Mono- and multivalent polymer active substance conjugates

One major research focus of our group is on multivalent interactions, such as the inhibition of inflammation and virus internalization.

Multivalent interactions (Fig. 1) are considerably stronger than the individual bonding of a corresponding number of monovalent ligands to a multivalent receptor and are often used in biological systems, particularly for cellular recognition processes and in signal transduction. In this research project multivalent ligands (saccharides and steroids) are being processed for oligomeric membrane receptors and their activity is being studied. (See also

Figure 1

Comparison of mono- and multivalent interactions.

Dendritic polyglycerols with a functional focal unit, X = SH, NH2, N3 or at the periphery may be selectively converted by a drug or ligand attachment to mono- or multifunctional polyglycerols, respectively (see Figure 2).

Figure 2

Mono- and multivalent polygylcerol-drug conjugates.

Due to their multivalent binding polyglycerol sulfates have recently been indentified as highly active heparine analogues with anti-inflammatory and artithrombic activity. The anti-inflamatory activity has been studied in detail and has revealed an extremely tight binding of PG sulfates to P and L seletines which prevents the adhesion of leucocytes to the inflamed tissue.

Furthermore, the conversion of dendritic polyglycerol with active substances like dyes and cytostatic drugs can significally improve the water solubility of these adducts. If an imine or hydrazone linker is used for binding it can be separated again under acidic conditions. This way selective release in tumor tissue (pH 5-6) can be achieved.

Figure 3

Conjugation of biologically active molecules to dendritic PG. Triggered release by pH-drop, enzyme activity, reductive environment, etc.

Within the framework of a cooperation with Dr. Felix Kratz from the Clinic for Tumor Biology, Freiburg, monovalent water soluble polyglycerol-cytostatic drug conjugates are being investigated. In addition to higher blood circulation times, the macromolecular active substance conjugates are more concentrated, preferably in tumor tissue due the EPR effect (enhanced permeation retention).

The increasing development of maleimide-bearing prodrugs and diagnostic dyes instigated us to synthesize thiolated nanocarriers with tuneable properties such as molecular weight, solubility, or targeting potential by selective functionalization of polyglycerol hydroxyl groups. Therefore, we developed a strategy to synthesize thiolated hyperbranched polyglycerols that can be used as a general, flexible method to couple diagnostic or therapeutic agents under physiological conditions. We reported the use of the thiolated PG scaffold for conjugation to maleimide-bearing prodrugs of doxorubicin or methotrexate which incorporate either a self-immolative para-aminobenzyloxycarbonyl (PABC) spacer coupled to dipeptide Phe–Lys or the tripeptide D-Ala–Phe–Lys as the protease substrate. Both prodrugs were cleaved by cathepsin B, an enzyme over-expressed by several solid tumors, to release doxorubicin or a methotrexate lysine derivate. An effective cleavage of PG–Phe–Lys–DOXO and PG–D-Ala–Phe–Lys–Lys–MTX and release of doxorubicin and methotrexate–lysine in the presence of the enzyme was observed. Cytotoxicity of the conjugates against human tumor cell lines showed that the activity of the drugs was primarily retained, which confirmed the macromolecular pro-drug concept.

In an ongoing investigation we prepared a series of conjugates of hyperbranched PG, 10 kDa with (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO–EMCH). The conjugates showed an acid-triggered release at pH 4.0 while only a marginal release was observed at pH 7.4. The antiproliferative activity assessed against two human tumor cell lines, i.e., AsPC1 LN (pancreatic carcinoma) and MDA-MB-231 LN (mamma carcinoma), showed a 2- to 10-fold lower cytotoxicity than the corresponding free drugs. With respect to antitumor efficacy, the conjugates manifested excellent antitumor effects with complete tumor remission up to day 30 without significant changes in body weight, even after administration of 3-fold the maximal tolerated dose for free doxorubicin. To determine the optimal size of the conjugates for achieving enhanced permeability and retention (EPR) mediated targeting and optimal cellular uptake of the drug, the molecular weight of the PG scaffold was varied systematically.


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