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Poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PEG-PLGA) block copolymers and their thermoresponsive hydrogels have been widely studied as injectable depots for the sustained release of therapeutics. The thermogelling temperatures of PEG-PLGA copolymer solutions and the properties of their corresponding hydrogels are highly sensitive to the polymer microstructure. Often, it can be difficult to precisely control the microstructure because of transesterification side reactions that occur during ring-opening polymerisation (ROP) of lactide (L) and glycolide (G) monomers under various conditions. Therefore, we undertook a detailed study to understand how different reaction conditions influenced the reaction kinetics, monomer sequence and side reactions for the synthesis of a PLGA-PEG-PLGA triblock copolymer. ROP was conducted under thermal control (100, 110 and 120 degrees C) or in the presence of the catalysts tin(ii) 2-ethylhexanoate (Sn(Oct)(2)) and tin(ii) trifluoromethanesulfonate (Sn(OTf)(2)) at various catalyst loadings. The reactions were monitored via proton nuclear magnetic resonance (H-1 NMR) spectroscopy, gel permeation chromatography (GPC) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI ToF MS). While ROP proceeded faster with catalysts than the uncatalysed reaction at an equivalent temperature, the onset of side reactions typically occurred at much lower monomer conversions and to a greater extent, resulting in a plateau in the molecular weight and broadening of the molecular weight distribution. For Sn(Oct)(2) catalysed ROP, an increase in the catalyst loading had negligible effect on the occurrence or extent of side reactions, but led to a surprising increase in the rate of polymerisation of L relative to G. Sn(OTf)(2) catalysed ROP resulted in significant side reactions from the start of the polymerisation. These results provide insights into the influence of polymerisation conditions on the microstructure of PLGA-PEG-PLGA triblock copolymers that may prove useful for their improved and controlled synthesis. Refereed/Peer-reviewed

Polymeric micelles are promising carriers for the delivery of poorly water-soluble drugs, providing enhanced drug solubility, blood circulation times, and bioavailability. Nevertheless, the storage and long-term stability of micelles in solution present challenges requiring the lyophilization and storage of formulations in the solid state, with reconstitution immediately prior to application. Therefore, it is important to understand the effects of lyophilization/reconstitution on micelles, particularly their drug-loaded counterparts. Herein, we investigated the use of β-cyclodextrin (β-CD) as a cryoprotectant for the lyophilization/reconstitution of a library of poly(ethylene glycol-b-ε-caprolactone) (PEG-b-PCL) copolymer micelles and their drug-loaded counterparts, as well as the effect of the physiochemical properties of different drugs (phloretin and gossypol). The critical aggregation concentration (CAC) of the copolymers decreased with increasing weight fraction of the PCL block (fPCL), plateauing at ~1 mg/L when the fPCL was >0.45. The blank (empty) and drug-loaded micelles were lyophilized/reconstituted in the absence and presence of β-CD (9% w/w) and analyzed via dynamic light scattering (DLS) and synchrotron small-angle X-ray scattering (SAXS) to assess for changes in aggregate size (hydrodynamic diameter, Dh) and morphology, respectively. Regardless of the PEG-b-PCL copolymer or the use of β-CD, the blank micelles displayed poor redispersibility (

Refereed/Peer-reviewed Engineered T-cell therapies have proven highly efficacious for the treatment of haematological cancers, but translation of this success to solid tumours has been limited, in part, due to difficulties in maintaining high doses at specific target sites. Hydrogel delivery systems that provide a sustained release of T-cells at the target site are emerging as a promising strategy. Therefore, in this study we aimed to develop an injectable hydrogel that gels in situ via efficient Diels-Alder cycloaddition (DAC) chemistry and provides a sustained release of T-cells through gradual hydrolysis of the hydrogel matrix. Hydrogels were prepared via the DAC between fulvene and maleimide functionalised poly(ethylene glycol) (PEG) derivatives. By adjusting the concentration and molecular weight of the functionalised PEGs in the hydrogel formulation the in vitro gelation time (Tgel), initial Young's modulus (E) and degradation time (Td) could be tailored from 15-150 min, 5-179 kPa and 7-114 h, respectively. Prior to gelation, the formulations could be readily injected through narrow gauge (26 G) needles with the working time correlating closely with the Tgel. A 5 wt% hydrogel formation with conjugated cyclic RGD motif was found to be optimal for the encapsulation and release of CD3+ T-cells with a near linear release profile and >70% cell viability over the first 4 d and release continuing out to 7 d. With their tuneable Tgel, Td and stiffness, the DAC hydrogels provide the opportunity to control the release period and profile of encapsulated cells

