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Using a synthetic oligopeptide, n-FKFEFKFEFKFE-c (KFE12), representative of a class of peptides that can undergo self-assembly into a three-dimensional matrix biomaterial, we show that the self-assembly occurs when solution conditions reduce intermolecular electrical double-layer repulsion below van der Waals attraction in accord with DLVO theory. This theory predicts that a critical coagulation concentration of counterions should be required to allow assembly and that this concentration should be inversely proportional to the valence of the counterion raised to the sixth power. Our experimental results show that KFE12, at low pH, exhibits critical coagulation concentrations in each of three different salt solutions, KCl, K2SO4, and K3Fe(CN)6, and that the relative values of these critical concentrations follow the predicted dependence upon anion valence. The theory further predicts that self-assembly should occur when the oligopeptide is electrically neutral even in the absence of exogenous salt. Our experimental results show that KFE12 indeed forms gels when neutralized with NaOH. Thus, we have gained fundamental theoretical understanding of how to control the assembly of this class of oligopeptide-based biomaterials.

Targeting drugs to specific cells by conjugating the drug to an antibody or ligand for a cell surface receptor currently requires that the receptor be uniquely over-expressed by the target cell (the target cell over-expresses a particular receptor in comparison with untargeted cells, which do display this receptor type but a lesser number of them). Here we develop a mathematical model to predict the behavior of multivalent ligand-drug constructs containing two different ligands for two different receptors, which would allow targeting cells that do not uniquely over-express any receptor. In this model, target cells express both receptors at a high level; whereas, untargeted cells express one receptor type at the high level but the other at a lower level. The model predicts that these heterovalent conjugates (containing two different types of ligands) can achieve specificity even when the target cell does not uniquely over-express any one receptor type. Using the current approach, constructs in which only one ligand type is used will bind as much to untargeted cells as to the target cells. Therefore, this combination strategy can enormously expand the number of applications for which cell surface receptor targeting of drugs is an appropriate option.

A challenge in tumor targeting is to deliver payloads to cancers while sparing normal tissues. A limited number of antibodies appear to meet this challenge as therapeutics themselves or as drug-antibody conjugates. However, antibodies suffer from their large size, which can lead to unfavorable pharmacokinetics for some therapeutic payloads, and that they are targeted against only a single epitope, which can reduce their selectivity and specificity. Here, we propose an alternative targeting approach based on patterns of cell surface proteins to rationally develop small, synthetic heteromultivalent ligands (htMVLs) that target multiple receptors simultaneously. To gain insight into the multivalent ligand strategy in vivo, we have generated synthetic htMVLs that contain melanocortin (MSH) and cholecystokinin (CCK) pharmacophores that are connected via a fluorescent labeled, rationally designed synthetic linker. These ligands were tested in an experimental animal model containing tumors that expressed only one (control) or both (target) MSH and CCK receptors. After systemic injection of the htMVL in tumor-bearing mice, label was highly retained in tumors that expressed both, compared with one, target receptors. Selectivity was quantified by using ex vivo measurement of Europium-labeled htMVL, which had up to 12-fold higher specificity for dual compared with single receptor expressing cells. This proof-of-principle study provides in vivo evidence that small, rationally designed bivalent htMVLs can be used to selectively target cells that express both, compared with single complimentary cell surface targets. These data open the possibility that specific combinations of targets on tumors can be identified and selectively targeted using htMVLs.receptor targeting | multivalency | cross-linking | gene expression profiling

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