Stimuli-sensitive polymer prodrug nanocarriers by reversible-deactivation radical polymerization.

Polymer prodrugs are based on the covalent linkage of therapeutic molecules to a polymer structure which avoids the problems and limitations commonly encountered with traditional drug-loaded nanocarriers in which drugs are just physically entrapped (e.g., burst release, poor drug loadings). In the past few years, reversible-deactivation radical polymerization (RDRP) techniques have been extensively used to design tailor-made polymer prodrug nanocarriers. This synthesis strategy has received a lot of attention due to the possibility of fine tuning their structural parameters (e.g., polymer nature and macromolecular characteristics, linker nature, physico-chemical properties, functionalization, etc.), to achieve optimized drug delivery and therapeutic efficacy. In particular, adjusting the nature of the drug-polymer linker has enabled the easy synthesis of stimuli-responsive polymer prodrugs for efficient spatiotemporal drug release. In this context, this review article will give an overview of the different stimuli-sensitive polymer prodrug structures designed by RDRP techniques, with a strong focus on the synthesis strategies, the macromolecular architectures and in particular the drug-polymer linker, which governs the drug release kinetics and eventually the therapeutic effect. Their biological evaluations will also be discussed.

Polymeric Nanozyme with SOD Activity Capable of Inhibiting Self- and Metal-Induced α-Synuclein Aggregation.

Despite decades of research, Parkinson's disease is still an idiopathic pathology for which no cure has yet been found. This is partly explained by the multifactorial character of most neurodegenerative syndromes, whose generation involves multiple pathogenic factors. In Parkinson's disease, two of the most important ones are the aggregation of α-synuclein and oxidative stress. In this work, we address both issues by synthesizing a multifunctional nanozyme based on grafting a pyridinophane ligand that can strongly coordinate CuII, onto biodegradable PEGylated polyester nanoparticles. The resulting nanozyme exhibits remarkable superoxide dismutase activity together with the ability to inhibit the self-induced aggregation of α-synuclein into amyloid-type fibrils. Furthermore, the combination of the chelator and the polymer produces a cooperative effect whereby the resulting nanozyme can also halve CuII-induced α-synuclein aggregation.

Coarse-Grained Model-Assisted Design of Polymer Prodrug Nanoparticles with Enhanced Cytotoxicity: A Combined Theoretical and Experimental Study.

To achieve drug release from polymer prodrug nanoparticles, the drug-polymer linker must be accessible for cleavage to release the drug, which can occur under certain physiological conditions (e.g., presence of specific enzymes). Supramolecular organization of polymer prodrug nanoparticles is crucial as it greatly affects the location of the linker, its surface exposure/solvation and thus its cleavage to release the drug. Since experimental access to these data is not straightforward, new methodologies are critically needed to access this information and to accelerate the development of more effective polymer prodrug nanoparticles, and replace the time-consuming and resource-intensive traditional trial-and-error strategy. In this context, we reported here the use of a coarse-grained model to assist the design of polymer prodrug nanoparticles with enhanced cytotoxicity. By choosing the solvent accessible surface area as the critical parameter for predicting drug release and hence cytotoxicity of polymer prodrug nanoparticles, we developed an optimized polymer-drug linker with enhanced hydrophilicity and solvation. Our hypothesis was then experimentally validated by the synthesis of the corresponding polymer prodrugs based on two different drugs (gemcitabine and paclitaxel), which demonstrated greater performances in terms of drug release and cytotoxicity on two cancer cell lines. Interestingly, our methodology can be easily applied to other polymer prodrug structures, which would contribute to the development of more efficient drug delivery systems via in silico screening.

Increasing the Hydrophilicity of Cyclic Ketene Acetals Improves the Hydrolytic Degradation of Vinyl Copolymers and the Interaction of Glycopolymer Nanoparticles with Lectins.