Refereed/Peer-reviewed The taste-masking of bitter-tasting active pharmaceutical ingredients is key to ensuring patient compliance when producing oral pharmaceutical formulations. This is generally achieved via the incorporation of pH-responsive, reverse enteric polymers, that prevent the dissolution of the formulation in the oral environment, but rapidly mediate it within the gastric environment. Reverse enteric polymers are commonly applied as coatings on oral dosage forms via spray atomisation (e.g., fluidised-bed spray coating), and generally exhibit the most efficient taste-masking. However, currently used reverse enteric coatings require high mass gains (% w/w) during coating to mediate taste-masking, and thereby exhibit delayed release within the gastric environment. Therefore, there remains a need for the development of new reverse enteric coatings, that can efficiently taste-mask at low mass gains and maintain rapid release characteristics within the gastric environment. Herein we report the synthesis and evaluation of a series of addition copolymers of 2-vinylpyridine and butyl methacrylate, methyl methacrylate and isobornyl methacrylate. The thermal, solubility, and water absorption properties of the copolymers were effectively tuned by altering the mol% fraction of the constitutive monomers. Based on their physical properties, selected copolymers were preliminarily evaluated for their compatibility with fluidised-bed spray coating, and effectiveness as taste-masking reverse enteric coatings. The copolymers poly[(2-vinylpyridine)-co-(butyl methacrylate)] (mol% ratio 40:60) and poly[(2-vinylpyridine)-co-(butyl methacrylate)-co-(methyl methacrylate)] (mol% ratio 40:50:10) were found to exhibit excellent taste-masking properties following fluidised-bed spray coating onto Suglets® sugar spheres. Suglets® bearing a film coating of either copolymer (5.2–6.5% w/w mass gain) were found to effectively impede the release of a model drug formulation for up to 72 h in a simulated salivary environment, and rapidly release it (

Efforts to mitigate the effects of feral cats through the management of remnant or reintroduced populations of threatened species, are often unsuccessful due to predation by control-averse feral cats, or 'problem individuals'. In order to target these animals, we have developed the Population Protecting Implant (PPI). PPIs are designed to be implanted subcutaneously in a native animal. If the animal is preyed upon, and the implant ingested by a feral cat, release of a toxic payload is triggered in the acidic stomach environment and the problem individual is eliminated. We introduce the first toxic implant incorporating the poison sodium fluoroacetate. Manufactured via fluidised-bed spray coating, toxic implants exhibited uniform reverse enteric coatings and low intra-batch variation. Toxic implants were found to exhibit favourable stability at subcutaneous pH in vitro, and rapidly release their toxic payload in vitro at gastric pH. However, limited stability was demonstrated in rats in vivo (similar to 39-230 d), due to the use of a filament scaffold to enable coating and was likely exacerbated by metachromatic interactions caused by 1080. This work highlights that future development of the PPIs should primarily focus on removal of the filament scaffold, to afford implants with increased in vivo stability. Refereed/Peer-reviewed

Ligation chemistries are often required to perform under stringent conditions that preserve the integrity and function of (bio)conjugates, including specific biological buffers. To explore the versatility of the pentafulvene-maleimide ligation for (bio)conjugations, we studied the stability of the coupling partners and their Diels-Alder cycloaddition (DAC) in buffers used commonly in biological assays, protein chemistry and bioconjugates. The stability of 6,6-dialkylpentafulvene and maleimide derivatives to a panel of buffers with pH values between 3.7 and 10.1 was monitored via(1)H NMR spectroscopy. While the pentafulvene displayed excellent stability, hydrolysis of the maleimide was observed in several cases, although the extent of hydrolysis did not correlate with pH. For almost all buffers the pentafulvene-maleimide DAC proceeded efficiently and at a significantly faster rate than maleimide hydrolysis under the conditions studied. The buffer composition nor pH appeared to have a significant effect on the kinetics of the DAC with second-order rate constants (k(2)) ranging from 0.14 to 0.33 M-1 s(-1) (23 +/- 1 degrees C). This study highlights the versatility of the pentafulvene-maleimide ligation to proceed under a wide range of conditions relevant for (bio)conjugations and that maleimide hydrolysis in aqueous system may be promoted or inhibited by certain buffers. Refereed/Peer-reviewed

