Local drug delivery systems (LDDS) are a promising method to overcome challenges associated with chemotherapeutic treatment of brain tumours, namely poor blood-brain barrier penetration. Here we report a poly(ethyleneglycol)-poly(lactide)-poly(caprolactone)-poly(lactide)-poly(ethyleneglycol) based injectable hydrogel, PELCLE, loaded with Doxorubicin (Dox) and Olaparib (Ola) as an LDDS against glioblastoma (GBM), a primary malignant brain tumour with a poor prognosis. The thermoresponsive properties of the hydrogel, which behaved as a liquid at room temperature and formed a gel at elevated temperatures, were not impacted by the inclusion of chemotherapeutics whereby two-week sustained release was recorded for both Dox and Ola. Drug potency was assessed against a panel of GBM cell lines, both a syngeneic mouse line and primary patient-derived lines, and the combination of Dox/Ola demonstrated synergistic effects at a range of drug: drug ratios. The application of radiotherapy (XRT) in combination with Dox/Ola improved treatment efficacy both in vitro and in vivo, with a significant increase in median survival observed when Dox/Ola PELCLE hydrogels were applied against a surgical resection model of GBM (syngeneic mouse model SB28) with and without the addition of adjuvant XRT (28 and 23 days, respectively, p < 0.01). Furthermore, a long-term survivor was noted in the group treated with the drug loaded HG and XRT, which was associated with a very small residual tumour, indicating the efficacy of this treatment against a GBM in vivo model.

Understanding the internalization of nanosized particles by mucosal epithelial cells is essential in a number of areas including viral entry at mucosal surfaces, nanoplastic pollution, as well as design and development of nanotechnology-type medicines. Here, we report our comparative study on pathways of cellular internalization in epithelial Caco-2 cells cultured in vitro as either a polarized, differentiated cell layer or as nonpolarized, nondifferentiated cells. The study reveals a number of differences in the extent that endocytic processes are used by cells, depending on their differentiation status and the nature of applied nanoparticles. In polarized cells, actin-driven and dynamin-independent macropinocytosis plays a prominent role in the internalization of both positively and negatively charged nanoparticles, contrary to its modest contribution in nonpolarized cells. Clathrin-mediated cellular entry plays a prominent role in the endocytosis of positive nanoparticles and cholesterol inhibition in negative nanoparticles. However, in nonpolarized cells, dynamin-dependent endocytosis is a major pathway in the internalization of both positive and negative nanoparticles. Cholesterol depletion affects both nonpolarized and polarized cells' internalization of positive and negative nanoparticles, which, in addition to the effect of cholesterol-binding inhibitors on the internalization of negative nanoparticles, indicates the importance of membrane cholesterol in endocytosis. The data collectively provide a new contribution to understanding endocytic pathways in epithelial cells, particularly pointing to the importance of the cell differentiation stage and the nature of the cargo.

Drug combinations have been shown to be highly effective in many cancer therapies but the ratios of the individual drugs must be adjusted carefully and formulated appropriately to ensure synergistic action.

Despite the success of polyethylene glycol-based (PEGylated) polyesters in the drug delivery and biomedical fields, concerns have arisen regarding PEG's immunogenicity and limited biodegradability. In addition, inherent limitations, including limited chemical handles as well as highly hydrophobic nature, can restrict their effectiveness in physiological conditions of the polyester counterpart. To address these matters, an increasing amount of research has been focused towards identifying alternatives to PEG. One promising strategy involves the use of bio-derived polyols, such as glycerol. In particular, glycerol is a hydrophilic, non-toxic, untapped waste resource and as other polyols, can be incorporated into polyesters via enzymatic catalysis routes.In the present study, a systematic screening is conducted focusing on the incorporation of 1,6-hexanediol (Hex) (hydrophobic diol) into both poly(glycerol adipate) (PGA) and poly(diglycerol adipate) (PDGA) at different (di)glycerol:hex ratios (30:70; 50:50 and 70:30mol/mol) and its effect on purification upon NPs formation. By varying the amphiphilicity of the backbone, we demonstrated that minor adjustments influence the NPs formation, NPs stability, drug encapsulation, and degradation of these polymers, despite the high chemical similarity. Moreover, the best performing materials have shown good biocompatibility in both in vitro and in vivo (whole organism) tests. As preliminary result, the sample containing diglycerol and Hex in a 70:30 ratio, named as PDGA-Hex 30%, has shown to be the most promising candidate in this small library analysed. It demonstrated comparable stability to the glycerol-based samples in various media but exhibited superior encapsulation efficiency of a model hydrophobic dye. This in-depth investigation provides new insights into the design and modification of biodegradable (di)glycerol-based polyesters, potentially paving the way for more effective and sustainable PEG-free drug delivery nano-systems in the pharmaceutical and biomedical fields.