Radical ring-opening polymerization (rROP) of cyclic ketene acetals (CKAs) with traditional vinyl monomers allows the synthesis of degradable vinyl copolymers. However, since the most commonly used CKAs are hydrophobic, most degradable vinyl copolymers reported so far degrade very slowly by hydrolysis under physiological conditions (phosphate-buffered saline, pH 7.4, 37 °C), which can be detrimental for biomedical applications. Herein, to design advanced vinyl copolymers by rROP with high CKA content and enhanced degradation profiles, we reported the copolymerization of 2-methylene-1,3,6-trioxocane (MTC) as a CKA with vinyl ether (VE) or maleimide (MI) derivatives. By performing a point-by-point comparison between the MTC/VE and MTC/MI copolymerization systems, and their counterparts based on 2-methylene-1,3-dioxepane (MDO) and 5,6-benzo-2-methylene-1,3-dioxepane (BMDO), we showed negligible impact on the macromolecular characteristics and similar reactivity ratios, suggesting successful substitution of MDO and BMDO by MTC. Interestingly, owing to the hydrophilicity of MTC, the obtained copolymers exhibited a faster hydrolytic degradation under both accelerated and physiological conditions. We then prepared MTC-based glycopolymers, which were formulated into surfactant-free nanoparticles, exhibiting excellent colloidal stability up to 4 months and complete degradation under enzymatic conditions. Importantly, MTC-based glyconanoparticles also showed a similar cytocompatibility toward two healthy cell lines and a much stronger lectin affinity than MDO-based glyconanoparticles.

A Polymer Prodrug Strategy to Switch from Intravenous to Subcutaneous Cancer Therapy for Irritant/Vesicant Drugs.

Chemotherapy is almost exclusively administered via the intravenous (IV) route, which has serious limitations (e.g., patient discomfort, long hospital stays, need for trained staff, high cost, catheter failures, infections). Therefore, the development of effective and less costly chemotherapy that is more comfortable for the patient would revolutionize cancer therapy. While subcutaneous (SC) administration has the potential to meet these criteria, it is extremely restrictive as it cannot be applied to most anticancer drugs, such as irritant or vesicant ones, for local toxicity reasons. Herein, we report a facile, general, and scalable approach for the SC administration of anticancer drugs through the design of well-defined hydrophilic polymer prodrugs. This was applied to the anticancer drug paclitaxel (Ptx) as a worst-case scenario due to its high hydrophobicity and vesicant properties (two factors promoting necrosis at the injection site). After a preliminary screening of well-established polymers used in nanomedicine, polyacrylamide (PAAm) was chosen as a hydrophilic polymer owing to its greater physicochemical, pharmacokinetic, and tumor accumulation properties. A small library of Ptx-based polymer prodrugs was designed by adjusting the nature of the linker (ester, diglycolate, and carbonate) and then evaluated in terms of rheological/viscosity properties in aqueous solutions, drug release kinetics in PBS and in murine plasma, cytotoxicity on two different cancer cell lines, acute local and systemic toxicity, pharmacokinetics and biodistribution, and finally their anticancer efficacy. We demonstrated that Ptx-PAAm polymer prodrugs could be safely injected subcutaneously without inducing local toxicity while outperforming Taxol, the commercial formulation of Ptx, thus opening the door to the safe transposition from IV to SC chemotherapy.

(Bio)degradable and Biocompatible Nano-Objects from Polymerization-Induced and Crystallization-Driven Self-Assembly.

Polymerization-induced self-assembly (PISA) and crystallization-driven self-assembly (CDSA) techniques have emerged as powerful approaches to produce a broad range of advanced synthetic nano-objects with high potential in biomedical applications. PISA produces nano-objects of different morphologies (e.g., spheres, vesicles and worms), with high solids content (∼10-50 wt %) and without additional surfactant. CDSA can finely control the self-assembly of block copolymers and readily forms nonspherical crystalline nano-objects and more complex, hierarchical assemblies, with spatial and dimensional control over particle length or surface area, which is typically difficult to achieve by PISA. Considering the importance of these two assembly techniques in the current scientific landscape of block copolymer self-assembly and the craze for their use in the biomedical field, this review will focus on the advances in PISA and CDSA to produce nano-objects suitable for biomedical applications in terms of (bio)degradability and biocompatibility. This review will therefore discuss these two aspects in order to guide the future design of block copolymer nanoparticles for future translation toward clinical applications.

