"Bind, cleave and leave": multiple turnover catalysis of RNA cleavage by bulge-loop inducing supramolecular conjugates.

Antisense sequence-specific knockdown of pathogenic RNA offers opportunities to find new solutions for therapeutic treatments. However, to gain a desired therapeutic effect, the multiple turnover catalysis is critical to inactivate many copies of emerging RNA sequences, which is difficult to achieve without sacrificing the sequence-specificity of cleavage. Here, engineering two or three catalytic peptides into the bulge-loop inducing molecular framework of antisense oligonucleotides achieved catalytic turnover of targeted RNA. Different supramolecular configurations revealed that cleavage of the RNA backbone upon sequence-specific hybridization with the catalyst accelerated with increase in the number of catalytic guanidinium groups, with almost complete demolition of target RNA in 24 h. Multiple sequence-specific cuts at different locations within and around the bulge-loop facilitated release of the catalyst for subsequent attacks of at least 10 further RNA substrate copies, such that delivery of only a few catalytic molecules could be sufficient to maintain knockdown of typical RNA copy numbers. We have developed fluorescent assay and kinetic simulation tools to characterise how the limited availability of different targets and catalysts had restrained catalytic reaction progress considerably, and to inform how to accelerate the catalytic destruction of shorter linear and larger RNAs even further.

Sequence-Specific Detection of Unlabeled Nucleic Acid Biomarkers Using a "One-Pot" 3D Molecular Sensor.

DNA and RNA biomarkers have not progressed beyond the automated specialized clinic due to failure in the reproducibility necessary to standardize robust and rapid nucleic acid detection at the point of care, where health outcomes can be most improved by early-stage diagnosis and precise monitoring of therapy and disease prognosis. We demonstrate here a new analytical platform to meet this challenge using functional 3D hydrogels engineered from peptide and oligonucleotide building blocks to provide sequence-specific, PCR-free fluorescent detection of unlabeled nucleic acid sequences. We discriminated at picomolar detection limits (<7 pM) "perfect-match" from mismatched sequences, down to a single nucleotide mutation, buried within longer lengths of the target. Detailed characterization by NMR, TEM, mass spectrometry, and rheology provided the structural understanding to design these hybrid peptide-oligonucleotide biomaterials with the desired sequence sensitivity and detection limit. We discuss the generic design, which is based on a highly predictable secondary structure of the oligonucleotide components, as a platform to detect genetic abnormalities and to screen for pathogenic conditions at the level of both DNA (e.g., SNPs) and RNA (messenger, micro, and viral genomic RNA).

Peptidyl-oligonucleotide conjugates demonstrate efficient cleavage of RNA in a sequence-specific manner.

Described here is a new class of peptidyl-oligonucleotide conjugates (POCs) which show efficient cleavage of a target RNA in a sequence-specific manner. Through phosphoramidate attachment of a 17-mer TΨC-targeting oligonucleotide to amphiphilic peptide sequences containing leucine, arginine, and glycine, zero-linker conjugates are created which exhibit targeted phosphodiester cleavage under physiological conditions. tRNA(Phe) from brewer's yeast was used as a model target sequence in order to probe different structural variants of POCs in terms of selective TΨC-arm directed cleavage. Almost quantitative (97-100%) sequence-specific tRNA cleavage is observed for several POCs over a 24 h period with a reaction half-life of less than 1 h. Nontargeted cleavage of tRNA(Phe) or HIV-1 RNA is absent. Structure-activity relationships reveal that removal of the peptide's central glycine residue significantly decreases tRNA cleavage activity; however, this can be entirely restored through replacement of the peptide's C-terminal carboxylic acid group with the carboxamide functionality. Truncation of the catalytic peptide also has a detrimental effect on POC activity. Based on the encouraging results presented, POCs could be further developed with the aim of creating useful tools for molecular biology or novel therapeutics targeting specific messenger, miRNA, and genomic viral RNA sequences.

Microfluidic-assisted fabrication of phosphatidylcholine-based liposomes for controlled drug delivery of chemotherapeutics.

