Designing Highly Stable Poly(sarcosine)-Based Telodendrimer Micelles with High Drug Content Exemplified with Fulvestrant

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 mu M) 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.Peer reviewe

Nucleic Acid-Loaded Lipid Nanoparticle Interactions with Model Endosomal Membranes

[Image: see text] Lipid nanoparticles (LNPs) are important delivery systems for RNA-based therapeutics, yet the mechanism of their interaction with endosomal membranes remains unclear. Here, the interactions of nucleic acid-loaded LNPs that contain an ionizable lipid with models of the early and late endosomal membranes are studied, for the first time, using different reflectometry techniques. Novel insight is provided with respect to the subphase pH, the stage of the endosome, and the nature of the nucleic acid cargo. It is found that the insertion of lipids from the LNPs into the model membrane is greatest at pH 6.5 and 5.5, whereas at higher pH, lipid insertion is suppressed with evidence instead for the binding of intact LNPs, demonstrating the importance of the pH in the fusion of LNPs undergoing the endosomal pathway. Furthermore, and independently of the pH, the effect of the early- versus late-stage endosomal models is minimal, suggesting that the increased fluidity and anionic nature of the late endosome has little effect on the extent of LNP interaction. Last, there is greater nucleic acid delivery from LNPs containing mRNA than Poly(A), indicating that the extent of interaction can be tuned according to the nature of the nucleic acid cargo. Such new information on the relative impact of factors influencing nucleic acid delivery by LNP interactions with endosomal membranes is important in the design and tuning of vehicles with improved nucleic acid delivery capacities

Delivery of temozolomide and N3-propargyl analog to brain tumors using an apoferritin nanocage

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 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

Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

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

Penetration and uptake of Nanoparticles in 3D tumour spheroids

Animal models are effective for assessing tumour localisation of nanosystems, but difficult to use for studying penetration beyond the vasculature. Here, we have used well-characterised HCT116 colorectal cancer spheroids to study the effect of nanoparticle (NP) physicochemical properties on penetration and uptake. Incubation of spheroids with Hoechst 33342 resulted in a dye gradient which facilitated discrimination between the populations of cells in the core and at the periphery of spheroids by flow cytometry. This approach was used to compare doxorubicin and liposomal doxorubicin (Caelyx®), and a range of model poly(styrene) nanoparticles of different sizes (30 nm, 50 nm, 100 nm) and with different surface chemistries (50 nm uniform plain, carboxylated, aminated and a range of NPs and polyethylene glycol modified NPs prepared from a promising new functionalized biodegradable polymer (poly(glycerol-adipate), PGA). Unmodified poly(styrene) nanoparticles (30 nm/50 nm) were able to penetrate to the core of HCT116 spheroids more efficiently than larger poly(styrene) nanoparticles (100 nm). Surprisingly, penetration of 30 and 50nm particles was as good as clinically relevant doxorubicin concentrations. However penetration was reduced with higher surface charge. PGA NPs of 100 nm showed similar penetration into spheroids as 50 nm poly(styrene) nanoparticles, which may be related to polymer flexibility. PEG surface modification of polymeric particles significantly improved penetration into the spheroid core. The new model combining the use of spheroids Hoechst staining and flow cytometry was a useful model for assessing NP penetration and gives useful insights into the effects of NPs physical properties when designing nanomedicines

In vitro co-culture model of medulloblastoma and human neural stem cells for drug delivery assessment

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 = 600 μm, CV% = 10%). Spheroids were treated with model cytotoxic drug etoposide (0.3–100 μM) and the viability of normal and tumour tissue quantified separately using flow cytometry and multiphoton microscopy. Etoposide levels of 10 μM 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

Microfluidic-assisted preparation of RGD-decorated nanoparticles: exploring integrin-facilitated uptake in cancer cell lines.

Funder: AstraZeneca; doi: http://dx.doi.org/10.13039/100004325Funder: University of Manchester; doi: http://dx.doi.org/10.13039/501100000770This study is about fine tuning the targeting capacity of peptide-decorated nanoparticles to discriminate between cells that express different integrin make-ups. Using microfluidic-assisted nanoprecipitation, we have prepared poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles with a PEGylated surface decorated with two different arginine-glycine-aspartic acid (RGD) peptides: one is cyclic (RGDFC) and has specific affinity towards αvβ3 integrin heterodimers; the other is linear (RGDSP) and is reported to bind equally αvβ3 and α5β1. We have then evaluated the nanoparticle internalization in two cell lines with a markedly different integrin fingerprint: ovarian carcinoma A2780 (almost no αvβ3, moderate in α5β1) and glioma U87MG (very high in αvβ3, moderate/high in α5β1). As expected, particles with cyclic RGD were heavily internalized by U87MG (proportional to the peptide content and abrogated by anti-αvβ3) but not by A2780 (same as PEGylated particles). The linear peptide, on the other hand, did not differentiate between the cell lines, and the uptake increase vs. control particles was never higher than 50%, indicating a possible low and unselective affinity for various integrins. The strong preference of U87MG for cyclic (vs. linear) peptide-decorated nanoparticles was shown in 2D culture and further demonstrated in spheroids. Our results demonstrate that targeting specific integrin make-ups is possible and may open the way to more precise treatment, but more efforts need to be devoted to a better understanding of the relation between RGD structure and their integrin-binding capacity

