Phase-changing Nanodroplets as Nanotheranostic Platform for Combination Cancer Therapy

Cancer is a cluster of diseases, and 1.8 million Americans are newly diagnosed each year. Treatment issues such as drug instability, the occurrence of severe side effects, as well as resistance make the need for solutions to improve conventional methods, like chemotherapy, apparent. Nano-sized drug-delivery platforms, particles loaded with therapeutic molecules that escape the immune system clearance and accumulate at the tumor site, were proposed as one of these solutions. Despite the expansion of the field, several aspects still need to be addressed: inconsistent delivery of the drugs, inability of measuring the effective dose being delivered to the tumor, lack of predictability of a response. Hence, the field of nanotheranostics was born, that combines drug-delivery nanoparticulate systems with imaging capabilities. By enabling a noninvasive visualization of delivered therapeutic molecules, nanotheranostics offer the opportunity of rapid optimization of drug delivery systems during in vivo testing. This insight into the kinetics and the fate of nanoparticle-encapsulated therapeutics can aid validate basic properties without needing to wait a long time for a pathological outcome. In the clinic, this feature would allow for a fast response in modifying a treatment course. This work describes a versatile nanotheranostic platform capable of triggered release of therapeutic molecules and strong ultrasound imaging contrast simultaneously. The activatable release is designed to minimize off-target effects, while the ultrasound contrast can enable visualization of the delivered dose to a region of interest. The particles described herein consist of a shell-core structure, with a perfluorocarbon iii core that can be externally vaporized by acoustical or optical stimuli. The activation generates highly echogenic microbubbles, together with the release of the loaded compounds. Notably, the release of the payload is correlated to the ultrasound magnitude after activation, enabling the basis for ultrasound dose-monitoring. The design, synthesis and characterization of perfluorocarbon nanodroplets aimed at several applications are described. These include co-delivery of hydrophobic and hydrophilic chemotherapeutics, delivery of a-PD-L1 immunecheckpoint inhibitors together with chemotherapeutics, as well as multimodal imaging capabilities. Overall, this work contributes towards expanding the utility of perfluorocarbon nanodroplets towards combinatorial therapies, and proposes necessary improvements for increased translatability of the technology

Time-Resolved Cryofixation Methods for the Study of Dynamic Cellular Events by Electron Microscopy: A Review

The preservation of cells for electron microscopy by chemical fixation is a lengthy process, requiring up to 30 minutes for cytoplasmic stabilisation. This time lag enables many changes to occur in specimens so that they may not reflect their living state when they are observed in electron microscopes. Many artefacts can be avoided by using cryofixation, which freezes specimens over a period that is measured in milliseconds, so that specimens are preserved by cryoimmobilisation. This time resolution can be used to study rapid processes in biology and chemistry because, although electron microscopes cannot observe dynamic cellular events directly, processes can be arrested after known time intervals so that transient stages are preserved and a series of time-lapse steps is acquired. Some experiments have involved freezing specimens which were maintained in controlled states and others have shown results after stimulation where structural differences are seen between one millisecond and the next. The experimental techniques that have been applied prior to freezing are electrical and chemical stimulation, electrophoresis, chemical relaxation after a temperature jump, electroporation, which is analogous to relaxation after applying a radio frequency electrical field, and flash photolysis methods. This review describes the origins and application of time-resolved freezing, which integrates electron microscopy with dynamic biochemical, physiological, and ultrastructural events