Volumetric Additive Manufacturing (VAM) represents a revolutionary advancement in the field of Additive Manufacturing, as it allows for the creation of objects in a single, cohesive process, rather than in a layer-by-layer approach. This innovative technique offers unparalleled design freedom and significantly reduces printing times. A current limitation of VAM is the availability of suitable resins with the required photoreactive chemistry and from sustainable sources. To support the application of this technology, we have developed a sustainable resin based on polyglycerol, a bioderived (e.g., vegetable origin), colourless, and easily functionisable oligomer produced from glycerol. To transform polyglycerol-6 into an acrylate photo-printable resin we adopted a simple, one-step, and scalable synthesis route. Polyglycerol-6-acrylate fulfils all the necessary criteria for volumetric printing (transparency, photo-reactivity, viscosity) and was successfully used to print a variety of models with intricate geometries and good resolution. The waste resin was found to be reusable with minimal performance issues, improving resin utilisation and minimising waste material. Furthermore, by incorporating dopants such as poly(glycerol) adipate acrylate (PGA-A) and 10,12-pentacosadyinoic acid (PCDA), we demonstrated the ability to print objects with a diverse range of functionalities, including temperature sensing probes and a polyester excipient, highlighting the potential applications of these new resins.

Enzymatic polymerization of glycerol-based polyesters has emerged as a selective and sustainable strategy for the production of degradable, amphiphilic, PEG-free polymeric materials. In the present work, we exploited the ability to produce copolyester variants of poly(glycerol adipate) (PGA), by adding a second functionalized diol in a one-pot fashion. The produced glycerol-based copolyesters, with a variety of amphiphilic balances and chemical functionalities along the backbone, have been screened as nanocarriers for drug delivery. The stability of the produced nanoparticles (NPs) was tested in relevant biological fluids and after freeze-drying treatment. The new variants outperformed PGA homopolymers in the penetration through a gel-like barrier as well as in their efficiency to encapsulate a poorly water-soluble model drug. All the tested polymeric NPs formulations have demonstrated biocompatibility in both in vitro and in vivo experiments, that utilize Caenorhabditis elegans nematodes. Taken together, these preliminary results highlight the importance of introducing new chemistry along polyester backbones while tuning the amphiphilic nature of the final polymers to improve the performance of NP systems.

Combinations of the topoisomerase II inhibitor doxorubicin and the poly (ADP-ribose) polymerase inhibitor olaparib offer potential drug–drug synergy for treatment of triple negative breast cancers (TNBC) both in free drug form and when delivered by oxidation-responsive nanoparticles.

ObjectiveTo improve the biological and toxicological properties of Mefenamic acid (MA), the galactosylated prodrug of MA named MefeGAL was included in polymeric solid dispersions (PSs) composed of poly(glycerol adipate) (PGA) and Pluronic® F68 (MefeGAL-PS). MefeGAL-PS was compared with polymeric solid formulations of MA (MA-PS) or a mixture of equal ratio of MefeGAL/MA (Mix-PS).MethodsThe in vitro and in vivo pharmacological and toxicological profiles of PSs have been investigated. In detail, we evaluated the anti-inflammatory (carrageenan-induced paw edema test), analgesic (acetic acid-induced writhing test) and ulcerogenic activity in mice after oral treatment. Additionally, the antiproliferative activity of PSs was assessed on in vitro models of colorectal and non-small cell lung cancer.ResultsWhen the PSs were resuspended in water, MefeGAL’s, MA’s and their mixture’s apparent solubilities improved due to the interaction with the polymeric formulation. By comparing the in-vivo biological performance of MefeGAL-PS with that of MA, MefeGAL and MA-PS, it was seen that MefeGAL-PS exhibited the same sustained and delayed analgesic and anti-inflammatory profile as MefeGAL but did not cause gastrointestinal irritation. The pharmacological effect of Mix-PS was present from the first hours after administration, lasting about 44 hours with only slight gastric mucosa irritation. In-vitro evaluation indicated that Mix-PS had statistically significant higher cytotoxicity than MA-PS and MefeGAL-PS.ConclusionsThese preliminary data are promising evidence that the galactosylated prodrug approach in tandem with a polymer-drug solid dispersion formulation strategy could represent a new drug delivery route to improve the solubility and biological activity of NSAIDs.

