Successfully translating anti-cancer nanomedicines from pre-clinical proof of concept to demonstration of therapeutic value in the clinic is challenging. Having made significant advances with drug ...delivery technologies, we must learn from other areas of oncology drug development, where patient stratification and target-driven design have improved patient outcomes. We should evolve our nanomedicine development strategies to build the patient and disease into the line of sight from the outset. The success of small molecule targeted therapies has been significantly improved by employing a specific decision-making framework, such as AstraZeneca's 5R principle: right target/efficacy, right tissue/exposure, right safety, right patient, and right commercial potential. With appropriate investment and collaboration to generate a platform of evidence supporting the end clinical application, a similar framework can be established for enhancing nanomedicine translation and performance. Building informative data packages to answer these questions requires the following: (I) an improved understanding of the heterogeneity of clinical cancers and of the biological factors influencing the behaviour of nanomedicines in patient tumours; (II) a transition from formulation-driven research to disease-driven development; (III) the implementation of more relevant animal models and testing protocols; and (IV) the pre-selection of the patients most likely to respond to nanomedicine therapies. These challenges must be overcome to improve (the cost-effectiveness of) nanomedicine development and translation, and they are key to establishing superior therapies for patients.
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Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their ...adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.
Here, we aimed to chemically modify PAMAM dendrimers using lysine as a site-selective anchor for successfully delivering mRNA while maintaining a low toxicity profile. PAMAM dendrimers were ...multi-functionalised by amidation reactions in a regioselective, quantitative and stepwise manner with carefully selected property-modifying surface groups. Alternatively, novel lysine-based dendrimers were prepared in the same manner with the aim to unlock their potential in gene delivery. The modified dendrimers were then formulated with Cy5-EGFP mRNA by bulk mixing via liquid handling robotics across different nitrogen to phosphate ratios. The resulting dendriplexes were characterised by size, charge, mRNA encapsulation, and mRNA binding affinity. Finally, their in-vitro delivery activity was systematically investigated across key cellular trafficking stages to relate chemical design to cellular effect. We demonstrate our findings in different cell lines and benchmarked relative to a commercially available transfection agent, jetPEI®. We demonstrate that specific surface modifications are required to generate small, reliable and well-encapsulated positively charged dendriplex complexes. Furthermore, we show that introduction of fusogenic groups is essential for driving endosomal escape and achieving cellular delivery and translation of mRNA in these cell lines.
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•PAMAM & lysine dendrimers were modified with functional groups for mRNA delivery•Automated formulation established optimal mRNA cargo loading conditions•In-vitro testing identified dendrimers with improved endosomal escape, particularly by p-toluylsulfonyl arginine groups•Dendrimers outperformed a commercial standard, jetPEI®•Effective dendrimers for mRNA delivery were well tolerated in vitro.
Polymeric micelles have been extensively used as nanocarriers for the delivery of chemotherapeutic agents, aiming to improve their efficacy in cancer treatment. However, the poor loading capacity, ...premature drug release, non-uniformity, and reproducibility still remain the major challenges. To create a stable polymeric micelle with high drug loading, a telodendrimer micelle was developed as a nanocarrier for fulvestrant, as an example of a drug that has extremely poor water solubility (sub-nanomolar range). Telodendrimers were prepared by the synthesis of hydrophilic linear poly(sarcosine) and growing a lysine dendron from the chain terminal amine by divergent synthesis. At the periphery of the dendritic block, either 4, 8, or 16 fulvestrant molecules were conjugated to the lysine dendron creating a hydrophobic block. Having drug molecules as a part of the carrier not only reduces the usage of the inert carrier materials but also prevents the drugs from leakage and premature release by diffusion. The self-assembled telodendrimer micelles demonstrated good colloidal stability (cmc < 2 μM) in buffer and were uniform in size. In addition, these telodendrimer micelles could solubilize additional fulvestrant yielding an excellent overall drug loading capacity of up to 77 wt % total drug load (summation of conjugated and encapsulated). Importantly, the size of the micelles could be tuned between 25 and 150 nm by controlling (i) the ratio between hydrophilic and hydrophobic blocks and (ii) the amount of encapsulated fulvestrant. The versatility of these telodendrimer-based micelle systems to both conjugated and encapsulated drugs with high efficiency and stability, in addition to possessing other tuneable properties, makes it a promising drug delivery system for a range of active pharmaceutical ingredients and therapeutic targets.
