations, accumulated in brain tumor, and could be used for specific targeting of several biomarkers simultaneously.

Figure 1

SDS–PAGE of mAb OX-26 and Fluorescein-Polycefin to demonstrate the conjugation of PMLA with the H-chain of the antibody, and the absence of multiple cross-linking of antibody. Lane N1, Fluorescein-Polycefin. Electrophoresis under nonreducing condition (DTT absent) showing the integral mAb after staining with Coomassie Brilliant Blue. Only the monomeric (150 kDa) but no multiple cross-linked mAbs (>150 kDa) are seen. Lanes R1–R5 and M, electrophoresis under reducing conditions (20 mM DTT) favoring dissociation into H- and L-chains. Detection of mAb OX-26 and its H-chains by Western blotting in lanes R1 and by Coomassie Brilliant Blue staining in lane R5. Detection by Western blotting in lane R2, by Fluorescein-specific fluorescence in lane R3, and by Coomassie Brilliant Blue in R4. Lane M, molecular mass markers. Bands in positions 150–165 kDa (R1–R5) refer to H-chain or a large fragment after a limited proteolytic nicking of the samples. Bands of approximately 25 kDa (R4 and R5) refer to L-chain. Fluorescence of the H-chain (fragment) in lane R3 indicates that this chain is conjugated with the scaffold.

Figure 2

Comparison of the membrane disrupting activities of the Polycefin constituents measured by the hemolysis assay. Concentrations of tested conjugates were adjusted to 1.5 × 10⁷ molecules per red blood cell. For referring numbers to conjugate compositions see Scheme 1 and text. Panel a, effects of the antibody and the oligonucleotides. 8, Polycefin; 3, Polycefin without antibody and oligonucleotides, 18, Polycefin without antibody; 19, Polycefin without oligonucleotides. Panel b, effect of mPEG₅₀₀₀ at different loads. Unsubstituted PMLA 1 and 5% (9), 10% (10) or 15% (11) mPEG₅₀₀₀. Panel c, effect of the hydrophobicity of the amino acid side chain. Glycine (12), l-alanine (13), or l-valine (14). Panel d, effect of different loads of valine. 13% (15), 34% (16), 55% (17) l-valine.

Figure 3

Cell viability of normal (HAST040) and malignant (U87MG and T98G) astrocyte cell culture after 48 h of Polycefin incubation. Different concentrations of Polycefin (1.4 μM and 14 μM) did not change the cell viability (p > 0.05) after 24 h (not shown) and 48 h treatment. 100% refers to conditions without treatment (control). Cell viability was measured twice in triplicates.

Figure 4

Drug delivery into cultured human glioma U87MG cells. A. U87MG glioma cells display surface staining with OX-26 antibody by indirect immunofluorescence demonstrating cross-reactivity with human antigen (left). Omission of primary antibody abolished staining (negative control, right). B. Left, 10 min of U87MG treatment with Fluorescein-labeled Polycefin. The location of Polycefin is indicated by green fluorescence near the cell membrane (arrows), and early endosomes are beginning to form. Right, endosomal formation after 20 min treatment with Polycefin. Maturing endosomes are visible inside the cells (arrows). C. Co-distribution of endosomal marker FM 4-64 (marker) with Fluorescein-Polycefin (30 min) in cultured U87MG cells 30 min after treatment. FM 4-64 stains endosomes (red color), and Polycefin is found in the same place (green color). Co-localization is revealed as yellow color (lower left). Confocal microscopy.

Figure 5

In vitro targeting and inhibition of laminin chain synthesis by Polycefin. Western blot analysis of laminin chain secretion into medium with or without Polycefin treatment. Conditioned media of both glioma cell lines, U87MG and T98G, contain α4 and β1 chains of laminin-8. Polycefin markedly inhibited secretion of both laminin chains, especially of β1 chain. Gel loading was normalized by the content of secreted fibronectin.

Figure 6

Xenogen IVIS 200 imaging of Polycefin in brain glioma. Polycefin variants 20 and Polycefin(-mAb) 21 are fluorescence labeled with Alexa Fluor 680, and the valine residue (Scheme 1) is replaced by l-leucine ethyl ester. Without targeting antibody, the drug still accumulates in tumor 72 h after intravenous injection (A). Complete Polycefin with mouse targeting antibody shows significantly higher tumor retention by both amount and area (B). Imaging was carried out after flushing perfused brains with sodium-buffered saline.

Scheme 1

Summary of the Synthesis of Dye-Polycefin (7)^a