Transport of Tat Peptide Cargoes in 3D Cellular Assemblies - Obtaining Efficient Penetration of Drugs into Tumors

Abstract

Hypoxia and deficient vascular supply to tumor tissues hinder the penetration of drugs required for therapy. Cell penetrating peptides (CPPs), including the Tat peptide, are used as delivery vectors for peptides, protein and DNA into cells. A proposed strategy to improve the delivery of chemotherapy is to conjugate CPPs with anticancer agents to enhance uptake within tumor tissues. Multicellular tumor spheroids (MCTS) may be a useful tool in developing such agents because they mimic relevant in-vivo characteristics such as hypoxia, heterogeneity, and cell packing. The aims of this research project were to characterize the spheroid model, determine qualitative and quantitative measures of penetration of Tat, and specifically to determine whether packing of cells into three-dimensional aggregates constitutes a delivery barrier to CPPs. Growth and viability of spheroids (HT-29 cell line) were measured by haemocytometry, confocal microscopy, and flow cytometry. Compaction of spheroids occurred from day 3-7, with the mature spheroids having low extracellular volume fraction. This provides a rationale to predict less rapid penetration as spheroids mature, and suggests that older (>≈7day) spheroids may better represent actual tumor tissue. Confocal microscopy shows a calcein-AM (live cell marker) fluorescent peak and propidium iodide (dead cell marker) local minimum at ≈45 micro metre depth, consistent with the geometrical model of a viable shell. Flow cytometry results show that with spheroid age, the percentage of hypoxic cells and size of necrotic region increases while the fraction of proliferating cells decreases. Accumulation of fluorescently tagged Tat peptide was observed by time-lapse confocal microscopy at different temperatures and for different ages of spheroids. Results demonstrated a statistically significant effect (ANOVA, p < 0.05) for exposure time and distance from spheroid surface. Penetration into the spheroid represents a balance between diffusion in the extracellular space versus binding to cell surface and subsequent internalization. A more rapid accumulation of labelled peptide in less developed (3-day) spheroids is attributed to their greater extracellular volume fraction. The distribution of various cellular labels depended on the cellular model used, with calcein-AM labelling greatest in cell monolayer > disaggregated spheroids > intact spheroids. The results show that intact spheroids mimic in-vivo tumor tissue in the presentation of a penetration barrier to the vital cells stains calcein-AM. Factors that may affect the transport of molecules into cells (temperature, concentration, incubation time and age of spheroids) were examined quantitatively through flow cytometry. The average uptake of labelled Tat peptide in cell monolayers was greater than the average cellular uptake in spheroids aged five and seven days but less than that in three day old spheroids (ANOVA, p < 0.05). Increase in temperature from 7 to 37°C increased the transport non-significantly by < 5%; the effect of incubation time (from 20 min to 3 h) was also non-significant. However, with increase in the Tat concentration, uptake significantly increased, where the mean fluorescence intensity was 2, 6 and 11 fold higher for 1, 5 and 25 μM Tat as compared to control sample.Flow cytometry and confocal microscopy studies are in agreement that Tat readily diffuses to deep cell layers in younger (3-day) spheroids. There is no indication of a "penetration barrier" in the recently-formed spheroid; and this could be partly attributed to the geometric arrangement of live proliferating cells in younger spheroids as compared to older ones. In older spheroids, only a small proportion of cells show uptake of labelled Tat, but the major reason for this is a decrease in the fraction of live proliferating cells.

Date of Award	1 Aug 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Alain Pluen (Supervisor), Harmesh Aojula (Supervisor) & David Alan Berk (Supervisor)