Effects of Polymer 3D Architecture, Size, and Chemistry on Biological Transport and Drug Delivery In Vitro and in Orthotopic Triple Negative Breast Cancer Models

© 2020 Wiley-VCH GmbH The size, shape, and underlying chemistries of drug delivery particles are key parameters which govern their ultimate performance in vivo. Responsive particles are desirable for triggered drug delivery, achievable through architecture change and biodegradation to control in vivo fate. Here, polymeric materials are synthesized with linear, hyperbranched, star, and micellar-like architectures based on 2-hydroxypropyl methacrylamide (HPMA), and the effects of 3D architecture and redox-responsive biodegradation on biological transport are investigated. Variations in “stealth” behavior between the materials are quantified in vitro and in vivo, whereby reduction-responsive hyperbranched polymers most successfully avoid accumulation within the liver, and none of the materials target the spleen or lungs. Functionalization of selected architectures with doxorubicin (DOX) demonstrates enhanced efficacy over the free drug in 2D and 3D in vitro models, and enhanced efficacy in vivo in a highly aggressive orthotopic breast cancer model when dosed over schedules accounting for the biodistribution of the carriers. These data show it is possible to direct materials of the same chemistries into different cellular and physiological regions via modulation of their 3D architectures, and thus the work overall provides valuable new insight into how nanoparticle architecture and programmed degradation can be tailored to elicit specific biological responses for drug delivery

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

OptoRheo: Simultaneous in situ micro-mechanical sensing and 3D imaging of live cell cultures

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

Functionalized block co-polymer pro-drug nanoparticles with anti-cancer efficacy in 3D spheroids and in an orthotopic triple negative breast cancer model

Amphiphilic block co-polymers composed of poly(ethylene glycol)-co-poly(lactide)-co-poly(2-((tert-butoxycarbonyl)amino)-3-propyl carbonate) (PEG-pLA-pTBPC) are synthesized in monomer ratios and arrangements to enable assembly into nanoparticles with different sizes and architectures. These materials are based on components in clinical use, or known to be biodegradable, and retain the same fundamental chemistry across 'AB' and 'BAB' block architectures. In MCF7 and MDA-MB-231 breast cancer cells, nanoparticles of < 100 nm are internalized most rapidly, by both clathrin-and caveolin-mediated pathways. In THP-1 cells, polymer architecture and length of the hydrophilic block is the most important factor in the rate of internalization. The organ distributions of systemically injected nanoparticles in healthy mice indicate highest accumulation of the BAB-blocks in lungs and liver and the lowest accumulation in these organs of a methoxyPEG5000-pLA-pTBPC polymer. Conjugation of doxorubicin via a serum-stable urea linker to the carbonate regions of PEG5000-pLA-pTBPC generates self-assembling nanoparticles which are more cytotoxic in 2D, and penetrate further in 3D spheroids of triple negative breast cancer cells, than the free drug. In an aggressive orthotopic triple negative breast cancer mouse model, the methoxyPEG5000-pLA-pTBPC is of similar potency to free doxorubicin but with no evidence of adverse effects in terms of body weight

Synthesis, characterisation and evaluation of hyperbranched N-(2-hydroxypropyl) methacrylamides for transport and delivery in pancreatic cell lines in vitro and in vivo

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

Versatile, Highly Controlled Synthesis of Hybrid (Meth)acrylate–Polyester–Carbonates and their Exploitation in Tandem Post-Polymerization–Functionalization

The use of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a mild catalyst for the ring-opening polymerization (ROP) of the pharma-friendly and biodegradable monomer lactide and a functionalizable tert-butyloxycarbonyl (BOC)-protected cyclic carbonate is explored. Successful and controlled ROP is demonstrated when employing a series of labile-ester (bis)(meth)acrylate initiators to produce macromonomers suitable for a range of post-polymerization modifications. Importantly, the use of DBU ensured retention of the BOC group of the carbonate monomer during the polymerization, thus facilitating the production of highly functionalizable hybrid materials unobtainable using the more reactive triazabicyclodecene (TBD). Subsequently, a variety of short homo- and copolymers are synthesized with good control over material properties and final polymer composition. Successful attainment of these short copolymers confirm that DBU can overcome the previously observed limitations of TBD related to its kinetic competition between ROP and transesterification side-reactions under these reaction conditions. Furthermore, the fidelity of the hydroxyl and (meth)acrylic end groups are maintained as confirmed by a series of secondary tandem reactions. The macromonomers are also utilized in reversible addition−fragmentation chain-transfer polymerization (RAFT) polymerization for the production of amphiphilic block or random copolymers with a hydrophilic comonomer, poly(ethyleneglycol)methacrylate. The amphiphilic copolymers produced via the tandem RAFT reaction demonstrate the ability to self-assemble into monodisperse nanoparticles in aqueous environments.</p

OptoRheo : Simultaneous in situ micro-mechanical sensing and imaging of live 3D biological systems

A new instrument named OptoRheo combines light sheet fluorescence microscopy and particle tracking microrheology for live imaging and micromechanical sensing of extracellular matrix-cell interactions. 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