Microfluidic enables precise control over the continuous mixing of fluid phases at the micrometre scale, aiming to optimize the processing parameters and to facilitate scale-up feasibility. The optimization of parameters to obtain monodispersed drug-loaded liposomes however is challenging. In this work, two phosphatidylcholines (PC) differing in acyl chain length were selected, and used to control the release of the chemotherapeutic agent doxorubicin hydrochloride, an effective drug used to treat breast cancer. Microfluidics was used to rapidly screen manufacturing parameters and PC formulations to obtain monodispersed unilamellar liposomal formulations with a reproducible size (i.e. < 200 nm). Cholesterol was included in all liposomal formulations; some formulations also contained DMPC(1,2-dimyristoyl-sn-glycero-3-phosphocholine) and/or DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine). Systematic variations in microfluidics total flow rate (TFR) settings were performed, while keeping a constant flow rate ratio (FRR). A total of six PC-based liposomes were fabricated using the optimal manufacturing parameters (TFR 500 µL/min, FRR 0.1) for the production of reproducible, stable liposome formulations with a narrow size distribution. Liposomes actively encapsulating doxorubicin exhibited high encapsulation efficiencies (>80%) for most of the six formulations, and sustained drug release profiles in vitro over 48 h. Drug release profiles varied as a function of the DMPC/DSPC mol content in the lipid bilayer, with DMPC-based liposomes exhibiting a sustained release of doxorubicin when compared to DSPC liposomes. The PC-based liposomes, with a slower release of doxorubicin, were tested in vitro, as to investigate their cytotoxic activity against three human breast cancer cell lines: the non-metastatic ER+/PR + MCF7 cells, the triple-negative aggressive MDA-MB 231 cells, and the metastatic HER2-overexpressing/PR + BT474 cells. Similar cytotoxicity levels to that of free doxorubicin were reported for DMPC5 and DMPC3 binary liposomes (IC50 ~ 1 µM), whereas liposomes composed of a single PC were less cytotoxic (IC50 ~ 3-4 µM). These results highlight that microfluidics is suitable for the manufacture of monodispersed and size-specific PC-based liposomes in a controlled single-step; furthermore, selected PC-based liposome represent promising nanomedicines for the prolonged release of chemotherapeutics, with the aim of improving outcomes for patients.

Inhibition of aggregation of amyloid-ß through covalent modification with benzylpenicillin; potential relevance to Alzheimer's disease.

The pathogenesis of Alzheimer's disease (AD) is correlated with the misfolding and aggregation of amyloid-beta protein (Aß). Here we report that the antibiotic benzylpenicillin (BP) can specifically bind to Aß, modulate the process of aggregation and supress its cytotoxic effect, initially via a reversible binding interaction, followed by covalent bonding between specific functional groups (nucleophiles) within the Aß peptide and the beta-lactam ring. Mass spectrometry and computational docking supported covalent modification of Aß by BP. BP was found to inhibit aggregation of Aß as revealed by the Thioflavin T (ThT) fluorescence assay and atomic force microscopy (AFM). In addition, BP treatment was found to have a cytoprotective activity against Aß-induced cell cytotoxicity as shown by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell toxicity assay. The specific interaction of BP with Aß suggests the possibility of structure-based drug design, leading to the identification of new drug candidates against AD. Moreover, good pharmacokinetics of beta-lactam antibiotics and safety on long-time use make them valuable candidates for drug repurposing towards neurological disorders such as AD.

Manufacturing drug co-loaded liposomal formulations targeting breast cancer: Influence of preparative method on liposomes characteristics and in vitro toxicity.

Developing more efficient manufacturing methods for nano therapeutic systems is becoming important, not only to better control their physico-chemical characteristics and therapeutic efficacy but also to ensure scale-up is cost-effective. The principle of cross-flow chemistry allows precise control over manufacturing parameters for the fabrication of uniform liposomal formulations, as well as providing reproducible manufacturing scale-up compared to conventional methods. We have herein investigated the use of microfluidics to produce PEGylated DSPC liposomes loaded with doxorubicin and compared their performance against identical formulations prepared by the thin-film method. The isoprenylated coumarin umbelliprenin was selected as a co-therapeutic. Umbelliprenin-loaded and doxorubicin:umbelliprenin co-loaded liposomes were fabricated using the optimised microfluidic set-up. The role of umbelliprenin as lipid bilayer fluidity modulation was characterized, and we investigated its role on liposomes size, size distribution, shape and stability compared to doxorubicin-loaded liposomes. Finally, the toxicity of all liposomal formulations was tested on a panel of human breast cancer cells (MCF-7, MDA-MB 231, BT-474) to identify the most potent formulation by liposomal fabrication method and loaded compound(s). We herein show that the microfluidic system is an alternative method to produce doxorubicin:umbelliprenin co-loaded liposomes, allowing fine control over liposome size (100-250 nm), shape, uniformity and doxorubicin drug loading (>80%). Umbelliprenin was shown to confer fluidity to model lipid biomembranes, which helps to explain the more homogeneous size and shape of co-loaded liposomes compared to liposomes without umbelliprenin. The toxicity of doxorubicin:umbelliprenin co-loaded liposomes was lower than that of free doxorubicin, due to the delayed release of doxorubicin from liposomes. An alternative, rapid and easy manufacturing method for the production of liposomes has been established using microfluidics to effectively produce uniform doxorubicin:umbelliprenin co-loaded liposomal formulations with proven cytotoxicity in human breast cancer cell lines in vitro.