Degradable Glycopolyester-like Nanoparticles by Radical Ring-Opening Polymerization.

A small library of degradable polyester-like glycopolymers was successfully prepared by the combination of radical ring-opening copolymerization of 2-methylene-1,3-dioxepane as a cyclic ketene acetal (CKA) with vinyl ether (VE) derivatives and a Pd-catalyzed thioglycoconjugation. The resulting thioglycopolymers were formulated into self-stabilized thioglyconanoparticles, which were stable up to 4 months and were enzymatically degraded. Nanoparticles and their degradation products exhibited a good cytocompatibility on two healthy cell lines. Interactions between thioglyconanoparticles and lectins were investigated and highlighted the presence of both specific carbohydrate/lectin interactions and nonspecific hydrophobic interactions. Fluorescent thioglyconanoparticles were also prepared either by encapsulation of Nile red or by the functionalization of the polymer backbone with rhodamine B. Such nanoparticles were used to prove the cell internalization of the thioglyconanoparticles by lung adenocarcinoma (A549) cells, which underlined the great potential of P(CKA-co-VE) copolymers for biomedical applications.

Supramolecular Organization of Polymer Prodrug Nanoparticles Revealed by Coarse-Grained Simulations.

Drug-polymer conjugates that can self-assemble into nanoparticles are promising drug delivery systems that improve the drug bioavailability and allow their controlled release. However, despite the possibility of reaching high drug loadings, the efficiency of the drug release, mediated by cleavage of the drug-polymer linker, is a key parameter to obtain significant anticancer activity. To overcome the limitations of experimental characterizations and to gain a better understanding of such systems, we conducted a coarse-grained molecular dynamics simulation study on four representative drug-polymer conjugates obtained by the "drug-initiated" method and studied their supramolecular organization upon self-assembly. The prodrugs were composed of either a gemcitabine or a paclitaxel anticancer drug, either a propanoate or a diglycolate linker, and a polyisoprene chain. Our simulations gave crucial information concerning the spatial organization of the different components (e.g., drug, linker, polymer, etc.) into the nanoparticles and revealed that the linkers are not fully accessible to the solvent. Notably, some cleavage sites were either poorly hydrated or partially solvated. These observations might account for the low efficiency of drug release from the nanoparticles, particularly when the linker is too short and/or not hydrophilic/solvated enough. We believe that our theoretical study could be adapted to other types of polymer prodrugs and could guide the design of new polymer prodrug nanoparticles with improved drug release efficiency.

Antibody-functionalized polymer nanoparticle leading to memory recovery in Alzheimer's disease-like transgenic mouse model.

Alzheimer's disease (AD) is a neurodegenerative disorder related, in part, to the accumulation of amyloid-ß peptide (Aß) and especially the Aß peptide 1-42 (Aß1-42). The aim of this study was to design nanocarriers able to: (i) interact with the Aß1-42 in the blood and promote its elimination through the "sink effect" and (ii) correct the memory defect observed in AD-like transgenic mice. To do so, biodegradable, PEGylated nanoparticles were surface-functionalized with an antibody directed against Aß1-42. Treatment of AD-like transgenic mice with anti-Aß1-42-functionalized nanoparticles led to: (i) complete correction of the memory defect; (ii) significant reduction of the Aß soluble peptide and its oligomer level in the brain and (iii) significant increase of the Aß levels in plasma. This study represents the first example of Aß1-42 monoclonal antibody-decorated nanoparticle-based therapy against AD leading to complete correction of the memory defect in an experimental model of AD.

Incomplete copolymer degradation of in situ chemotherapy.