Sustainably derived poly(glycerol adipate) (PGA) has been deemed to deliver all the desirable features expected in a polymeric scaffold for drug-delivery, including biodegradability, biocompatibility, self-assembly into nanoparticles (NPs) and a functionalisable pendant group. Despite showing these advantages over commercial alkyl polyesters, PGA suffers from a series of key drawbacks caused by poor amphiphilic balance. This leads to weak drug-polymer interactions and subsequent low drug-loading in NPs, as well as low NPs stability. To overcome this, in the present work, we applied a more significant variation of the polyester backbone while maintaining mild and sustainable polymerisation conditions. We have investigated the effect of the variation of both hydrophilic and hydrophobic segments upon physical properties and drug interactions as well as self-assembly and NPs stability. For the first time we have replaced glycerol with the more hydrophilic diglycerol, as well as adjusting the final amphiphilic balance of the polyester repetitive units by incorporating the more hydrophobic 1,6-n-hexanediol (Hex). The properties of the novel poly(diglycerol adipate) (PDGA) variants have been compared against known polyglycerol-based polyesters. Interestingly, while the bare PDGA showed improved water solubility and diminished self-assembling ability, the Hex variation demonstrated enhanced features as a nanocarrier. In this regard, PDGAHex NPs were tested for their stability in different environments and for their ability to encode enhanced drug loading. Moreover, the novel materials have shown good biocompatibility in both in vitro and in vivo (whole organism) experiments.

Biomechanical cues from the extracellular matrix (ECM) are essential for directing many cellular processes, from normal development and repair, to disease progression. To better understand cell-matrix interactions, we have developed a new instrument named 'OptoRheo' that combines light sheet fluorescence microscopy with particle tracking microrheology. OptoRheo lets us image cells in 3D as they proliferate over several days while simultaneously sensing the mechanical properties of the surrounding extracellular and pericellular matrix at a sub-cellular length scale. OptoRheo can be used in two operational modalities (with and without an optical trap) to extend the dynamic range of microrheology measurements. We corroborated this by characterising the ECM surrounding live breast cancer cells in two distinct culture systems, cell clusters in 3D hydrogels and spheroids in suspension culture. This cutting-edge instrument will transform the exploration of drug transport through complex cell culture matrices and optimise the design of the next-generation of disease models.

Abstract AIMS Synergistic combinations of doxorubicin and gemcitabine have been assessed for post-surgical treatment of IDH1 wild-type glioblastoma (GBM). As a complementary drug carrier, hyperbranched and micellar nanoparticles are being investigated to enable enhanced drug loading and sustained drug release. METHOD 2D and 3D spheroid in vitro models have been generated from patient-derived glioblastoma cells resected from tumour core and invasive margin. Drug combinations were screened using synergy quantification from the Chou and Talalay method and synergy mechanisms investigated using measures of caspase 3/6-mediated apoptosis and γH2AX-mediated DNA damage. Polymeric nanoparticles (PEG-PCL) loaded with Cy5 were tested for internalisation and subsequent endocytosis pathway determination. Both single drug and drug combinations are currently being incorporated into PEG-PCL nanoparticles for in vitro assessment of biocompatibility, cellular uptake and cytotoxicity. RESULTS Data demonstrate synergism with doxorubicin and gemcitabine combinations is obtained in a molar ratio de- pendent manner. Consistent with this, enhanced apoptosis and DNA damage is observed in a synergistic manner. PEG-PCL nanoparticles demonstrate effcient cellular uptake and are internalised via dynamin-dependent caveolae and clathrin endocytotic pathways. Interestingly, nanoparticle internalisation is substantially in- creased (approx. 50%) in invasive GBM cells relative to proliferative core cells. CONCLUSIONS Doxorubicin and gemcitabine has been identified as a synergistic drug combination with the potential for sustained drug release when combined with PEG-PCL nanoparticle formulations. Together, the data has provided a platform of knowledge that will enable translation to pre-clinical in vivo models.