Resveratrol-mediated cleavage of amyloid ß1-42 peptide: potential relevance to Alzheimer's disease.

Aggregation of amyloid ß1-42 (Aß1-42) peptide within the brain is considered one of the main causes of the neuropathological changes associated with Alzheimer's disease. Resveratrol is a well-known antioxidant but has also been reported to bind to Aß1-42 peptide, thereby reducing aggregation. However, little is known of the precise mechanism by which resveratrol reduces Aß1-42 peptide aggregation. Using the thioflavin-T assay, the ability of resveratrol to reduce the extent of Aß1-42 peptide aggregation was investigated. The findings of the present study demonstrate that interaction of resveratrol with Aß1-42 peptide resulted in the cleavage of Aß1-42 peptide into smaller fragments, as detected by matrix assisted laser desorption ionization-time of flight mass spectrometry. Atomic force microscopy analyses revealed Aß1-42 peptide, under control conditions, aggregated into oligomers, protofibrils, and fibrils, whereas there was a distinct lack of these structures when Aß1-42 peptide was incubated with resveratrol. Following 10 days incubation of Aß1-42 peptide with resveratrol, particles with a mean z-height of 1.940 nm (range 0.675-3.275 nm) were observed, which are characteristic of shorter peptide species. In cell-based studies, resveratrol significantly reduced the cytotoxicity of Aß1-42 peptide toward SH-SY5Y human neuroblastoma cells, suggesting a protective effect of the polyphenol. We therefore propose a novel mechanism by which resveratrol disrupts Aß1-42 aggregation by mediating fragmentation of Aß1-42 into smaller peptides, which have no propensity to aggregate further.

Pentapeptides for the treatment of small cell lung cancer: Optimisation by Nind-alkyl modification of the tryptophan side chain.

The pentapeptide, tert-Prenyl4th-NH2 (DMePhe-DTrp-Phe-DTrp(N-tert-prenyl)-Leu-NH2), has recently been reported by our group to exhibit properties of substance P (SP) antagonist G against small cell lung cancer (SCLC). In this study, we undertook a systematic structure activity investigation to optimise this lead compound to improve its in vitro anti-tumour activity and biocompatibility. A series of d-tryptophan (D-Trp) derivatives were synthesised, with a range of aliphatic N-alkyl chains (methyl to pentyl) on the indole nitrogen (Nind). These were incorporated into the pentapeptide sequence by substitution of the Nind-tert-prenylated D-Trp 4th residue with the Nind-alkylated D-Trp derivatives. These pentapeptides were significantly more potent than tert-Prenyl4th-NH2, with the Nind-butyl modification generating the most cytotoxic peptides. Compared to tert-Prenyl4th-NH2, a single butyl modification on the 4th D-Trp residue (Butyl4th-NH2) showed a ∼3 fold enhancement in cytotoxicity in either the chemo-naive H69 or the DMS79 (originating from a patient treated with chemotherapeutics and radiation therapy) SCLC cell lines. In addition, the di-butylated sequence on the 2nd and 4th D-Trp residues (Butyl2nd,4th-NH2) gave ∼4.5 times higher cytotoxicity against the H69 cell line and a ∼2 fold increase against the DMS79 cell line, compared to tert-Prenyl4th-NH2. The favoured position for butyl modification was the 4th D-Trp residue, as the Butyl2nd-NH2 peptide gave lower cytotoxicity on both cell lines. Butylated peptide sequences, when exposed to neat mouse plasma for 24 h at 37 °C, were found to resist degradation with >80% remaining intact compared to ∼58% for tert-Prenyl4th-NH2. The degradation pathway in plasma occurs via de-amidation of the C-terminus, confirmed by mass spectrometry and RP-HPLC analysis. The butyl modification also conferred resistance to metabolism when tested using S9 liver fraction from mouse. The optimum analogue responsive against the DMS79 cell line was the Butyl4th-NH2 pentapeptide, which revealed a concentration dependent increase in apoptosis: the level of late apoptotic cells rose from ∼36% at 2 µM to ∼96% at 6 µM, as determined by flow cytometry, compared to the unmodified peptide that showed no such effect. Concluding, the butyl substitutions offered the best perspective for high cytotoxicity, induction of apoptosis and metabolic compatibility thereby comprising an improved broad spectrum SP antagonist candidate for treatment of SCLC.