The development of delivery systems capable of tumor targeting represents a promising strategy to overcome issues related to nonspecific effects of conventional anticancer therapies. Currently, one ...of the most investigated agents for cancer targeting is hyaluronic acid (HA), since its receptor, CD44, is overexpressed in many cancers. However, most of the studies on CD44/HA interaction have been so far performed in cell-free or genetically modified systems, thus leaving some uncertainty regarding which cell-related factors influence HA binding and internalization (collectively called “uptake”) into CD44-expressing cells. To address this, the expression of CD44 (both standard and variants, designated CD44s and CD44v, respectively) was evaluated in human dermal fibroblasts (HDFs) and a large panel of cancer cell lines, including breast, prostate, head and neck, pancreatic, ovarian, colorectal, thyroid, and endometrial cancers. Results showed that CD44 isoform profiles and expression levels vary across the cancer cell lines and HDF and are not consistent within the cell origin. Using composite information of CD44 expression, HA binding, and internalization, we found that the expression of CD44v can negatively influence the uptake of HA, and, instead, when cells primarily expressed CD44s, a positive correlation was observed between expression and uptake. In other words, CD44shigh cells bound and internalized more HA compared to CD44slow cells. Moreover, CD44shigh HDFs were less efficient in uptaking HA compared to CD44shigh cancer cells. The experiments described here are the first step toward understanding the interplay between CD44 expression, its functionality, and the underlying mechanism(s) for HA uptake. The results show that factors other than the amount of CD44 receptor can play a role in the interaction with HA, and this represents an important advance with respect to the design of HA-based carriers and the selection of tumors to treat according to their CD44 expression profile.
This paper describes the synthesis of star polymers designed for future drug-delivery applications. A generation-5 lysine dendrimer was used as a macroinitiator for the ring-opening polymerization of ...the sarcosine N-carboxyanhydride monomer to produce 32-arm star polymers with narrow molar mass distributions and desirable hydrodynamic size control. Fluorescent dye-labeled polymers were dosed in mice to measure plasma pharmacokinetics. Long circulation times were observed, representing ideal properties for biophysical targeting of tumors. In vivo efficacy of one of these star polymers conjugated to the therapeutic molecule SN-38 was evaluated in mice bearing SW620 xenografted tumors to demonstrate high antitumor activity and low body weight loss compared to the SN-38 prodrug irinotecan and this shows the potential of these delivery systems. As a further build, we demonstrated that these star polymers can be easily chain-end-functionalized with useful chemical moieties, giving opportunities for future receptor-targeting strategies. Finally, we describe the synthetic advantages of these star polymers that make them attractive from a pharmaceutical manufacturing perspective and report characterization of the polymers with a variety of techniques.
Lipid nanoparticles have great potential for delivering nucleic-acid-based therapeutics, but low efficiency limits their broad clinical translation. Differences in transfection capacity between ...in vitro models used for nanoparticle pre-clinical testing are poorly understood. To address this, using a clinically relevant lipid nanoparticle (LNP) delivering mRNA, we highlight specific endosomal characteristics in in vitro tumor models that impact protein expression. A 30-cell line LNP-mRNA transfection screen identified three cell lines having low, medium, and high transfection that correlated with protein expression when they were analyzed in tumor models. Endocytic profiling of these cell lines identified major differences in endolysosomal morphology, localization, endocytic uptake, trafficking, recycling, and endolysosomal pH, identified using a novel pH probe. High-transfecting cells showed rapid LNP uptake and trafficking through an organized endocytic pathway to lysosomes or rapid exocytosis. Low-transfecting cells demonstrated slower endosomal LNP trafficking to lysosomes and defective endocytic organization and acidification. Our data establish that efficient LNP-mRNA transfection relies on an early and narrow endosomal escape window prior to lysosomal sequestration and/or exocytosis. Endocytic profiling should form an important pre-clinical evaluation step for nucleic acid delivery systems to inform model selection and guide delivery-system design for improved clinical translation.
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Lipid nanoparticles are promising vectors for delivery of macromolecular therapeutics, such as mRNA. Pre-clinical testing of vectors relies on cell models of disease, such as cancer. Here, using high-content endocytic profiling in different cell types, we have identified key endocytic features that are critical to effective cytosolic delivery of mRNA.