Polymersomes are an exciting modality for drug delivery due to their structural similarity to biological cells and their ability to encapsulate both hydrophilic and hydrophobic drugs. In this regard, the current work aimed to develop multifunctional polymersomes, integrating dye (with hydrophobic Nile red and hydrophilic sulfo-cyanine5-NHS ester as model drugs) encapsulation, stimulus responsiveness, and surface-ligand modifications. Polymersomes constituting poly(N-2-hydroxypropylmethacrylamide)-b-poly(N-(2-(methylthio)ethyl)acrylamide) (PHPMAm-b-PMTEAM) are prepared by aqueous dispersion RAFT-mediated polymerization-induced self-assembly (PISA). The hydrophilic block lengths have an effect on the obtained morphologies, with short chain P(HPMAm)16 affording spheres and long chain P(HPMAm)43 yielding vesicles. This further induces different responses to H2O2, with spheres fragmenting and vesicles aggregating. Folic acid (FA) is successfully conjugated to the P(HPMAm)43, which self-assembles into FA-functionalized P(HPMAm)43-b-P(MTEAM)300 polymersomes. The FA-functionalized P(HPMAm)43-b-P(MTEAM)300 polymersomes entrap both hydrophobic Nile red (NR) and hydrophilic Cy5 dye. The NR-loaded FA-linked polymersomes exhibit a controlled release of the encapsulated NR dye when exposed to 10 mM H2O2. All the polymersomes formed are stable in human plasma and well-tolerated in MCF-7 breast cancer cells. These preliminary results demonstrate that, with simple and scalable chemistry, PISA offers access to different shapes and opens up the possibility of the one-pot synthesis of multicompartmental and responsive polymersomes.

The therapeutic efficacy of nanomedicines is highly dependent on their access to target sites in the body, and this in turn is markedly affected by their size, shape and transport properties in tissue. Although there have been many studies in this area, the ability to design nanomaterials with optimal physicochemical properties for in vivo efficacy remains a significant challenge. In particular, it is often difficult to quantify the detailed effects of cancer drug delivery systems in vivo as tumour volume reduction, a commonly reported marker of efficacy, does not always correlate with cytotoxicity in tumour tissue. Here, we studied the behaviour in vivo of two specific poly(2-hydroxypropyl methacrylamide) (pHPMA) pro-drugs, with hyperbranched and chain-extended branched architectures, redox-responsive backbone components, and pH-sensitive linkers to the anti-cancer drug doxorubicin. Evaluation of the biodistribution of these polymers following systemic injection indicated differences in the circulation time and organ distribution of the two polymers, despite their very similar hydrodynamic radii (∼10 and 15 nm) and architectures. In addition, both polymers showed improved tumour accumulation in orthotopic triple-negative breast cancers in mice, and decreased accumulation in healthy tissue, as compared to free doxorubicin, even though neither polymer-doxorubicin pro-drug decreased overall tumour volume as much as the free drug under the dosing regimens selected. However, the results of histopathological examinations by haematoxylin and eosin, and TUNEL staining indicated a higher population of apoptotic cells in the tumours for both polymer pro-drug treatments, and in turn a lower population of apoptotic cells in the heart, liver and spleen, as compared to free doxorubicin treatment. These data suggest that the penetration of these polymer pro-drugs was enhanced in tumour tissue relative to free doxorubicin, and that the combination of size, architecture, bioresponsive backbone and drug linker degradation yielded greater efficacy for the polymers as measured by biomarkers than that of tumour volume. We suggest therefore that the effects of nanomedicines may be different at various length scales relative to small molecule free drugs, and that penetration into tumour tissue for some nanomedicines may not be as problematic as prior reports have suggested. Furthermore, the data indicate that dual-responsive crosslinked polymer-prodrugs in this study may be effective nanomedicines for breast cancer chemotherapy, and that endpoints beyond tumour volume reduction can be valuable in selecting candidates for pre-clinical trials.

ABA block copolymers synthesized by a combination of SET-LRP and photoinduced LRP techniques form self-assemblies of varied morphologies in an aqueous environment and show low cell toxicity.

AbstractBiomechanical cues from the extracellular matrix (ECM) are essential for directing many cellular processes, from normal development and repair, to disease progression. To better understand cell-matrix interactions, we have developed a new instrument named ‘OptoRheo’ that combines light sheet fluorescence microscopy with particle tracking microrheology. OptoRheo lets us image cells in 3D as they proliferate over several days while simultaneously sensing the mechanical properties of the surrounding extracellular and pericellular matrix at a sub-cellular length scale. OptoRheo can be used in two operational modalities (with and without an optical trap) to extend the dynamic range of microrheology measurements. We corroborated this by characterising the ECM surrounding live breast cancer cells in two distinct culture systems, cell clusters in 3D hydrogels and spheroids in suspension culture. This cutting-edge instrument will transform the exploration of drug transport through complex cell culture matrices and optimise the design of the next-generation of disease models.