N-tert-Prenylation of the indole ring improves the cytotoxicity of a short antagonist G analogue against small cell lung cancer.

Natural prenylated indoles have been proposed as potential anticancer agents. To exploit this discovery for developing new peptide therapeutics, we report the first studies whereby incorporation of prenylated indoles into primary sequences has been achieved. We developed a route to synthesise Nα-Fmoc-protected tryptophan derivatives in which the prenyl group is linked to the N-indole core, using Pd(ii)-mediated C-H functionalisation of 2-methyl-2-butene. Based on the Substance P antagonist G (SPG), a well-known Small Cell Lung Cancer (SCLC) anticancer agent, we designed a new penta-peptide sequence to include a prenyl moiety on one of the tryptophan residues. The N-tert-prenylated tryptophan analogue was assembled into the pentameric peptide using standard solid phase peptide synthesis or liquid phase synthesis by fragment coupling. In vitro screening showed that the N-tert-prenylation of the indole ring on the tryptophan residue located near the C-terminal of the penta-peptide enhanced the cytotoxicity against H69 (IC50 = 2.84 ± 0.14 µM) and DMS79 (IC50 = 4.37 ± 0.44 µM) SCLC cell lines when compared with the unmodified penta-peptide (H69, IC50 = 30.74 ± 0.30 µM and DMS79, IC50 = 23.00 ± 2.07 µM) or the parent SPG sequence (IC50 > 30 µM, both cell lines). SCLC almost invariably relapses with therapy-resistant disease. The DMS79 cell line was established from a patient following treatment with a number of chemotherapeutics (cytoxan, vincristine and methotrexate) and radiation therapy. Treating DMS79 tumour-bearing nude mice provided a human xenograft model of drug resistance to test the efficacy of the prenylated peptide. A low dose (1.5 mg kg-1) of the prenylated peptide was found to reduce tumour growth by ∼30% (P < 0.05) at day 7, relative to the control group receiving vehicle only. We conclude that the availability of the Fmoc-Trp(N-tert-prenyl)-OH amino acid facilitates the synthesis of prenylated-tryptophan-containing peptides to explore their therapeutic potential.

Effects of different isoforms of apoE on aggregation of the α-synuclein protein implicated in Parkinson's disease.

Parkinson's disease is a progressive brain disorder due to the degeneration of dopaminergic neurons in the substantia nigra. The accumulation of aggregated forms of α-synuclein protein into Lewy bodies is one of the characteristic features of this disease although the pathological role of any such protein deposits in causing neurodegeneration remains elusive. Here, the effects of different apolipoprotein E isoforms (apoE2, apoE3, apoE4) on the aggregation of α-synuclein in vitro were examined using thioflavin T assays and also an immunoassay to detect the formation of multimeric forms. Our results revealed that the aggregation of α-synuclein is influenced by apoE concentration. At low concentrations of apoE (<15nM), all of the isoforms were able to increase the aggregation of α-synuclein (50µM), with apoE4 showing the greatest stimulatory effect. This is in contrast to a higher concentration (>15nM) of these isoforms, where a decrease in the aggregation of α-synuclein was noted. The data show that exceptionally low levels of apoE may seed α-syn aggregation, which could potentially lead to the pathogenesis of α-synuclein-induced neurodegeneration. On the other hand, higher levels of apoE could potentially lower the degree of α-synuclein aggregation and confer protection. The differential effects noted with apoE4 could explain why this particular isoform results in an earlier age of onset for Parkinson's disease.