Microfluidic-assisted preparation of RGD-decorated nanoparticles: exploring integrin-facilitated uptake in cancer cell lines

From Springer Nature via Jisc Publications RouterHistory: received 2020-02-23, accepted 2020-08-06, registration 2020-08-17, pub-electronic 2020-09-02, online 2020-09-02, collection 2020-12Publication status: PublishedFunder: AstraZeneca; doi: http://dx.doi.org/10.13039/100004325Funder: University of Manchester; doi: http://dx.doi.org/10.13039/501100000770Abstract: This study is about fine tuning the targeting capacity of peptide-decorated nanoparticles to discriminate between cells that express different integrin make-ups. Using microfluidic-assisted nanoprecipitation, we have prepared poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles with a PEGylated surface decorated with two different arginine-glycine-aspartic acid (RGD) peptides: one is cyclic (RGDFC) and has specific affinity towards αvβ3 integrin heterodimers; the other is linear (RGDSP) and is reported to bind equally αvβ3 and α5β1. We have then evaluated the nanoparticle internalization in two cell lines with a markedly different integrin fingerprint: ovarian carcinoma A2780 (almost no αvβ3, moderate in α5β1) and glioma U87MG (very high in αvβ3, moderate/high in α5β1). As expected, particles with cyclic RGD were heavily internalized by U87MG (proportional to the peptide content and abrogated by anti-αvβ3) but not by A2780 (same as PEGylated particles). The linear peptide, on the other hand, did not differentiate between the cell lines, and the uptake increase vs. control particles was never higher than 50%, indicating a possible low and unselective affinity for various integrins. The strong preference of U87MG for cyclic (vs. linear) peptide-decorated nanoparticles was shown in 2D culture and further demonstrated in spheroids. Our results demonstrate that targeting specific integrin make-ups is possible and may open the way to more precise treatment, but more efforts need to be devoted to a better understanding of the relation between RGD structure and their integrin-binding capacity

Amphiphilic tri- and tetra-block co-polymers combining versatile functionality with facile assembly into cytocompatible nanoparticles

In order for synthetic polymers to find widespread practical application as biomaterials, their syntheses must be easy to perform, utilising freely available building blocks, and should generate products which have no adverse effects on cells or tissue. In addition, it is highly desirable that the synthesis platform for the biomaterials can be adapted to generate polymers with a range of physical properties and macromolecular architectures, and with multiple functional handles to allow derivatisation with 'actives' for sensing or therapy. Here we describe the syntheses of amphiphilic tri-and tetra-block copolymers, using diazabicyclo[5.4.0]undec-5-ene (DBU) as a metal-free catalyst for ring-opening polymerisations of the widely-utilised monomer lactide combined with a functionalised protected cyclic carbonate. These syntheses employed PEGylated macroinitiators with varying chain lengths and architectures, as well as a labile-ester methacrylate initiator, and produced block copolymers with good control over monomer incorporation, molar masses, side-chain and terminal functionality and physico-chemical properties. Regardless of the nature of the initiators, the fidelity of the hydroxyl end group was maintained as confirmed by a second ROP chain extension step, and polymers with acryloyl/methacryloyl termini were able to undergo a second tandem reaction step, in particular thiol-ene click and RAFT polymerisations for the production of hyperbranched materials. Furthermore, the polymer side-chain functionalities could be easily deprotected to yield an active amine which could be subsequently coupled to a drug molecule in good yields. The resultant amphiphilic copolymers formed a range of unimolecular or kinetically-trapped micellar-like nanoparticles in aqueous environments, and the non-cationic polymers were all well-tolerated by MCF-7 breast cancer cells. The rapid and facile route to such highly adaptable polymers, as demonstrated here, offers promise for a range of bio materials applications

Endocytic profiling of cancer cell models reveals critical factors influencing lipid nanoparticle mediated mRNA delivery and protein expression

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 is poorly understood. To address this, using a clinically relevant lipid nanoparticle (LNP) delivering mRNA we highlight specific endosomal characteristics in in vitro tumour models that impact on protein expression. A 30-cell line LNP-mRNA transfection screen identified three cells lines having low, medium and high transfection that correlated with protein expression when they were analysed in tumour models. Endocytic profiling of these cell lines identified major differences in endolysosomal morphology, localisation, endocytic uptake, trafficking, recycling, and endolysosomal pH, identified using a novel pH probe. High transfecting cells showed rapid LNP uptake and trafficking through an organised endocytic pathway to lysosomes or rapid exocytosis. Low transfecting cells demonstrated slower endosomal LNP trafficking to lysosomes, and defective endocytic organisation and acidification. Our data establishes 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