The crystallinity of polymers strongly affects their properties. For block copolymers, whereby two crystallisable blocks are covalently tethered to one another, the molecular weight of the individual blocks and their relative weight fraction are important structural parameters that control their crystallisation. In the case of block copolymer micelles, these parameters can influence the crystallinity of the core, which has implications for drug encapsulation and release. Therefore, in this study, we aimed to determine how the microstructure of poly(ethylene glycol-b-caprolactone) (PEG-b-PCL) copolymers contributes to the crystallinity of their hydrophobic PCL micelle cores. Using a library of PEG-b-PCL copolymers with PEG number-average molecular weight (M-n) values of 2, 5, and 10 kDa and weight fractions of PCL (f(PCL)) ranging from 0.11 to 0.67, the thermal behaviour and morphology were studied in blends, bulk, and micelles using differential scanning calorimetry (DSC), wide-angle X-ray diffraction (WXRD), and Synchrotron wide-angle X-ray scattering (WAXS). Compared to PEG and PCL homopolymers, the block copolymers displayed reduced crystallinity in the bulk phase and the individual blocks had a large influence on the crystallisation of one another. The f(PCL) was determined to be the dominant contributor to the extent and order of crystallisation of the two blocks. When f(PCL) < 0.35, the initial crystallisation of PEG led to an amorphous PCL phase. At f(PCL) values between 0.35 and 0.65, PEG crystallisation was followed by PCL crystallisation, whereas this behaviour was reversed when f(PCL) > 0.65. For lyophilised PEG-b-PCL micelles, the crystallinity of the core increased with increasing f(PCL), although the core was predominately amorphous for micelles with f(PCL) < 0.35. These findings contribute to understanding the relationships between copolymer microstructure and micelle core crystallinity that are important for the design and performance of micellar drug delivery systems, and the broader application of polymer micelles. Refereed/Peer-reviewed

A facile, highly efficient, and metal-free synthesis of well-defined polyester-based core cross-linked star (CCS) polymers with yields of up to 96 % was achieved via an organic catalyst (i.e., methanesulfonic acid) mediated ring-opening polymerization (ROP) at room temperature, through either a two-pot or a one-pot, two-step strategy. CCS polymers with narrow molecular weight distributions (PDI ≤ 1.3) and macroinitiator (MI) conversions of 90−96% were prepared using poly(ε- caprolactone) (PCL) MIs with molecular weights ranging from 9.9 to 36.2 kDa and [4,4′-bioxepane]-7,7′-dione (BOD) as the cross-linker. Furthermore, transesterification was identified as being responsible for the small percentage of unincorporated low molecular weight polymer remaining and star−star couplings in the star formation. Compared to CCS polymers synthesized via the methanesulfonic acidmediated ROP, CCS polymers prepared via ROP mediated by high transesterification rate catalysts (i.e., stannous octoate (Sn(Oct)2)) suffer from much lower star purity (ca. 70%) and star−star coupled products due to more prominent transesterification side-reactions. Refereed/Peer-reviewed

Refereed/Peer-reviewed Globally, invasive mammalian predators are responsible for the depredation and extinction of threatened species. The development of innovative control methods that target the most impactful of these predators is crucial to ensure the successful conservation of threatened species. Feral cats (Felis catus) are one of the most damaging invasive mammalian predators, particularly in Australia, where they are estimated to kill more than 1.5 billion mammals, birds, and reptiles annually. Mammals are disproportionally depredated by feral cats, and efforts to reintroduce or conserve remnant populations of threatened mammal species are hampered by shortfalls in current control methods. To address this challenge, we have prototyped the population-protecting implant (PPI), a subcutaneous implant that is designed to target the most dangerous and efficient predators, so called ‘problem individuals’. Conceptually, the implant has a reverse enteric coating that is stable in the subcutaneous environment of a prey animal but dissolves rapidly in the gastric environment of a predator, resulting in the release of a toxic payload and death of the predator. In this work we report a novel approach for the manufacture of PPIs, that combines hot-melt extrusion and fluidised-bed spray coating, to respectively afford implants with accurate drug loading and a uniform reverse-enteric coating. Most of the implants bearing a 300 μm coating were found to effectively contain their payload for more than 280 d in vitro, in a simulated subcutaneous environment (pH 7.4, 37 °C) but release it within 2.5 h in vitro under simulated gastric conditions (pH 1.5, 37 °C). However, two failures were observed at 64 d and 200 d, likely due to the increased osmotic pressure caused by the soluble core. This work highlights a revised implant design that may improve implant stability in vivo and facilitate the translation of the PPI to field use, for the conservation of threatened species. We also introduce a novel and facile approach to determine the loading of non-chromophoric payloads in hot-melt extruded systems that is highly relevant for the determination of active pharmaceutical ingredients in drug-delivery formulations.