Biophysical investigation of novel cryoprotectants

This thesis aims to examine the critical molecular properties that lead to good cryoprotective performance and use this knowledge to test novel non-toxic compounds which can be optimized to use as cryoprotectants (CPAs). The key properties needed to make a good CPA are: low toxicity; the ability to pass through membranes to get inside cells; the ability to form glasses at relatively high sub-zero temperatures; and the ability to inhibit ice recrystallisation during thawing (the growth of larger ice crystals from smaller ones). This thesis presents a systematic investigation of these important properties, for both traditional CPAs and novel compounds with cryoprotective potential. Cryopreservation trials with some of these compounds are carried out to assess their cryoprotective potential. First, this thesis presents a Langmuir monolayer study of the effects of four common cryoprotective agents (dimethyl sulfoxide, ethylene glycol, glycerol and dimethyl formamide) on phospholipid monolayers. Four different phospholipids (DOPC, DPPC, POPC, POPE) are studied to determine if the head group and level of chain saturation influence the interactions. It is shown that the phospholipid species can have a significant effect. DMSO showed interesting lipid specific effects - causing expansion of DPPC monolayers but compression for POPC monolayers, while having little effect for DOPC and POPE. The results highlight the importance of studying more than one model lipid system as well as the need to study concentrations relevant to cryopreservation. Second, this thesis investigates the potential of carbohydrate-based surfactants such as n- octyl(thio)glycosides to be a novel and accessible class of penetrating CPAs. A series of eight n-octyl (thio)glycosides (1alpha/beta - 4alpha/beta) with D-glucose or D-galactose-configured head groups and varying anomeric configurations were evaluated for glass transition behavior and membrane permeability. Of these, n-octyl beta-D-glucopyranoside (2beta) exhibited high glass transition temperature (Tg), both as a neat sample and 20wt-% aqueous solution. Membrane permeability studies of this compound revealed cellular uptake at concentrations relevant to the inhibition of intracellular ice formation, thus presenting a promising lead candidate for further biophysical and cryopreservation studies. Third, this thesis presents similar studies on four trehalose derivatives in order to understand their cryoprotective potential. Permeability trials on these molecules showed that trehalose-di acetate has no cell permeability, whereas both trehalose di pivotate and trehalose tetra acetate have moderate permeability. The most promising candidate with high glass transition temperature and relatively good permeability was trehalose di pivotate, so this compound was used for further cryopreservation studies on THP-1 cells. Finally, this thesis presents the development of a novel microfluidic cell trapping device for cell permeability studies, which has considerable advantages over traditional techniques. Two microfluidic cell trapping structures were produced, combining direct laser writing (DLW) and soft lithography techniques. These structures were used for the hydrodynamic capturing of single human monocyte (THP-1) cells and tested by studying the cell volume kinetics upon the addition of DMSO. Compared to existing technologies, the method allowed: (i) rapid capture of single cells without surface functionalization; (ii) the rapid exchange of solvent to generate osmotic gradients across the cell membrane; and (iii) real-time imaging of cells during the shrinkage and swelling phases due to the imaging stability provided by the traps. To date, the search for new CPAs has been reliant on educated guesswork and trial and error. This thesis systematically investigates the critical molecular properties relevant for cryopreservation and provides a systematic method for assessing novel molecules as alternative cryoprotectants

Controlled particle production by membrane emulsification for mammalian cell culture and release

Existing commercially available microcarriers are very efficient at encouraging cell attachment and proliferation. However, recovery of the cells is problematic as it requires the use of proteolytic enzymes which are damaging to critical cell adhesion proteins. From this perspective, temperature responsive polymers appear to be a valid option. The current innovative study is to produce and engineer microcarriers in terms of particle size, surface coating and properties, as well as thermo-responsiveness for cell release. All these benefits are based on particle production by membrane emulsification to provide a highly controlled particle size. The polymer of choice is poly N-isopropylacrylamide (pNIPAM) because of the sharpness of its phase transition, biocompatibility and transition temperature close to the physiological value. These characteristics make pNIPAM a very attractive material for Tissue Engineering applications. Cells are cultured on the hydrophobic surface at 37°C and can be readily detached without using proteolytic enzymes from the surface by lowering the temperature to room temperature. The Dispersion Cell (MicroPore Technologies Ltd, UK) was successfully employed for the production of W/O emulsions. The generated monomer droplets were additionally solidified by applying a free radical polymerisation to manufacture solid pNIPAM microspheres. Additionally, calcium alginate particles were also generated and further functionalised with amine terminated pNIPAM to form temperature responsive core-shell particles by simply taking advantage of the electrostatic interactions between the carboxyl groups of the alginate and amino groups of the modified pNIPAM. Controlled particle production was achieved by varying process parameters and changing the recipe formulation (e.g. monomer concentration, surfactant concentration, pore size and inter-pore spacing, injection rate, shear stress applied at the membrane s surface). The manufactured particles were then analysed in terms of particle size and size distribution, chemical composition, surface analysis, shrinkage ratio and thermo-responsiveness and further sterilised and used for cell culture and release experiments. Swiss Albino 3T3 fibroblastic cells (ATCC, USA) were utilised to show proof-of-concept for this technology. Cell attachment and proliferation were assessed and successfully demonstrated qualitatively and quantitatively. pNIPAM solid particles, uncoated and with different protein coatings were shown to allow a limited degree of cell attachment and proliferation compared to a commercially available microcarrier. On a different approach, uncoated core-shell structures demonstrated improved capabilities for cell attachment and proliferation, similar to commercially available microcarriers. Having in mind the potential of temperature responsive polymers and the aim of this innovative study, cell detachment from the generated microcarriers was evaluated and compared to a commercially available temperature responsive surface. Necessary time for detachment was recorded and detached cells were recovered and reseeded onto tissue culture plastic surfaces in order to evaluate the replating and reattachment capabilities of the recovered cells. Successful cell detachment was achieved when using the core-shell structures as cell microcarriers, but the necessary time of detachment was of an order higher than that for the commercial temperature responsive surface

