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Background: Plant derived saponins or other glycosides are widely used for their anti-inflammatory, antioxidant, and anti-viral properties in therapeutic medicine. In this study, we focus on understanding the function of the less known steroidal saponin from the roots of Liriope muscari L.H.Bailey – saponin C (also known as DT-13) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages in comparison to the well-known saponin ginsenoside Rk1 and dexamethasone. Results: We proved that DT-13 reduces LPS-induced inflammation by inhibiting nitric oxide (NO) production, interleukin-6 (IL-6) release, cycloxygenase-2 (COX-2) and tumour necrosis factor-alpha (TNF-α) gene expression, and nuclear factor kappa-B (NFκB) translocation into the nucleus. It also inhibits the inflammasome component NOD-like receptor family pyrin domain containing protein 3 (NLRP3) regulating the inflammasome activation. This was supported by the inhibition of interleukin 1 beta (IL-1β) and caspase-1 levels. Conclusion: This study demonstrates the anti-inflammatory effect of saponins on LPS-stimulated macrophages. For the first time, an in vitro study shows the attenuating effect of DT-13 on NLRP3-inflammasome formation. In comparison to the existing anti-inflammatory drug, dexamethasone, and the triterpenoid saponin from ginseng plant, Rk1, DT-13 more efficiently inhibits inflammation in the applied cell culture model. Therefore, DT-13 may serve as a lead compound for the development of new more effective anti-inflammatory drugs with minimized side effects.

Simultaneous visualization and concentration quantification of molecules in biological tissue is an important though challenging goal. The advantages of fluorescence lifetime imaging microscopy (FLIM) for visualization, and electron paramagnetic resonance (EPR) spectroscopy for quantification are complementary. Their combination in a multiplexed approach promises a successful but ambitious strategy because of spin label‐mediated fluorescence quenching. Here, we solved this problem and present the molecular design of a dual label (DL) compound comprising a highly fluorescent dye together with an EPR spin probe, which also renders the fluorescence lifetime to be concentration sensitive. The DL can easily be coupled to the biomolecule of choice, enabling in vivo and in vitro applications. This novel approach paves the way for elegant studies ranging from fundamental biological investigations to preclinical drug research, as shown in proof‐of‐principle penetration experiments in human skin ex vivo., A novel multi‐label synthetic platform for a functional dual fluorescence–spin label probe enables the non‐destructive simultaneous quantification and visualization of molecules in biological tissue. Multiplexed FLIM and EPR spectroscopy avoids analytical inconsistencies between both techniques. Beside tissue applications, molecular spectroscopic studies of biomolecular conformation, structure, dynamics, and microenvironment are feasible.

The introduction of cleavable motifs by dynamic covalent chemistry is widely applied in the design of drug delivery systems (DDS) to introduce controlled release properties. Since the cleavable moieties can be triggered by various exogenous or endogenous stimuli, the choice of the linker has substantial implications on the performance of the DDS. Here, we present a pair of theranostic polymer conjugates (TPC) to study the influence of the cleavable bond on the cell-mediated drug release by a facile in vitro fluorescence assay. The TPC represent model DDS that consist of dendritic polyglycerol as polymeric carrier labeled with an indodicarbocyanine (IDCC) dye and the chemotherapeutic drug doxorubicin (Dox) conjugated through different cleavable linkers. Cleavage of the conjugate can be mediated by either acidic environment or protease activity. The spatial proximity of the IDCC dye and the fluorescent drug led to effective quenching of Dox fluorescence when bound to the carrier. The stimuli-induced linker cleavage was correlated with the recovery of fluorescence giving real-time information about the stimuli-dependent drug release. By tracking the fluorescence recovery in a cell-based high throughput microplate assay, we were able to obtain characteristic release profiles of Dox for different cell lines. Here, we found that the pH-cleavable linker was more suitable for drug delivery applications since the enzyme-sensitive system suffered premature release due to the presence of extracellular proteases. This had a pronounced effect on the treatment of a multidrug-resistant cell line where an intracellular drug release is crucial to overcome the resistance mechanisms. We want to highlight that the modular synthetic approach combined with the cell-based assay has potential to extend the common in vitro methods to evaluate DDS performance and suitability as the design can be easily employed for diverse carrier/linker systems as well as various cell lines.

