Rational Design of Responsive Prodrug-based Nanomedicines for Antitumor Therapy

Abstract

A major issue of conventional chemotherapy is the lack of selective delivery which results in high systemic exposure and severe side effects. The limitations of small molecular weight drugs for cancer therapy prompted the development of diverse nanocarrier systems for targeted drug delivery. The impressive progress in nanomaterial science and increased understanding of the nano-bio interface allowed the progression of these systems. However, the targeted delivery remains challenging due to obstacles that are encountered during the drug delivery process, particularly, in the tumor microenvironment (TME). (Multi)stimuliresponsive nanocarriers have the potential to overcome the faced barriers by taking advantage of altered pathological characteristics in the TME and/or intracellular signals. The motivation of the presented work was to incorporate rational design features into novel responsive nanomedicines to address the limitations of conventional chemotherapeutic drugs and tackle issues of current drug delivery systems (DDS). For this purpose, prodrugs of the chemotherapeutic agent doxorubicin (Dox) were combined with three nanocarrier designs including polymer-drug conjugates, a nanoemulsion (NE), and nanogels (NGs). The Dox prodrugs comprised cleavable motifs which introduce a responsiveness towards endogenous stimuli into the nanocarriers. The nanocarrier architectures with different sizes and compositions were evaluated in terms of controlled drug release, drug-carrier compatibility, carrier degradability, and transport restrictions in the TME, all of which are important aspects for an efficient delivery process. The choice of the cleavable linkage strongly affects the specificity of the desired responsive behavior. To address this aspect, Dox prodrugs with pH- or protease-cleavable bonds were evaluated regarding their impact on the intracellular drug release. Activatable fluorescence probes were utilized to follow the drug release from polymer-drug conjugates in real-time. This assessment of the linker formed the basis for the rational design of two prodrug-based nanomedicines with adjusted cleavage properties. First, a pH-sensitive Dox prodrug was entrapped in a NE to form a DDS with explicit intracellular drug release. The second design was based on dual-responsive nanogels as multistage delivery systems with specific extracellular response to protease and acid-mediated intracellular payload release. Initially, we evaluated the impact of the cleavable linkage on the drug release using theranostic polymer conjugates (TPC) with activatable fluorescence probes. The TPC represent model DDS that consist of dendritic polyglycerol (dPG) as polymeric carrier labeled with an indodicarbocyanine (IDCC) dye that quenches the fluorescence of Dox, conjugated through a cleavable linker. Cleavage of the conjugates was mediated either by acidic pH or protease activity. By tracking the fluorescence recovery in a cell-based microplate assay, we were able to obtain characteristic release profiles of Dox for different cell lines. Here, the pH-cleavable linker was found to be cleaved mainly intracellularly, whereas the protease-sensitive system suffered from extracellular drug release. The intracellular release was crucial to treat multidrug-resistant cells and overcome their resistance mechanisms. It can be highlighted that the modular synthetic approach, combined with the cell-based assay, has potential to extend the common in vitro methods to evaluate DDS performance. The results of this study motivated us to develop a pH-sensitive Dox prodrug (C16-Dox) to efficiently dissolve the drug in the nanodroplets of an oil/water NE. By attaching a hydrophobic alkyl chain (C16), Dox was provided with an amphiphilic character for increased drug-carrier compatibility. pH-sensitive properties of the prodrug allowed the intracellular release of the drug from the NE by recovering the hydrophilic parent drug. The new formulation of Dox (NE-C16-Dox) was compared with free Dox in a murine breast cancer model. Enhanced delivery to tumor tissue and reduction of systemic toxicity allowed the administration of a higher Dox dose in the NE formulation as compared to the free drug. The high dosage significantly inhibited the primary tumor growth and prevented the formation of distant lung metastasis without signs of side effects. The improved chemotherapeutic index compared to free Dox indicates that NE-C16-Dox is a promising formulation for breast cancer treatment At last, we combined protease- and pH-sensitive moieties into a multistage nanocarrier to enhance drug transport in tumor tissue. Matrix metalloproteinase (MMP)-sensitive NGs (pNGs) were developed which consists of a dPG scaffold crosslinked with a fluorogenic peptide. The crosslinker integrates biodegradability to the nanocarrier mediated by proteases in the TME. The intrinsic reporter moiety of the crosslinker allowed us to study the influence of different pNG compositions on the degradation profile in detail. One pNG candidate was chosen to conjugate the therapeutic drug Dox through a pH-sensitive linkage to dPG. The degradable multistage pNGs demonstrated deeper penetration into multicellular tumor spheroids (MCTS) as compared to their non-degradable counterparts. Hence, the triggered size reduction of the pNGs by enzymatic degradation facilitated the infiltration of the nanocarrier into dense tissue and thereby promoted the delivery of the therapeutic cargo.