Glioblastoma multiforme (GBM) is a grade IV astrocytoma, which is the most aggressive form of brain tumor. The standard of care for this disease includes surgery, radiotherapy and temozolomide (TMZ) ...chemotherapy. Poor accumulation of TMZ at the tumor site, tumor resistance to drug, and dose-limiting bone marrow toxicity eventually reduce the success of this treatment. Herein, we have encapsulated >500 drug molecules of TMZ into the biocompatible protein nanocage, apoferritin (AFt), using a “nanoreactor” method (AFt-TMZ). AFt is internalized by transferrin receptor 1-mediated endocytosis and is therefore able to facilitate cancer cell uptake and enhance drug efficacy. Following encapsulation, the protein cage retained its morphological integrity and surface charge; hence, its cellular recognition and uptake are not affected by the presence of this cargo. Additional benefits of AFt include maintenance of TMZ stability at pH 5.5 and drug release under acidic pH conditions, encountered in lysosomal compartments. MTT assays revealed that the encapsulated agents displayed significantly increased antitumor activity in U373V (vector control) and, remarkably, the isogenic U373M (MGMT expressing TMZ-resistant) GBM cell lines, with GI50 values <1.5 μM for AFt-TMZ, compared to 35 and 376 μM for unencapsulated TMZ against U373V and U373M, respectively. The enhanced potency of AFt-TMZ was further substantiated by clonogenic assays. Potentiated G2/M cell cycle arrest following exposure of cells to AFt-TMZ indicated an enhanced DNA damage burden. Indeed, increased O6-methylguanine (O6-MeG) adducts in cells exposed to AFt-TMZ and subsequent generation of γH2AX foci support the hypothesis that AFt significantly enhances the delivery of TMZ to cancer cells in vitro, overwhelming the direct O6-MeG repair conferred by MGMT. We have additionally encapsulated >500 molecules of the N3-propargyl imidazotetrazine analog (N3P), developed to combat TMZ resistance, and demonstrated significantly enhanced activity of AFt-N3P against GBM and colorectal carcinoma cell lines. These studies support the use of AFt as a promising nanodelivery system for targeted delivery, lysosomal drug release, and enhanced imidazotetrazine potency for treatment of GBM and wider-spectrum malignancies.
As a malignant tumour of lymphatic origin, B-cell lymphoma represents a significant challenge for drug delivery, where effective therapies must access malignant cells in the blood, organs and ...lymphatics while avoiding off-target toxicity. Subcutaneous (SC) administration of nanomedicines allows preferential access to both the lymphatic and blood systems and may therefore provide a route to enhanced drug exposure to lymphomas.
Here we examine the impact of SC dosing on lymphatic exposure, pharmacokinetics (PK), and efficacy of AZD0466, a small molecule dual Bcl-2/Bcl-xL inhibitor conjugated to a ‘DEP®’ G5 poly-l-lysine dendrimer.
PK studies reveal that the plasma half-life of the dendrimer-drug conjugate is 8-times longer than that of drug alone, providing evidence of slow release from the circulating dendrimer nanocarrier. The SC dosed construct also shows preferential lymphatic transport, with over 50% of the bioavailable dose recovered in thoracic lymph. Increases in dose (up to 400 mg/kg) are well tolerated after SC administration and studies in a model of disseminated lymphoma in mice show that high dose SC treatment outperforms IV administration using doses that lead to similar total plasma exposure (lower peak concentrations but extended exposure after SC).
These data show that the DEP® dendrimer can act as a circulating drug depot accessing both the lymphatic and blood circulatory systems. SC administration improves lymphatic exposure and facilitates higher dose administration due to improved tolerability. Higher dose SC administration also results in improved efficacy, suggesting that drug delivery systems that access both plasma and lymph hold significant potential for the treatment of haematological cancers where lymphatic and extranodal dissemination are poor prognostic factors.
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•AZD0466: a G5 polylysine dendrimer conjugate, acts as a circulating reservoir for the dual Bcl-2/Bcl-xL inhibitor AZD4230.•Subcutaneous administration of AZD0466 improves lymphatic exposure and allows higher doses due to improved tolerability.•Drug delivery systems that access both blood and lymph are a promising strategy to better treat lymph resident diseases.
•Physiologically relevant in vitro medulloblastoma model is described.•Human-based normal and tumour tissue were cultured together in three dimensions.•A universally applicable cell labelling and ...analysis technique was developed.•Flow cytometry and multiphoton microscopy were used to quantify each population.•Etoposide was used to model local brain-targeted chemotherapy.
Physiologically relevant in vitro models can serve as biological analytical platforms for testing novel treatments and drug delivery systems. We describe the first steps in the development of a 3D human brain tumour co-culture model that includes the interplay between normal and tumour tissue along with nutrient gradients, cell-cell and cell-matrix interactions. The human medulloblastoma cell line UW228-3 and human foetal brain tissue were marked with two supravital fluorescent dyes (CDCFDASE, Celltrace Violet) and cultured together in ultra-low attachment 96-well plates to form reproducible single co-culture spheroids (d=600μm, CV%=10%). Spheroids were treated with model cytotoxic drug etoposide (0.3–100μM) and the viability of normal and tumour tissue quantified separately using flow cytometry and multiphoton microscopy. Etoposide levels of 10μM were found to maximise toxicity to tumours (6.5% viability) while stem cells maintained a surviving fraction of 40%. The flexible cell marking procedure and high-throughput compatible protocol make this platform highly transferable to other cell types, primary tissues and personalised screening programs. The model's key anticipated use is for screening and assessment of drug delivery strategies to target brain tumours, and is ready for further developments, e.g. differentiation of stem cells to a range of cell types and more extensive biological validation.
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