Hyperbranched polymers have many promising features for drug delivery, owing to their ease of synthesis, multiple functional group content, and potential for high drug loading with retention of solubility. Here we prepared hyperbranched N-(2-hydroxypropyl)methacrylamide (HPMA) polymers with a range of molar masses and particle sizes, and with attached dyes, radiolabel or the anticancer drug gemcitabine. Reversible addition-fragmentation chain transfer (RAFT) polymerisation enabled the synthesis of pHPMA polymers and a gemcitabine-comonomer functionalised pHPMA polymer pro-drug, with diameters of the polymer particles ranging from 7-40 nm. The non-drug loaded polymers were well-tolerated in cancer cell lines and macrophages, and were rapidly internalised in 2D cell culture and transported efficiently to the centre of dense pancreatic cancer 3D spheroids. The gemcitabine-loaded polymer pro-drug was found to be toxic both to 2D cultures of MIA PaCa-2 cells and also in reducing the volume of MIA PaCa-2 spheroids. The non-drug loaded polymers caused no short-term adverse effects in healthy mice following systemic injection, and derivatives of these polymers labelled with 89Zr-were tracked for their distribution in the organs of healthy and MIA PaCa-2 xenograft bearing Balb/c nude mice. Tumour accumulation, although variable across the samples, was highest in individual animals for the pHPMA polymer of ∼20 nm size, and accordingly a gemcitabine pHPMA polymer pro-drug of ∼18 nm diameter was evaluated for efficacy in the tumour-bearing animals. The efficacy of the pHPMA polymer pro-drug was very similar to that of free gemcitabine in terms of tumour growth retardation, and although there was a survival benefit after 70 days for the polymer pro-drug, there was no difference at day 80. These data suggest that while polymer pro-drugs of this type can be effective, better tumour targeting and enhanced in situ release remain as key obstacles to clinical translation even for relatively simple polymers such as pHPMA.

Poly(lactic-co-glycolic acid) (PLGA) is a versatile synthetic copolymer that is widely used in pharmaceutical applications. This is because it is well-tolerated in the body, and copolymers of varying physicochemical properties are readily available via ring-opening polymerization. However, native PLGA polymers are hard to track as drug delivery carriers when delivered to subcellular spaces, due to the absence of an easily accessible "handle" for fluorescent labeling. Here we show a one-step, scalable, solvent-free, synthetic route to fluorescent blue (2-aminoanthracene), green (5-aminofluorescein), and red (rhodamine-6G) PLGA, in which every polymer chain in the sample is fluorescently labeled. The utility of initiator-labeled PLGA was demonstrated through the preparation of nanoparticles, capable of therapeutic subcellular delivery to T-helper-precursor-1 (THP-1) macrophages, a model cell line for determining in vitro biocompatibility and particle uptake. Super resolution confocal fluorescence microscopy imaging showed that dye-initiated PLGA nanoparticles were internalized to punctate regions and retained bright fluorescence over at least 24 h. In comparison, PLGA nanoparticles with 5-aminofluorescein introduced by conventional nanoprecipitation/encapsulation showed diffuse and much lower fluorescence intensity in the same cells and over the same time periods. The utility of this approach for in vitro drug delivery experiments was demonstrated through the concurrent imaging of the fluorescent drug doxorubicin (λex = 480 nm, λem = 590 nm) with carrier 5-aminofluorescein PLGA, also in THP-1 cells, in which the intracellular locations of the drug and the polymer could be clearly visualized. Finally, the dye-labeled particles were evaluated in an in vivo model, via delivery to the nematode Caenorhabditis elegans, with bright fluorescence again apparent in the internal tract after 3 h. The results presented in this manuscript highlight the ease of synthesis of highly fluorescent PLGA, which could be used to augment tracking of future therapeutics and accelerate in vitro and in vivo characterization of delivery systems prior to clinical translation.