In situ carmustine wafers containing 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) are commonly used for the treatment of recurrent glioblastoma to overcome the brain-blood barrier. In theory, this chemotherapy diffuses into the adjacent parenchyma and the excipient degrades in maximum 8 weeks but no clinical data confirms this evolution, because patients are rarely operated again. A 75-year-old patient was operated twice for recurrent glioblastoma, and a carmustine wafer was implanted during the second surgery. Eleven months later, a third surgery was performed, revealing unexpected incomplete degradation of the wafer. 1H-Nuclear Magnetic Resonance was performed to compare this wafer to pure BCNU and to an unused copolymer wafer. In the used wafer, peaks corresponding to hydrophobic units of the excipient were no longer noticeable, whereas peaks of the hydrophilic units and traces of BCNU were still present. These surprising results could be related to the formation of a hydrophobic membrane around the wafer, thus interfering with the expected diffusion and degradation processes. The clinical benefit of carmustine wafers in addition to the standard radio-chemotherapy remains limited, and in vivo behavior of this treatment is not completely elucidated yet. We found that the wafer may remain after several months. Alternative strategies to deal with the blood-brain barrier, such as drug-loaded liposomes or ultrasound-opening, must be explored to offer larger drug diffusion or allow repetitive delivery.

Pulmonary Surfactant Protein A-Mediated Enrichment of Surface-Decorated Polymeric Nanoparticles in Alveolar Macrophages.

Surfactant protein A (SP-A), a lung anti-infective protein, is a lectin with affinity for sugars found on fungal and micrococcal surfaces such as mannose. We synthesized a mannosylated poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) copolymer and used it to produce nanoparticles with a polyester (PLGA/PLA) core and a PEG shell decorated with mannose residues, designed to be strongly associated with SP-A for an increased uptake by alveolar macrophages. Nanoparticles made of the copolymers were obtained by nanoprecipitation and displayed a size of around 140 nm. The presence of mannose on the surface was demonstrated by zeta potential changes according to pH and by a strong aggregation in the presence of concanavalin A. Mannosylated nanoparticles bound to SP-A as demonstrated by dynamic light scattering and transmission electron microscopy. The association with SP-A increased nanoparticle uptake by THP-1 macrophages in vitro. In vivo experiments demonstrated that after intratracheal administration of nanoparticles with or without SP-A, SP-A-coated mannosylated nanoparticles were internalized by alveolar macrophages in greater proportion than SP-A-coated nonmannosylated nanoparticles. The data demonstrate for the first time that the pool of nanoparticles available to lung cells can be changed after surface modification, using a biomimetic approach.

Nitroxide-mediated radical ring-opening copolymerization: chain-end investigation and block copolymer synthesis.

Well-defined, degradable copolymers are successfully prepared by nitroxide-mediated radical ring opening polymerization (NMrROP) of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) or methyl methacrylate (MMA), a small amount of acrylonitrile (AN) and cyclic ketene acetals (CKAs) of different structures. Phosphorous nuclear magnetic resonance allows in-depth chain-end characterization and gives crucial insights into the nature of the copoly-mer terminal sequences and the living chain fractions. By using a small library of P(OEGMA-co-AN-co-CKA) and P(MMA-co-AN-co-CKA) as macroinitiators, chain extensions with styrene are performed to furnish (amphiphilic) block copolymers comprising a degradable segment.

Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery.

Design and functionalization strategies for multifunctional nanocarriers (e.g., nanoparticles, micelles, polymersomes) based on biodegradable/biocompatible polymers intended to be employed for active targeting and drug delivery are reviewed. This review will focus on the nature of the polymers involved in the preparation of targeted nanocarriers, the synthesis methods to achieve the desired macromolecular architecture, the selected coupling strategy, the choice of the homing molecules (vitamins, hormones, peptides, proteins, etc.), as well as the various strategies to display them at the surface of nanocarriers. The resulting morphologies and the main colloidal features will be given as well as an overview of the biological activities, with a special focus on the main in vivo achievements.

