Poly(malic acid) nanoconjugates containing various antibodies and oligonucleotides for multitargeting drug delivery

Abstract

Nanoconjugates are emerging as promising drug-delivery vehicles because of their multimodular structure enabling them to actively target discrete cells, pass through biological barriers and simultaneously carry multiple drugs of various chemical nature. Nanoconjugates have matured from simple devices to multifunctional, biodegradable, nontoxic and nonimmunogenic constructs, capable of delivering synergistically functioning drugs in vivo. This review mainly concerns the Polycefin family of natural-derived polymeric drug-delivery devices as an example. This type of vehicle is built by hierarchic conjugation of functional groups onto the backbone of poly(malic acid), an aliphatic polyester obtained from the microorganism Physarum polycephalum. Particular Polycefin variants target human brain and breast tumors implanted into animals specifically and actively and could be detected easily by noninvasive imaging analysis. Delivery of antisense oligonucleotides to a tumor-specific angiogenic marker using Polycefin resulted in significant inhibition of tumor angiogenesis and increase of animal survival.

Figure 1. Production and purification of poly(malic acid) from Physarum polycephalum

β-poly(L-malic acid) (PMLA) is produced by plasmodia of the slime mold. The polymer is isolated from the culture broth and purified by passages over DEAE-cellulose, ethanol precipitation of its Ca²⁺-salt, size fractionation on Sephadex G25 and acidification over Amberlite 120H⁺. Lyophillized PMLA is dry, white and of Mn (number-averaged molecular weight) 50–100 kDa. The yield from 20 l fermentation of glucose/C0₂ is 20 g polymer.

Figure 2. Synthesis of Polycefin nanoconjugate with various functional modules

Note the hierarchy in the choice of conjugation reactions. The platform consists of biodegradable highly purified β-poly(L-malic acid) from Physarum polycephalum. The carboxyl groups are first activated by esterification with N-hydroxysuccinimide. The hierarchy involves a first series of conjugation reactions with more durable residues, in other words, with polyethylene glycol (PEG), leucine ethylester and the thiol-containing spacer, 2-mercaptoethylamine. In the second series of conjugation reactions, the biochemically fragile molecules, monoclonal antibodies (mAbs) and antisense oligonucleotides (AONs), are conjugated by forming thioether and disulfide bonds, respectively. An optional fluorescent reporter molecule can be similarly conjugated. Nonreacted free thiol groups are then blocked chemically to prevent uncontrolled reactions. The modules are: (A) morpholino AON (to laminin α4 chain) and (B) (to laminin β1 chain) conjugated to the scaffold by disulfide bonds cleavable by the cytoplasmic glutathione to release the free drugs; (C & D) mAbs for cancer cell targeting and receptor-mediated endocytosis; (E) PEG for protection; (F) thiol-masking groups; (G) optional fluorescent reporter dye (fluorescein, Oregon Green or Alexa Fluor 680) for imaging and (H) stretches of conjugated leucine ethyl ester to provide lipophilicity for the disruption of endosomal membranes.

Figure 3. Time course of whole mouse imaging with Xenogen IVIS 200 system after intravenous Polycefin administration into human brain tumor-bearing mice

At time zero (0 min), Polycefin was injected into the tail vein. In the next 24 h, it is gradually removed by kidney clearance. By 24 h, only brain tumor and kidneys show significant Polycefin accumulation.

Figure 4. Imaging with different organs 24 h after intravenous administration of Polycefin variants

(A & C) Imaging of mouse organs after treatment with Polycefin that has no targeting mAb attached. There is a slight accumulation in the brain tumor, some drug in the spleen and the majority of Polycefin is found in the kidney. (B & D) Polycefin variant with antimouse TfR mAb shows significant accumulation in the tumor, liver and kidney. Organs have been perfused with phosphate-buffered saline before imaging to remove unbound drug in the vessels. mAb: Monoclonal antibody; TfR: Transferrin receptor.