N-Hydroxyethyl acrylamide was used as a functional initiator for the enzymatic ring-opening polymerisation of ε-caprolactone and δ-valerolactone. N-Hydroxyethyl acrylamide was found not to undergo self-reaction in the presence of Lipase B from Candida antarctica under the reaction conditions employed. By contrast, this is a major problem for 2-hydroxyethyl methacrylate and 2-hydroxyethyl acrylate which both show significant transesterification issues leading to unwanted branching and cross-linking. Surprisingly, N-hydroxyethyl acrylamide did not react fully during enzymatic ring-opening polymerisation. Computational docking studies helped us understand that the initiated polymer chains have a higher affinity for the enzyme active site than the initiator alone, leading to polymer propagation proceeding at a faster rate than polymer initiation leading to incomplete initiator consumption. Hydroxyl end group fidelity was confirmed by organocatalytic chain extension with lactide. N-Hydroxyethyl acrylamide initiated polycaprolactones were free-radical copolymerised with PEGMA to produce a small set of amphiphilic copolymers. The amphiphilic polymers were shown to self-assemble into nanoparticles, and to display low cytotoxicity in 2D in vitro experiments. To increase the green credentials of the synthetic strategies, all reactions were carried out in 2-methyl tetrahydrofuran, a solvent derived from renewable resources and an alternative for the more traditionally used fossil-based solvents tetrahydrofuran, dichloromethane, and toluene.

Silver nanoparticles (AgNP) are widely exploited for their effective antimicrobial activity against a range of pathogens. Their high efficacy in this regard has seen the global demand for AgNP in consumer products steadily increase in recent years, necessitating research into novel low environmental impact synthesis approaches. Here we present a new synthetic methodology to produce polymer-AgNP composite microparticles using supercritical carbon dioxide (scCO(2)) and avoiding use of any petrochemically derived solvents. Poly(methyl methacrylate)-poly(4-vinylpyridine) (PMMA-b-P4VP) block copolymers were synthesised via RAFT-mediated dispersion polymerisation in scCO(2), with in situ thermal degradation of various amounts of a CO2-soluble silver complex. Selective interaction of the silver with the pyridinyl moieties of the block copolymer allowed the formation of AgNP, dispersed within the block copolymer microparticles, leading to homogeneous composites. The by-products of the reaction were also removed by extracting with a flow of CO2 to yield a clean dry product in a single process. The composites were found to be non-cytotoxic and proved to have good antimicrobial activity against two bacterial strains. Though no significant activity was seen for at least the first 24 hours, inhibition of bacterial growth afterwards proved to be extremely persistent, with inhibition observed even after 15 days. Finally, the microparticulate nature of the synthesised composites was exploited and tested for compatibility in the Laser Sintering (LS) 3D printing process. Composite microparticles were fused to produce solid objects, without aggregation of the AgNP. With further optimisation, these composites could prove to be an incredibly versatile 'ink' that may be used within additive manufacturing and 3D printing to rapidly produce bespoke medical devices with inherent antimicrobial activity.

Abstract AIMS A hyaluronic acid-based hydrogel with mechanical properties comparable to human brain tissue is being investigated as an implantable drug-delivery vehicle for post-surgical treatment of IDH1 wild-type glioblastoma (GBM). Hydrogels are to be loaded with polymeric pro-drug nanoparticles to enable controlled drug release and increased drug penetration through brain parenchyma for enhanced treatment of residual disease within and beyond the invasive margin. METHOD A panel of patient-derived glioblastoma cell lines resected from tumour core and invasive margin, have been used to generate tumour spheroids. Chemotherapeutic drug screening has been performed to select lead compounds for formulation and gel loading. Synergies of drug combinations have been studied for potential combination loading. Upcoming work will investigate nanoparticle internalisation, spheroid penetration and prodrug nanoparticle potency. Nanoparticle loaded hydrogels are then to be studied for validation of effective in vitro application. RESULTS Current results demonstrate that patient-derived cell lines can generate robust, reproducible tumour spheroids with diameters approx. 350-400 µm. Patient-to-patient differences in drug potencies were observed, with differences also noted between counterpart core and marginal cells from the same patients, illustrating the importance of using patient-derived cells and distinct regional cells to study heterogenous disease such as GBM. Drug combination experiments indicated synergy of topoisomerase I inhibitor (Irinotecan) with PARP inhibitor (Ola-parib). Interestingly, combination of PARP inhibition with topoisomerase II inhibitors (Doxorubicin, Etoposide) was found not synergistic. CONCLUSION Together, data gathered on single drug and drug-drug combinations will inform upcoming formulation and in vitro application of hydrogels planned, with eventual translation to pre-clinical in vivo models.