Cloaking cytolytic peptides for liposome-based detection and potential drug delivery.

Potent cytolytic peptides with specific tethering and cloaking sites have been synthesised and used to release payload from liposomes in a quantitative manner. A functionally located cloaking site has been modified specifically by simple conjugation without adversely affecting the cytolytic properties of the peptide. The cytolytic activity of modified peptides was then efficiently (>98%) cloaked and uncloaked by ligand-protein or hapten-antibody interactions. The principle of a dual response peptide has been demonstrated using an avidin-cloaked pH-sensitive peptide. Biospecific cloaking/uncloaking provided a new sensitive (approximately 12 pmol) homogeneous diagnostic and also appears potentially suited to bioresponsively targeted release of antimicrobial, anticancer and other drugs now delivered using liposomes.

On the cellular uptake and membrane effect of the multifunctional peptide, TatLK15.

Recently, the multifunctional peptide TatLK15 resulting from the fusion of the cell penetrating peptide Tat and the amphipathic peptide LK15 was shown to be efficient at mediating siRNA and shRNA delivery in leukemia cells to silence the bcr-abl oncoprotein. The present study focused on TatLK15 peptide cellular uptake and defining conditions for its use within a range of doses exhibiting minimal toxicity. The initial part of the study carried out in solution confirmed that the insertion of a glycine bridge allowed retention of the LK15 α-helicity, and fluorescence correlation spectroscopy did not reveal preferential conformations at the studied concentrations. In the second part, TatLK15 uptake mechanisms appeared peptide dose- and cell line- dependent as well as requiring membrane potential. Below a critical dose, TatLK15 toxicity appeared limited for approximately three hours as demonstrated by the combined use of lactate dehydrogenase release, MTT assays, and time-dependent observation of membrane-impermeant dye uptake using high content screening apparatus. Furthermore, toxicity was observed to occur rapidly at higher peptide doses. Finally, a comparison between TatLK15 and another Tat amphipathic peptide construct suggested that α-helix content should be viewed as a key element in the development of similar peptides.

Reduction of doxorubicin resistance in P-glycoprotein overexpressing cells by hybrid cell-penetrating and drug-binding peptide.

Drug efflux by the membrane transporter P-glycoprotein (P-gp) plays a key role in multidrug resistance (MDR). In order to bypass P-gp, thus overcoming MDR, a hybrid peptide comprising a cell penetrating peptide (Tat) and a drug binding motif (DBM) has been developed to noncovalently bind and deliver doxorubicin (Dox) into MDR cells. The uptake of Dox into the leukemia cell line K562 and its P-gp overexpressing subline KD30 increased in the presence of DBM-Tat peptide. Confocal microscopy indicated that DBM-Tat associated Dox was directed to a perinuclear area of KD30 cells, while this was not observed in parent K562 cells. When KD30 cells were pretreated with the endosomotropic agent chloroquine (CLQ), peptide associated Dox redistributed into the cytosol, indicating that endocytosis was the predominant uptake route. Altered drug uptake kinetics observed by cellular accumulation assay also supported an endocytic uptake. In the presence of CLQ, DBM-Tat was able to enhance the cytotoxicity of Dox by 68.4% at 5 microM peptide concentration in KD30 cells but there were only minor effects on Dox cytotoxicity in K562 cells even in the presence of CLQ. Thus, combining Dox with DBM-Tat reduces P-gp mediated drug efflux, without a requirement for drug modification or inhibiting P-gp function.

Delivery of therapeutic shRNA and siRNA by Tat fusion peptide targeting BCR-ABL fusion gene in Chronic Myeloid Leukemia cells.