Figure 5. Time course of mouse imaging with Xenogen IVIS 200 system after intravenous Polycefin administration into human brain tumor-bearing mice

(A) Whole body imaging. Polycefin gradually disappears from circulation over 48 h time course. After 48 h, Polycefin accumulations remain only in the subcutaneous tumor and kidney. After PBS perfusion, most of Polycefin remains in the tumor. (B) Organ imaging 48 h after Polycefin administration. The drug accumulation is detected only in breast tumor and kidney as the drug-clearing organ. PBS: Phosphate-buffered saline.

Figure 6. Fluorescence imaging of brain from nude mice bearing human U87MG glioma

24 h after intravenous injection of free Alexa Fluor 680 and various Alexa Fluor 680-labeled Polycefin variants, only the tumor but not other areas of the brain contain fluorescent drug. Free drug does not show any accumulation. The highest drug accumulation in the tumor is observed with the tandem configuration in Polycefin (mTfR, 2C5) bearing two antibodies targeting mouse endothelium (anti-mTfR) and tumor cell (antinucleosomal monoclonal antibody 2C5 reacting with tumor cell surface antigen). Polycefin variants with one antibody (antimouse TfR or antitumor surface antigen) show less accumulation. A total of 100 μl of Alexa Fluor 680 (0.6 μM) or labeled Polycefin variants at concentrations of 3 μM antisense oligonucleotides were injected intravenously. mTfR: Mouse transferrin receptor. Reproduced with permission from [95].

Figure 7. Schematic illustrating Polycefin reaction with different cells in a human U87MG brain tumor xenograft model in mice

Polycefin contains mAbs to mouse TfR and tumor cell surface antigen. Binding of antimouse TfR to mouse endothelium causes drug internalization and transcytosis, whereby it becomes available to glioma cells that bind Polycefin through the attached 2C5 antibody. mAb: Monoclonal antibody; TfR: Transferrin receptor.

Figure 8. In vivo target inhibition by Polycefin and animal survival

(A) Survival of Polycefin-treated (0.5 mg/kg body weight) and control rats. After intracranial administration of four doses of Polycefin, the animal survival time was significantly increased (p < 0.0004) compared with saline-treated or Polycefin(-mAb)-treated rats. (B) Immunofluorescent analysis of xenotransplanted brain tumors in rats with antihuman mAbs to laminin α4 or β1 chains. After Polycefin treatment, the number of tumor vessels positive for either laminin chain was markedly diminished. Therefore, Polycefin inhibited the expression of both its targets and their incorporation into basement membrane by human tumor cells. Asterisks denote tumor-adjacent (normal) brain area. This area has significantly decreased cellularity (revealed by blue nuclear staining with 4′,6-diamidino-2-phenylindole [DAPI]) compared with highly cellular tumor at the left. No vascular staining is observed in the tumor-adjacent area with both mAbs because the antibodies only recognized human laminin chains produced in this case by tumor astrocytes. Left lower panel, a H&E-stained tumor section showing a sharp boundary between highly cellular tumor (to the left) and surrounding brain parenchyma (asterisk) with significantly fewer cells. Right lower panel, staining of a serial section with a pAb to laminin α4 chain recognizing human and rat protein that reveals all vessels. Note increased vascularity and cellularity of the tumor as opposed to hypocellular surrounding tissue (asterisk) that has only scattered vessels (arrows), v, large blood vessel marked for orientation purposes. H&E: Hematoxylin and eosin; mAb: Monoclonal antibody; pAb: Polyclonal antibody. Reproduced with permission from [30].

Figure 9. Human brain tumor in a nude mouse without and after intravenous Polycefin treatment

(A) Multicellular glioma structure developed after inoculation of U87MG cell line intracranially into the nude mouse that died at day 32. (B) Massive necrosis that replaced glioma structure after eight intravenous treatments with Polycefin of a nude mouse sacrificed at day 74.