Thermosensitive polymer prodrug nanoparticles prepared by an all-aqueous nanoprecipitation process and application to combination therapy.

Despite their great versatility and ease of functionalization, most polymer-based nanocarriers intended for use in drug delivery often face serious limitations that can prevent their clinical translation, such as uncontrolled drug release and off-target toxicity, which mainly originate from the burst release phenomenon. In addition, residual solvents from the formulation process can induce toxicity, alter the physico-chemical and biological properties and can strongly impair further pharmaceutical development. To address these issues, we report polymer prodrug nanoparticles, which are prepared without organic solvents via an all-aqueous formulation process, and provide sustained drug release. This was achieved by the "drug-initiated" synthesis of well-defined copolymer prodrugs exhibiting a lower critical solution temperature (LCST) and based on the anticancer drug gemcitabine (Gem). After screening for different structural parameters, prodrugs based on amphiphilic diblock copolymers were formulated into stable nanoparticles by all-aqueous nanoprecipitation, with rather narrow particle size distribution and average diameters in the 50-80 nm range. They exhibited sustained Gem release in human serum and acetate buffer, rapid cellular uptake and significant cytotoxicity on A549 and Mia PaCa-2 cancer cells. We also demonstrated the versatility of this approach by formulating Gem-based polymer prodrug nanoparticles loaded with doxorubicin (Dox) for combination therapy. The dual-drug nanoparticles exhibited sustained release of Gem in human serum and acidic release of Dox under accelerated pathophysiological conditions. Importantly, they also induced a synergistic effect on triple-negative breast cancer line MDA-MB-231, which is a relevant cell line to this combination.

Polymer prodrug nanoparticles based on naturally occurring isoprenoid for anticancer therapy.

The synthesis of a novel class of polymer prodrug nanoparticles with anticancer activity is reported by using squalene, a naturally occurring isoprenoid, as a building block by the reversible addition-fragmentation (RAFT) technique. The RAFT agent was functionalized by gemcitabine (Gem) as anticancer drug, and the polymerization of squalenyl-methacrylate (SqMA) led to well-defined macromolecular prodrugs comprising one Gem at the extremity of each polymer chain. The amphiphilic nature of the resulting Gem-PSqMA conjugates allowed them to self-assemble into long-term stable and narrowly dispersed nanoparticles with significant anticancer activity in vitro on various cancer cell lines. To confer stealth properties on these nanoparticles, their PEGylation was successfully performed, as confirmed by X-ray photoelectron spectroscopy (XPS) and complement activation assay. It was also shown that the PEGylated nanoparticles could be internalized in cancer cells to a greater extent than their non-PEGylated counterparts.

Degradable and comb-like PEG-based copolymers by nitroxide-mediated radical ring-opening polymerization.

Three cyclic ketene acetals, 2-methylene-1,3-dioxepane (MDO), 5,6-benzo-2-methylene-1,3-dioxepane (BMDO), and 2-methylene-4-phenyl-1,3-dioxolane (MPDL), have been copolymerized with oligo(ethylene glycol) methyl ether methacrylate and a small amount of acrylonitrile (or styrene) at 90 °C by nitroxidemediated radical ring-opening polymerization, as a convenient way to prepare degradable PEG-based copolymers for biomedical applications. MPDL was the best candidate, enabling high monomer conversions to be reached and well-defined PEG-based copolymers with adjustable amount of ester groups in the main chain to be synthesized, leading to nearly complete hydrolytic degradation (5% KOH aqueous solution, ambient temperature). The noncytotoxicity of the obtained copolymers was shown on three different cell lines (i.e., fibroblasts, endothelial cells and macrophages), representing a promising approach for the design of degradable precursors for PEGylation and bioconjugation via the NMP technique.

Facile synthesis of multicompartment micelles based on biocompatible poly(3-hydroxyalkanoate).