Gene silencing by RNA interference (RNAi) is a promising therapeutic approach for a wide variety of diseases for which the biological cause is known. The main challenge remains the ineffective RNAi delivery inside the cells. Non-viral gene delivery vectors have low immunogenicity compared to viral vectors, but are constrained by their reduced transfection efficiency. Silencing of the bcr-abl gene expression by RNAi confers therapeutic potential in Chronic Myeloid Leukemia (CML), but is limited by the cytotoxicity of the existing delivery methods. Here, we present evidence that the fusion between the cell penetrating peptide (CPP) HIV-Tat (49-57) and the membrane lytic peptide (LK15), Tat-LK15, mediates high transfection efficiency in delivering short hairpin RNA (shRNA) and small interfering RNA (siRNA) targeting the BCR-ABL oncoprotein in K562 CML cells. Our results show that shRNA complexes induce a more stable gene silencing of bcr-abl when compared to silencing mediated by siRNA complexes. In addition, silencing of the BCR-ABL oncoprotein by both shRNA and siRNA delivered by Tat-LK15 is more efficient and longer lasting than that achieved using Lipofectamine and more importantly without considerable cytotoxicity. In these terms Tat-LK15 can be an alternative to DNA/siRNA delivery in difficult-to-transfect leukemic cells.

Improved Tat-mediated plasmid DNA transfer by fusion to LK15 peptide.

The use of cell penetrating peptides (CPPs), such as Tat-derived peptide, to deliver DNA into cells is limited as evidenced by the low transfection efficiency of their DNA complexes. Here, we demonstrate that covalent attachment of membrane active peptide LK15 to Tat peptide improves gene transfer. Our results demonstrate that Tat peptide was able to form complexes with DNA, but their transfection efficiency was insufficient as assessed by luciferase assay. The attachment of LK15 to Tat significantly improved the physiochemical properties of the DNA complexes, rendered the complexes membrane active and enhanced the gene expression in HT29 and in HT1080 cultured cells. The enhanced transfection ability of Tat-LK15 compared to Tat is likely to be due mainly to the higher uptake of DNA. Finally, we evaluated the penetration and transfection ability of Tat and Tat-LK15 in multicellular tumour spheroids (MCTS) to mimic in vivo delivery to tumours. The results showed that the penetration and transfection ability of Tat and Tat-LK15/DNA complexes were limited to the rim of HT29 spheroids. Taken together, our data shows improvement in the transfection efficiency of Tat peptide by covalent attachment to LK15. Further advancements are needed before any potential applications in tissues as the penetration into the core of MCTS remains severely restricted.

Enhancement of gene transfer using YIGSR analog of Tat-derived peptide.

Cell penetrating peptide based gene carriers are notably known for low level of gene transfer. To remedy this, as laminin receptor (LR) has been previously linked to tumor metastasis, the LR-binding domain (YIGSR) as well as a scrambled sequence (SGIYR) were added to Tat-derived peptide sequence (YIGSR-Tat and SGIYR-Tat respectively). Peptides cellular uptake was assessed with high-LR (HT1080) and low-LR (HT29) cell lines by flow cytometry. Their ability to form complexes with DNA was examined using YOPRO-1 fluorescence assay and their transfection efficiencies evaluated using a luciferase reporter gene assay. DNA complexes were formed at (+/-) charge ratios as low as 2:1. While no conclusion could be drawn on the effect of YIGSR sequence on peptides uptake in both cell lines, a significant improvement in gene transfection in HT1080 cells was achieved using YIGSR-Tat compared to Tat and SGIYR-Tat. Additionally this increased efficiency was inhibited by excess free YIGSR. No significant difference in transfection efficiency was observed between Tat, SGIYR-Tat and YIGSR-Tat based complexes in HT29 cells. These studies demonstrate that attachment of receptor-binding ligand (YIGSR) to Tat-derived peptide can improve the efficiency of gene transfer in LR-positive cells (HT1080).

YOYO as a dye to track penetration of LK15 DNA complexes in spheroids: use and limits.

To elucidate the reasons underlying the poor penetration of non-viral vectors in tissues, relating transport properties to physico-chemical parameters of vectors may be crucial. These properties can be influenced by the presence of multiples labels that are used. Therefore utilizing a vector with minimum of labels preferably not more than one is important to studying penetration in tissues. The cell impermeant bisintercalating dye YOYO-1 was found suitable to both monitor the formation of complexes between DNA and an amphipathic peptide LK15 and, to track their penetration in HCT116 spheroids by confocal microscopy. The results revealed a limited decrease of fluorescence ascribed to the high affinity of YOYO-1 to bind DNA. The residual fluorescence of complexes can be exploited to monitor penetration into spheroids, after correction for YOYO-1 attenuation, and to revealing hyaluronidase-induced reduced binding. Hence high affinity dyes such as YOYO-1 with inefficiently quenched fluorescence may be important to establish a relation between novel medicines characteristics and penetration in tissues.