Refereed/Peer-reviewed Introduced predators are a significant threat to global biodiversity and are responsible for most of all modern bird, reptile, and mammal extinctions. In Australia, the introduced feral cat (Felis catus) kills 459 million mammals annually and leaves many species facing extinction. Attempted reintroductions of threatened mammal species often fail due to the persistence of intractable feral cats termed "problem individuals" and the swift depredation of the reintroduced population. Biomaterial implants could hold the key to targeting problem individuals. Herein, we report the development of the population-protecting implant, a subcutaneous implant for native mammals. The implant is intended to be inert within the subcutaneous environment for the life of the native mammal and to release a toxic payload in the gastric environment of a feral cat when ingested during a predation event. By toxifying and causing the death of the feral cat, the problem individual is eliminated, and the remaining population of native mammals is protected from further predation. To achieve this, an innovative reverse enteric coating was developed for use as a biomaterial, which exhibited a previously unreported level of pH selectivity for solubility at gastric pH. Large batches of implants were manufactured via fluidized-bed spray coating with a uniform reverse enteric coating and low intrabatch variability. Implants with a 300 μm coating afforded significant stability and retention of the payload at subcutaneous pH in vitro, with rapid release of the payload observed at gastric pH. In addition, implants exhibited favorable stability in vivo in rats, with no observed difference in biocompatibility compared to conventional radio-frequency identification microchips. This work demonstrates a proof of concept of an innovative type of implant and serves as the basis for its future development and translation into the field.

Refereed/Peer-reviewed The five-year survival rate for women with ovarian cancer is very poor despite radical cytoreductive surgery and chemotherapy. Although most patients initially respond to platinum-based chemotherapy, the majority experience recurrence and ultimately develop chemoresistance, resulting in fatal outcomes. The current administration of cytotoxic compounds is hampered by dose-limiting severe adverse effects. There is an unmet clinical need for targeted drug delivery systems that transport chemotherapeutics selectively to tumor cells while minimizing off-target toxicity. G protein-coupled receptors (GPCRs) are the largest family of membrane receptors, and many are overexpressed in solid tumors, including ovarian cancer. This review summarizes the progress in engineered nanoparticle research for drug delivery for ovarian cancer and discusses the potential use of GPCRs as molecular entry points to deliver anti-cancer compounds into ovarian cancer cells. A newly emerging treatment paradigm could be the personalized design of nanomedicines on a case-by-case basis.

Injectable hydrogels are promising materials for the sustained delivery of therapeutics, bioactives and cell therapies. Hydrogels that display suitable processing times and rapid gelling kinetics upon injection are particularly desirable as they can limit burst release of their payload. Therefore, we aimed to develop hydrogels that could be readily processed at ambient temperature, gelled rapidly at physiological temperature, and possessed tuneable properties (e.g., degradation and release profiles). To achieve this, a unique dual cross-linking mechanism was investigated, employing a combination of pentafulvene-maleimide Diels-Alder cycloaddition (DAC) chemistry and thermoresponsive physical cross-linking with a functionalised poly(ethylene glycol -b -propylene glycol -b-ethylene glycol) (PEG-PPG-PEG; Pluronic F-127) copolymer. A combination of temperature dependent nuclear magnetic resonance spectroscopy, dynamic light scattering, and rheological measurements were conducted to interrogate the gelation mechanism. Whereas the reaction between aqueous solutions of maleimide functionalised Pluronic and pentafulvene functionalised PEG star polymer resulted in hydrogels with gelation times (tgel) between 30 and 55 mins at ambient temperature, significantly faster tgel values (5-10 mins) were observed at physiological temperatures (37 degrees C). The relatively slow gelation at ambient temperature provided good working times and injectability through narrow gauge (261/2 G) needles. While the initial Young's modulus (E) of the hydrogels was highly dependent on the gelation temperature, the E remained dynamic in response to further temperature changes post-gelation. The hydrogels displayed relatively low swelling (20-30% mass increases) and degradation times (td) up to 16 d, which provided for the sustained release of latex particles (cell mimic) over a period of 7-14 d. With their tuneable gelation kinetics and properties, the thermoresponsive DAC hydrogels have potential for the controlled release of therapeutics. Refereed/Peer-reviewed