In this paper, a straightforward method to produce poly(3-hydroxyalkanoate)-based multicompartment micelles (MCMs) is presented. Thiol-ene addition is used to graft sequentially perfluorooctyl chains and poly(ethylene glycol) oligomers onto poly(3-hydroxyoctanoate-co-hydroxyundecenoate) oligomers backbone. Well-defined copolymers are obtained as shown by ¹H NMR and size-exclusion chromatography. After nanoprecipitation in water, novel PHA-based MCMs are evidenced by cryo-transmission electron microscopy. Moreover, the cytocompatibility of MCMs is demonstrated in vitro via cell viability assay.

Vinyl copolymers with faster hydrolytic degradation than aliphatic polyesters and tunable upper critical solution temperatures.

Vinyl polymers are the focus of intensive research due to their ease of synthesis and the possibility of making well-defined, functional materials. However, their non-degradability leads to environmental problems and limits their use in biomedical applications, allowing aliphatic polyesters to still be considered as the gold standards. Radical ring-opening polymerization of cyclic ketene acetals is considered the most promising approach to impart degradability to vinyl polymers. However, these materials still exhibit poor hydrolytic degradation and thus cannot yet compete with traditional polyesters. Here we show that a simple copolymerization system based on acrylamide and cyclic ketene acetals leads to well-defined and cytocompatible copolymers with faster hydrolytic degradation than that of polylactide and poly(lactide-co-glycolide). Moreover, by changing the nature of the cyclic ketene acetal, the copolymers can be either water-soluble or can exhibit tunable upper critical solution temperatures relevant for mild hyperthermia-triggered drug release. Amphiphilic diblock copolymers deriving from this system can also be formulated into degradable, thermosensitive nanoparticles by an all-water nanoprecipitation process.

Biodegradable nanoparticles meet the bronchial airway barrier: how surface properties affect their interaction with mucus and epithelial cells.

Despite the wide interest raised by lung administration of nanoparticles (NPs) for the treatment of various diseases, little information is available on their effect toward the airway epithelial barrier function. In this study, the potential damage of the pulmonary epithelium upon exposure to poly(lactide-co-glycolide) (PLGA) NPs has been assessed in vitro using a Calu-3-based model of the bronchial epithelial barrier. Positively and negatively charged as well as neutral PLGA NPs were obtained by coating their surface with chitosan (CS), poloxamer (PF68), or poly(vinyl alcohol) (PVA). The role of NP surface chemistry and charge on the epithelial resistance and mucus turnover, using MUC5AC as a marker, was investigated. The interaction with mucin reduced the penetration of CS- and PVA-coated NPs, while the hydrophilic PF68-coated NPs diffused across the mucus barrier leading to a higher intracellular accumulation. Only CS-coated NPs caused a transient but reversible decrease of the trans-epithelial electrical resistance (TEER). None of the NP formulations increased MUC5AC mRNA expression or the protein levels. These in vitro results highlight the safety of PLGA NPs toward the integrity and function of the bronchial airway barrier and demonstrate the crucial role of NP surface properties to achieve a controlled and sustained delivery of drugs via the pulmonary route.

Nanotechnologies for Alzheimer's disease: diagnosis, therapy, and safety issues.

Alzheimer's disease (AD) represents the most common form of dementia worldwide, affecting more than 35 million people. Advances in nanotechnology are beginning to exert a significant impact in neurology. These approaches, which are often based on the design and engineering of a plethora of nanoparticulate entities with high specificity for brain capillary endothelial cells, are currently being applied to early AD diagnosis and treatment. In addition, nanoparticles (NPs) with high affinity for the circulating amyloid-ß (Aß) forms may induce "sink effect" and improve the AD condition. There are also developments in relation to in vitro diagnostics for AD, including ultrasensitive NP-based bio-barcodes, immunosensors, as well as scanning tunneling microscopy procedures capable of detecting Aß(1-40) and Aß(1-42). However, there are concerns regarding the initiation of possible NP-mediated adverse events in AD, thus demanding the use of precisely assembled nanoconstructs from biocompatible materials. Key advances and safety issues are reviewed and discussed.