In vivo tumor targeting by a NGR-decorated micelle of a recombinant diblock copolypeptide
Graphical abstract
NGR-functionalized amphiphilic block copolymers, which self-assemble into spherical micelles, are able to selectively target the CD13 receptor and accumulate in tumor tissue.
Introduction
Hydrophilic polymer carriers have gained much attention in the past few decades for the delivery of anti-cancer agents to solid tumors because they can: (1) increase the solubility of hydrophobic drugs; (2) increase their plasma half-life; (3) increase accumulation of drug in tumor; (4) reduce their accumulation in healthy organs and thereby reduce systemic toxicity; and can (5) allow incorporation of targeting moieties [1], [2]. While initial research in this area focused on polymer-drug conjugates [1], [3], recent work has focused on amphiphilic block copolymers that can self-assemble into nano–meso scale structures [4], [5], [6], which are capable of both sequestering drug and presenting a targeting ligand or a sub-cellular localization signal on their exterior.
We [7], [8] and others [5], [9], [10] are interested in developing block copolymers that self-assemble into amphiphilic nanoscale structures that facilitate the presentation of ligands specific for tumor-associated antigens. In this context, we are particularly interested in using multivalent polymer micelles to target receptors that are preferentially upregulated in the tumor vasculature and perivascular cells. The NGR/aminopeptidase-N (CD13) ligand–receptor system is of particular interest for tumor targeting for the following reasons [11], [12]. First, CD13 is a membrane-bound enzyme that is associated with angiogenic tumor vessels [12], [13]. Second, the NGR peptide has been shown previously to target macromolecular constructs to angiogenic vasculature in tumors through a putative interaction with CD13, making it of interest for targeting antiangiogenic agents to the tumor vasculature [14]. Third, the NGR tripeptide shows low-affinity in linear form suggesting potential benefit to increasing avidity through the multivalency effect [15].
To design a nanoscale delivery system that is capable of targeting CD13, we focused our attention on a class of recombinant repetitive polypeptides termed elastin-like polypeptides (ELPs). ELPs consist of a Val-Pro-Gly-Xaa-Gly repeat (Xaa = any amino acid besides Pro) and exhibit inverse phase transition behavior at a specific transition temperature (Tt); ELPs are soluble in water at T < Tt and become insoluble at T > Tt [16], [17]. We and others have previously shown that diblock ELPs (ELP block co-polymers, ELPBCs) consisting of a hydrophilic ELP block and a hydrophobic ELP block are temperature triggered amphiphiles; the ELPBC is a hydrophilic unimer that self-assembles into monodisperse spherical micelles above a critical micelle temperature (CMT) due to selective desolvation and collapse of the hydrophobic block [7], [17], [18]. These ELPBC micelles have several attractive features for the design of macromolecular drug carriers. First, ELPBCs are monodisperse, which provides exquisite control over their size and hence the size and coordination number of the self-assembled supramolecular structure. Second, they can be easily expressed at high levels in E. coli and are conveniently purified by inverse transition cycling (ITC) [19], a method that exploits the ELP phase transition to purify them in large quantities directly from cell lysates without chromatography. Third, we have shown that ELPBCs are remarkably tolerant to the presentation of linear peptide ligands at their N-terminus, as we have successfully incorporated linear peptides without disrupting self-assembly [7]. Fourth, we have shown that micelle self-assembly enables multivalent presentation of these linear peptides for high affinity targeting of cell surface receptors [8]. These advantages make ELPBCs a useful system for developing ligand-functionalized nanoparticles for drug delivery and provide the motivation for this study.
Herein, we report on the ability of multivalent NGR-functionalized ELPBC nanoparticles to target CD13 in the tumor vasculature following systemic administration. We examined the self-assembly properties using dynamic light scattering (DLS) and used two different methods—intravital laser scanning confocal fluorescence microscopy (ILSCFM) and immunofluorescence (IF)—to examine in vivo tumor targeting by these NGR-functionalized nanoparticles. Each of these approaches provided insights into different aspects of tumor accumulation of ELPBCs. ILSCFM of tumors implanted in a dorsal skin-fold window chamber provided qualitative data regarding the spatial distribution and quantitative data regarding plasma half-life, accumulation, and extravasation of the micelle in the tumor. IF provided a complementary methodology that was useful in identifying colocalization of the NGR–ELPBC with specific histological markers present in the tumor. To the best of our knowledge, with the exception of a recently published study by Allen et al. [20], this is the first study that quantifies the real-time 3D accumulation of both functionalized and non-functionalized monodisperse polymeric micelles, and the findings of these studies have implications for the field of vascular targeting of tumors using this class of peptide ligand.
Section snippets
Nomenclature
ELPs are described by the nomenclature ELP[VxAyGz]m, where m refers to the number of pentapeptide repeats and x, y, and z refer to the relative fraction of valine, alanine, and glycine in the guest residue position along the length of the protein. The block copolymers used in this study have the composition ELP[V1A8G7]m/ELP[V]n which we abbreviate as (ELP − m/n) where m = 64 and n = 90.
ELP cloning and expression
The gene encoding the ELP-64/90 block copolymer was available from a previous study [17], and was modified to
ELPBC design, expression and physical characterization
We designed an NGR-ELPBC using an existing gene for ELP-64/90. This diblock ELPBC encoded four contiguous segments: a hydrophilic ligand (NGR), a flexible GGS peptide linker, a hydrophilic ELP segment and a hydrophobic ELP segment followed by a trailer peptide (WPC) for conjugation of fluorophore or drug to the Cys (C) residue. The ELP-64/90 diblock copolymer was successfully expressed with a yield of 200 mg/L of culture. SDS-PAGE showed that the resulting ELPBC unimers had the expected MW as
Discussion
The results presented herein show that multivalent presentation of NGR by ELPBC self-assembly selectively targets tumor vasculature through the effect of multivalent targeting of CD13. Analysis of the dorsal fold window chamber images acquired by ILSCFM showed greater vascular retention and extravascular accumulation of NGR-ELPBC constructs in tumor tissue compared to normal tissue. In contrast, this effect was not observed with non-ligand ELPBC constructs. Histological staining supported these
Conclusion
Multivalent presentation of an NGR peptide ligand by self-assembled polypeptide micelles is shown to selectively target the tumor vasculature by multivalent targeting of CD13 and to increase the delivery of the micelles to tumors compared with non-functionalized micelles. Analysis of the dorsal fold window chamber images acquired by ILSCFM showed greater vascular retention and extravascular accumulation of NGR-ELPBC constructs in tumor tissue compared to normal tissue. Concurrently,
Acknowledgements
We thank Dr. Jun Chen for her guidance on immunostaining technique and Dr. C. Alexander Valencia for his advice on cell-based assays. This work was funded by NIH 5R01 EB 007205.
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