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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.

Improving our understanding of how design choices in materials synthesis impact biological outcomes is of critical importance in the development of nanomedicines. Here, we show that fluorophore labeling of polymer nanomedicine candidates significantly alters their transport and cell association in multi-cellular tumor spheroids and their penetration in breast cancer xenografts, dependent on the type of the fluorophore and their positioning within the macromolecular structure. These data show the critical importance of the biomaterials structure and architecture in their tissue distribution and intracellular trafficking, which in turn govern their potential therapeutic efficacy. The broader implication of these findings suggests that when developing materials for medical applications, great care should be taken early on in the design process as relatively simple choices may have downstream impacts that could potentially skew preclinical biology data.

Additional file 2: Fig. S1. a Radio-TLC of purified antibody conjugates showing successful radiolabelling of DFO-Miltuximab® with 89Zirconium. b Radio-TLC of purified antibody conjugates showing successful radiolabelling of DOTA-Miltuximab® with 177Lutetium. Fig. S2. Immunoreactivity of a,c DFO-Miltuximab® and b,d DOTAMiltuximab® to cell surface GPC-1 on DU-145 cells measured via flow cytometry (a,b) and ELISA (c,d). For some tests, antibody was “mock” radiolabelled, i.e. treated in the same way as the radiolabelled antibody, but without the addition of radiolabel, to estimate the effect of radiolabelling conditions on immunoreactivity. Fig. S3. Quantitative flow cytometry analysis of DU-145 cells using MIL-38 binding for quantification. MIL-38 was used with the QIFIKIT antigen density analysis kit to determine GPC-1 density on the cell surface of a. prostate cancer cell line DU-145 and b. GPC1 negative lymphoma cell line Raji. Fig. S4. No effect of DOTA-Miltuximab® alone on in vivo tumour growth. DU-145-RFP-Luc cells (5x106 ) in matrigel were injected subcutaneously into the right flank of BALB/c/nude mice. When tumours reached ~100mm3 , mice were injected with saline (n=6) or DOTA-Miltuximab® (n=6) (80ug) intravenously. All mice were euthanised approximately 2 weeks thereafter. a Mean weekly mouse tumour volume. d Individual mouse tumour volume at endpoint. Data expressed as Mean ± SEM and statistical analysis performed using an unpaired t test. *p < 0.05. Fig. S5. Representative H&E staining of the mouse brain, heart, lung, liver, kidney, spleen, small intestine and testis tissue a 3 days, b 5 days, c 7 days and d 27 days post 6MBq [ 177Lu]Lu-DOTA-Miltuximab® treatment or e 27 days post DOTA-Miltuximab® treatment. Fig. S6. Individual weekly mouse tumour volumes of mice treated with a DOTAMiltuximab® (n=8) b 3MBq [ 177Lu]Lu-DOTA-Miltuximab® (n=9) or c 10MBq [ 177Lu]LuDOTA-Miltuximab® (n=9) treated DU-145 xenograft mice.

Additional file 1. Supplementary Methods.

The development of therapeutic resistance to targeted anticancer therapies remains a significant clinical problem, with intratumoral heterogeneity playing a key role. In this context, improving the therapeutic outcome through simultaneous targeting of multiple tumor cell subtypes within a heterogeneous tumor is a promising approach. Liposomes have emerged as useful drug carriers that can reduce systemic toxicity and increase drug delivery to the tumor site. While clinically used liposomal drug formulations show marked therapeutic advantages over free drug formulations, ligand-functionalized liposomes that can target multiple tumor cell subtypes may further improve the therapeutic efficacy by facilitating drug delivery to a broader population of tumor cells making up the heterogeneous tumor tissue. Ligand-directed liposomes enable the so-called active targeting of cell receptors via surface-attached ligands that direct drug uptake into tumor cells or tumor-associated stromal cells, and so can increase the selectivity of drug delivery. Despite promising preclinical results demonstrating improved targeting and anti-tumor effects of ligand-directed liposomes, there has been limited translation of this approach to the clinic. Key challenges for translation include the lack of established methods to scale up production and comprehensively characterize ligand-functionalized liposome formulations, as well as the inadequate recapitulation of in vivo tumors in the preclinical models currently used to evaluate their performance. Herein, we discuss the utility of recent ligand-directed liposome approaches, with a focus on dual-ligand liposomes, for the treatment of solid tumors and examine the drawbacks limiting their progression to clinical adoption.

Gold nanoclusters (Au NCs) have become a promising nanomaterial for cancer therapy because of their biocompatibility and fluorescent properties. In this study, the effect of ultrasmall protein-stabilized 2 nm Au NCs on six types of mammalian cells (fibroblasts, B-lymphocytes, glioblastoma, neuroblastoma, and two types of prostate cancer cells) under electromagnetic radiation is investigated. Cellular association of Au NCs in vitro is concentration-dependent, and Au NCs have low intrinsic toxicity. However, when Au NC-incubated cells are exposed to a 1 GHz electromagnetic field (microwave radiation), cell viability significantly decreases, thus demonstrating that Au NCs exhibit specific microwave-dependent cytotoxicity, likely resulting from localized heating. Upon i.v. injection in mice, Au NCs are still present at 24 h post administration. Considering the specific microwave-dependent cytotoxicity and low intrinsic toxicity, our work suggests the potential of Au NCs as effective and safe nanomedicines for cancer therapy. Refereed/Peer-reviewed

Studying the interactions between nanoengineered materials and biological systems plays a vital role in the development of biological applications of nanotechnology and the improvement of our fundamental understanding of the bio-nano interface. A significant barrier to progress in this multidisciplinary area is the variability of published literature with regards to characterizations performed and experimental details reported. Here, we suggest a 'minimum information standard' for experimental literature investigating bio-nano interactions. This standard consists of specific components to be reported, divided into three categories: material characterization, biological characterization and details of experimental protocols. Our intention is for these proposed standards to improve reproducibility, increase quantitative comparisons of bio-nano materials, and facilitate meta analyses and in silico modelling. Refereed/Peer-reviewed