The potential use embryonic shoot apices as explants for cryopreservation of selected recalcitrant-seeded species.

Master of Science in Biological Sciences. University of KwaZulu-Natal, Durban, 2017.Abstract available in PDF file

Development of explants potentially suitable for cryopreservation of the recalcitrant-seeded species Theobroma cacao L. and Barringtonia racemosa (L.) roxb.

Thesis (M.Sc.)-University of KwaZulu-Natal, 2008.The two species investigated in this study were Theobroma cacao and Barringtonia racemosa. Theobroma cacao has worldwide economic importance, as cocoa (the main ingredient in chocolate) is produced from the seeds of this tree; while B. racemosa has several applications in herbal medicine. The seeds of both T. cacao and B. racemosa are highly recalcitrant and therefore not amenable to storage for any significant periods. The long-term conservation of the germplasm of these species may only be feasible via cryopreservation. The aims of the present study were to: 1) optimize in vitro regeneration protocols for different types of explants that have the potential to be cryopreserved while maintaining the genetic integrity of these two species; and 2) develop cryopreservation protocols for selected explants. For T. cacao, protocols were established for bud-break and multiplication for both in vitro - and greenhouse-derived nodal explants, as well as a rooting medium for shoots derived from axillary buds. Parameters investigated towards the cryopreservation of axillary shoots, from greenhouse nodal segments, and nodal segments from in vitro plantlets, included the size of the explant and pre-treatments for cryopreservation. Nodal segments (6 - 7 mm) and axillary shoots (2 - 4 mm) needed to be soaked in 0.5% (w/v) ascorbic acid for 10 min to minimise phenolic production and subsequent tissue death, and surface-sterilized by soaking in 1% Ca(OCl)2 solution for 5 min to reduce microbial contamination. Subsequent cryopreservation attempts involved only in vitro nodal segments because of the lack of success in achieving elongation of excised axillary buds. Vitrification and slow freezing methods, with or without the application of cryoprotectants, did not achieve successful cryopreservation. Attempts to establish a protocol for producing somatic embryos, as an alternate to axillary shoots and in vitro nodal segments, resulted in the production of globular embryogenic callus for both leaf and cotyledon explants. Cryopreservation of these explants was not investigated in the scope of this study. The study on B. racemosa focused on the development of a somatic embryogenesis protocol. Segments of embryonic axes produced globular-stage embryos when placed on MS medium supplemented with 30 g 1-1 sucrose, 1.0 g 1-1 casein hydrolysate, 2.0 mg 1-1 2,4-D, 0.1 mg 1-1 BAP and 8.0 g 1-1 agar. Various strategies were employed to obtain embryo germination, which included 1) different time intervals on callus initiation medium; 2) the use of different auxins (IAA, NAA and 2,4-D) in combination with the cytokinins BAP and kinetin; 3) desiccation and 4) cold treatments. Although somatic embryo germination was not achieved, globular embryos proceeded with development to the cotyledonary stage when cold-treated for 8 h at 4°C. This study provides some fundamental bases for further investigation towards achieving long-term conservation for both T. cacao and B. racemosa. However, the use of meristems as explants for cryopreservation is suggested to be the way forward for the cryopreservation of both species