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Coordination chemistry in doubly stabilized micelles for the tageted delivery of cytostatic drugs in cancer therapy

Periodic Reporting for period 1 - Lx Micelles (Coordination chemistry in doubly stabilized micelles for the tageted delivery of cytostatic drugs in cancer therapy)

Reporting period: 2015-06-01 to 2017-05-31

Summary of the context and overall objectives of the project
The overall aim of the project is to develop a nanomedicine formulation for cancer therapy. The treatment of cancer with chemotherapeutic drugs is frequently associated with severe side effects limiting their tolerated dose. This often results in insufficient therapeutic effects. The application of such drugs in micelles may improve their biodistribution, but in vivo studies showed fast drug release in the blood stream due to extraction of the drug from the micelles and micellar destabilization, even after micellar crosslinking. This demonstrates that micellar stabilization prevents premature drug release only to a limited extent. We therefore aim for prolonged drug retention and intracellular release by introducing the stable, yet reversible LX linker in the core of bioresorbable micelles based on poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA). This coordinative platinum linker will have two functions: (1) it will facilitate the stable conjugation between the drug and the polymer molecules constituting the micelle and (2) it will act as a stabilizer between the polymer molecules constituting the micelle. We postulate that double stabilization of the micelles (π-π stacking and LX linker mediated coordination between polymer molecules) together with the anchoring of drug molecules via the LX linker results in excellent stability of the drug-loaded micelles in circulation. Due to the EPR effect the micelles will accumulate in tumor tissue, where the micelles will be destabilized intracellularly and the drugs will be released from the linker in the cytoplasm after competitive glutathione displacement. Here, the drug can exert its function and induce tumor cell death.

Conclusion of the action
At the end of the action, several objectives have been achieved. A new type of micelle has been developed which contains anchors in the micellar core (in the form of thioether groups) for coordination with the LX linker. Strong indications have been obtained for LX mediated coordinative core-crosslinking as polymers formed smaller micelles at lower concentrations in the presence of the LX. LX crosslinked micelles were loaded with the hydrophobic drug curcumin (which has various effects, including anti-cancer activities) via the self-assembly method. In the presence of 10 mM dithioerythritol (DTE), mimicking the reductive intracellular environment, the release from LX micelles was significantly faster than from LX micelles in the absence of DTE and faster than the release from normal micelles (which contained no LX linker). This suggests that LX crosslinked micelles can potentially be destabilized intracellularly to induce a fast drug release, whereas they show little drug release in the absence of a reducing agent (such as in the blood stream). Also experiments with drug-LX conjugates have been initiated.
The cytotoxic effect of curcumin-loaded LX micelles on MCF-7 cancer cell viability was higher than that of free curcumin dissolved in 0.1 % DMSO (for the same amount of curcumin, ~8 μg/ml) and that of a saturated solution of curcumin in water. At a higher curcumin concentration (~50 μg/ml), curcumin-loaded non-LX micelles as well as free curcumin in DMSO induced more cytotoxicity than curcumin-loaded LX micelles. The lower cytotoxicity of curcumin-loaded LX micelles may be due to the slower release of curcumin from the LX micelles and/or differences in cellular uptake and intracellular release. Unloaded LX micelles were more cytotoxic than normal micelles, which may be due to the platinum atom of the LX linker.
In vivo studies could not be started within the limited time frame of this project. Therefore, the hypothesis that the LX PEG-P(HPMA-MTB) micelles are able to increase the amount of drug deposited at the target tissue and to reduce side effects at non-target sites compared to systems lacking the LX linker could not yet be validated. On the other hand, there are also n
The following work has been performed during the LX micelles project:
- Synthesis and characterization of HPMA-MTB monomer.
- Synthesis and characterization of PEG-P(HPMA-MTB) polymers.
- Preparation and characterization of PEG-P(HPMA-MTB) micelles.
- Loading of the hydrophobic anti-cancer drug curcumin in PEG-P(HPMA-MTB) micelles and investigation of the release behaviour.
- In vitro cell experiments with unloaded and curcumin-loaded PEG-P(HPMA-MTB) micelles.

The most important results are summarized below.
- It was found that LX mediated coordinative core-crosslinking results in a:
Lower CMC and smaller micellar size.
Lower drug loading.
More sustained, but non-quantitative release.
- LX micelles can be destabilized via competitive displacement, resulting in significantly faster drug release.
- The cytotoxic effect of curcumin-loaded LX micelles on MCF-7 cancer cell viability is higher than that of free curcumin dissolved in 0.1 % DMSO (for the same amount of curcumin, ~8 μg/ml) and that of a saturated solution of curcumin in water.
- At a higher curcumin concentration (~50 μg/ml), curcumin-loaded non-LX micelles induce more cytotoxicity than curcumin-loaded LX micelles.
- Unloaded LX micelles are more cytotoxic than normal micelles.

To date, the results of the ‘LX micelles’ project have been disseminated via presentations at national and international conferences. Depending on the outcomes of the experiments which are currently underway (most notably those with drug-LX conjugates), a patent application may be filed. Furthermore, a paper is in preparation. Various collaborations resulted in the publication of 3 papers on other subjects, whereas 3 more papers are in preparation.
The uniqueness of the approach applied in the LX micelles project resides in the dual functionality of the LX linker: (1) it facilitates coordinative crosslinks between the polymer molecules constituting the micelle and (2) it facilitates the stable coordination between the drug and the polymer molecules constituting the micelle. So far, concept (1) has been demonstrated and experiments involving concept (2) are underway and will be finished in due time.
Although the project has not yet gone beyond the state of the art yet (coordinative crosslinking in the micellar core has been demonstrated previously), it is expected that the micellar core-crosslinking together with the anchoring of drug molecules via LX will result in an excellent stability of the drug-loaded micelles in circulation. This method has the potential to significantly increase the amount of drug deposited at the target tissue and to reduce the side effects at non-target sites. As such, the dual functionality of the LX linker in PEG-P(HPMA-MTB) micelles may still constitute a significant step forward in cancer therapy, which clearly will be of significant clinical, social and economic interest.
Cumulative release of curcumin from non-LX and LX crosslinked PEG-P(HPMA-MTB) micelles
Synthesis of HPMA-MTB (top), (PEG)2-ABCPA macro-initiator (middle) & PEG-P(HPMA-MTB) (bottom)
Formation of non-LX PEG-P(HPMA-MTB) micelles (top) and LX PEG-P(HPMA-MTB) micelles (bottom)
Curcumin in H2O (left). Curcumin-loaded non-LX (middle) and LX (right) PEG-P(HPMA-MTB) micelles