Hair follicles can serve as an effective reservoir for dermal drug delivery upon the topical application of particulate substances. Here, the follicular penetration of an indodicarbocyanine-labelled thermoresponsive nanogel (189 nm) having a cloud point temperature of 34°C and linked via an acid-labile linker to the model drug indocarbocyanine was investigated. In total, 227 hair follicles of porcine ear skin were examined after topical application of the thermoresponsive nanogels at room temperature (21°C), physiological skin surface temperature (32°C) and core body temperature (37°C) for the follicular penetration depths of indodicarbocyanine and indocarbocyanine using confocal laser scanning microscopy. The results showed a significantly increased mean follicular penetration of the carrier to a depth of 298.8±85.8 μm after incubation at 37°C compared to samples incubated at 21°C and 32°C with mean follicular penetration depths of 202.7±81.7 μm and 219.4±52.9 μm, respectively (p

Physiological barriers inside of tumor tissue often result in poor interstitial penetration and heterogeneous intratumoral distribution of nanoparticle-based drug delivery systems (DDS). Novel, matrix metalloproteinase (MMP)-sensitive peptide-crosslinked nanogels (pNGs) as multistage DDS are reported with a beneficial size reduction property to promote the process of deep tissue penetration. Methods: The presented pNGs are based on a dendritic polyglycerol (dPG) scaffold crosslinked by a modified MMP-sensitive fluorogenic peptide. The crosslinker integrates degradability in response to proteases present in the tumor microenvironment. Surfactant-free, inverse nanoprecipitation is employed to prepare the nanogels using strain-promoted click chemistry. The size and crosslinking density of the pNGs are controlled by the functionalization degree of dPG with cyclooctyne groups and by the peptide crosslinker fraction. The intrinsic reporter moiety of the crosslinker was used to study the influence of pNG compositions on the degradation profile. The therapeutic drug Doxorubicin was conjugated through a pH-sensitive linkage to dPG to form a multistage DDS. The penetration behavior of the pNGs was studied using agarose matrix and multicellular tumor spheroids (MCTS). Results: Nanogel sizes were controlled in the range of 150-650 nm with narrow size distributions and varying degrees of crosslinking. The pNGs showed stability in PBS and cell media but were readily degraded in the presence of MMP-7. The crosslinking density influenced the degradation kinetic mediated by MMP-7 or cells. Stable conjugation of DOX at physiological pH and controlled drug release at acidic pH were observed. The digestions of nanogels lead to a size reduction to polymer-drug fragments which efficiently penetrated into agarose gels. Moreover, the degradable multistage pNGs demonstrated deeper penetration into MCTS as compared to their non-degradable counterparts. Thus, degradable pNGs were able to deliver their cargo and efficiently reduce the cell viability in MCTS. Conclusion: The triggered size reduction of the pNGs by enzymatic degradation can facilitate the infiltration of the nanocarrier into dense tissue, and thereby promote the delivery of its cargo.

Nanogels (or nano-sized hydrogels) have been extensively investigated as an effective drug delivery system due to their various advantageous properties. Among them, stimuli responsive ‘smart’ nanogels, which have the ability to respond to various external stimuli, such as pH, redox, temperature, enzymes, and light, are the most attractive in the area of controlled anti-cancer drug delivery. In this book chapter, we review and discuss recent progress in the synthesis and applications of polymer-based stimuli-responsive nanogels for anti-cancer therapy and their future prospects.

Aim: To develop an acid-sensitive lipidated, doxorubicin (Dox) prodrug (C16-Dox) to be entrapped in lipid nanoemulsion (NE-C16-Dox) as a nanocarrier to treat breast cancer models (in vitro and in vivo). Results: We report the efficacy of NE-C16-Dox in in vitro experiments, as well as the improved chemotherapeutic index and tumor-control efficacy compared with treatment with free Dox in an in vivo murine 4T1 breast cancer model. In addition, NE-C16-Dox allowed the use of a higher dose of Dox, acceptable biocompatibility and a significant reduction in lung metastasis. Conclusion: Taken together, these results indicate that NE-C16-Dox is promising for breast cancer treatment, thus creating possibilities to translate these nanotechnology concepts to clinical applications.

Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening.