Role of the Polymer Microstructure in Controlling Colloidal and Thermo-Responsive Properties of Nano-Objects Prepared Via RAFT Polymerization in a Non-polar Medium.

After having demonstrated their potential in biomedical applications, thermo-responsive block copolymers that are able to self-assemble into nano-objects in response to temperature modifications are becoming more and more appealing in other sectors, such as the oil and gas and lubricant fields. Reversible addition-fragmentation chain transfer (RAFT) polymerization-induced self-assembly has been demonstrated as a valuable strategy for producing nano-objects from modular block copolymers in non-polar media, required for the mentioned applications. Although the influence of the nature and size of the thermo-responsive block of these copolymers on the properties of the nano-objects is extensively studied in the literature, the role of the solvophilic block is often neglected. In this work, we elucidate the role of the main microstructural parameters, including those of the solvophilic portion, of block copolymers produced by RAFT polymerization in the hydrocarbon blend decane/toluene 50:50 v/v on the thermo-responsive behavior and colloidal properties of the resulting nano-objects. Two long-aliphatic chain monomers were employed for the synthesis of four macromolecular chain transfer agents (macroCTAs), with increasing solvophilicity according to the number of units (n) or length of the alkyl side chain (q). Subsequently, the macroCTAs were chain-extended with different repeating units of di(ethylene glycol) methyl ether methacrylate (p), leading to copolymers that are able to self-assemble below a critical temperature. We show that this cloud point can be tuned by acting on n, p, and q. On the other hand, the colloidal stability, expressed in terms of area of the particle covered by each solvophilic segment, is only a function of n and q, which provides a way for controlling the size distribution of the nano-objects and to decouple it from the cloud point.

A Graphene-Based Supramolecular Nanoreactor for the Fast Synthesis of Imines in Water.

The confinement of organic synthesis within waterborne nanoreactors is regarded with increasing attention to improve its yield and reduce the environmental impact. However, many catalysts, such as graphene, are barely dispersible in aqueous media and many chemical reactions cannot be performed in the presence of water due to thermodynamic limitations. Therefore, there is an urgent need to develop novel strategies to carry out these processes in more sustainable conditions. To pursue this goal, in this work, a waterborne supramolecular nanoreactor is developed. The system comprises a polymeric micelle obtained from the self-assembly of pyrrole-based amphiphilic block copolymers. The active catalytic component is represented by few graphene layers, functionalized with pyrrole to enhance their interaction with the micelle core and hence their nanoencapsulation. Using this nanoreactor, it is possible to synthesize imines starting from primary amines and aldehydes or ketones with high yield and in short time (Y = 90% after 5 min) at room temperature. Moreover, an efficient strategy to recycle the reactor is proposed, thus increasing the potential of this technology.

Zwitterionic Polyester-Based Nanoparticles with Tunable Size, Polymer Molecular Weight, and Degradation Time.

Biodegradable polymer nanoparticles are an important class of materials used in several applications for their unique characteristics. In particular, the ones stabilized by zwitterionic materials are gaining increased interest in medicine as alternative to the more common ones based on poly(ethylene glycol) thanks to their superior stability and ability to avoid both the accelerated blood clearance and allergic reactions. In this work, a novel class of zwitterionic based NPs has been produced, and a method to independently control the nanoparticle size, degradation time, and polymer molecular weight has been developed and demonstrated. This has been possible by the synthesis and the fine-tuning of zwitterionic amphiphilic block copolymers obtained via the combination of ring-opening polymerization and reversible addition-fragmentation chain transfer polymerization. The final results showed that when two block copolymers contain the same number of caprolactone units, the one with longer oligoester lateral chains degrades faster. This phenomenon is in sharp contrast with the one seen so far for the common linear polyester systems where longer chains result in longer degradation times, and it can be used to better tailor the degradation behavior of the nanoparticles.

Synthesis and characterization of pH-sensitive drinkable nanoparticles for oral delivery of ibuprofen.

Ibuprofen (IBU) is a widespread drug used to treat both acute and chronic disorders. It is generally taken orally but the free drug can induce the irritation of the gastric mucosa due to its acid nature. In literature, different approaches have been adopted to prevent the release in the stomach, such as physical entrapment with film-coated tablets and drug-conjugates. Nevertheless, these solutions have many disadvantages, including the fast release of the drug and the difficulty to swallow the tablet, especially for children who may vomit or refuse the tablet. For this reason, in this work, novel formulations are proposed that do not require the encapsulation of the drug into a solid form and, in turn, their assumption as a pill. IBU has been linked to different types of methacrylates via ester bond in order to produce pH-responsive macromolecular monomers. The novelty is related to the use of these drug-conjugates macromonomer for the production of nanoparticles (NPs) via emulsion polymerization (EP), using water as solvent. The final emulsion is able to load up to 30 mg ml-1 of IBU, so less than 10 ml is required to be assumed to reach the minimum therapeutic dose of the drug (200 mg). Finally, the release of IBU from these novel drinkable formulations has been investigated in the gastric and intestinal simulated fluids to show the preferential release of IBU from the NPs in basic conditions. A comparison with an existing oral suspension has been performed to highlight the slower release in acid environment of these new formulations. Afterwards, the IBU loaded NPs were tested in vitro showing lower toxicity compared to the free drug.

Hydrazone linked doxorubicin-PLA prodrug nanoparticles with high drug loading.

An optimal drug delivery system should be characterized by biocompatibility, biodegradability, high drug loading and favorable drug release profile. To achieve this goal a hydrazone linked doxorubicin-poly(lactic acid) prodrug (PLA-DOX) was synthesized by the functionalization of a short polymer chain produced by ring opening polymerization. The hydrophobic prodrug generated in this way was nanoprecipitated using a block copolymer to form polymeric nanoparticles (NPs) with a quantitative loading efficiency and a high and tunable drug loading. The effects of the concentration of the PLA-DOX prodrug and surfactant were studied by dynamic light scattering showing a range of NP size between 50 and 90 nm and monodispersed size distributions with polydispersity indexes lower then 0.27 up to a maximum DOX concentration of 27% w/w. The release profile of DOX from these NPs, tested at different pH conditions, showed a higher release rate in acidic conditions, consistent with the nature of the hydrazone bond which was used to conjugate the drug to the polymer. In vitro cytotoxicity studies performed on BV2 microglia-like cell line highlighted a specific cytotoxic effect of these NPs suggesting the maintenance of the drug efficacy and a modified release profile upon encapsulation of DOX in the NPs.

Biocompatible Polymer Nanoformulation To Improve the Release and Safety of a Drug Mimic Molecule Detectable via ICP-MS.

Fluorescent poly(ε-caprolactone)-based nanoparticles (NPs) have been synthesized and successfully loaded with a titanium organometallic compound as a mimic of a water-insoluble drug. The nature of this nanovector enabled us to combine the quantification of the metal in tissues after systemic administration in healthy immunocompetent mice by inductively coupled plasma mass spectroscopy (ICP-MS) followed by the visualization of NPs in organ sections by confocal microscopy. This innovative method of nanodrug screening has enabled us to elucidate the crucial parameters of their kinetics. The organometallic compound is a good mimic of most anticancer drugs, and this approach is an interesting starting point to design the relevance of a broad range of nanoformulations in terms of safety and targeted delivery of the cargoes.

Use of RAFT macro-surfmers for the synthesis of transparent aqueous colloids with tunable interactions.

We propose a new method to produce fluorinated nanoparticles (NPs) based on ab initio reversible addition-fragmentation chain transfer (RAFT) emulsion polymerization without the use of toxic surfactants. NP size, surface charge, and chemistry can be controlled via the adoption of different macromolecular transfer agents produced via RAFT polymerization of amphiphilic monomers. Thanks to this versatility, interparticle interactions can be easily tuned by changing solvent composition and temperature. In addition, the refractive index and density of the solvent can simultaneously match those of the NPs by adding sodium polytungstate, an organic salt widely used for density gradient centrifugation. These colloids may be used as model systems for the study of self-assembly and aggregation in aqueous media when optical methods are required.

Internalization of nanopolymeric tracers does not alter characteristics of placental cells.

In the cell therapy scenario, efficient tracing of transplanted cells is essential for investigating cell migration and interactions with host tissues. This is fundamental to provide mechanistic insights which altogether allow for the understanding of the translational potential of placental cell therapy in the clinical setting. Mesenchymal stem/stromal cells (MSC) from human placenta are increasingly being investigated for their potential in treating patients with a variety of diseases. In this study, we investigated the feasibility of using poly (methyl methacrylate) nanoparticles (PMMA-NPs) to trace placental MSC, namely those from the amniotic membrane (hAMSC) and early chorionic villi (hCV-MSC). We report that PMMP-NPs are efficiently internalized and retained in both populations, and do not alter cell morphofunctional parameters. We observed that PMMP-NP incorporation does not alter in vitro immune modulatory capability of placental MSC, a characteristic central to their reparative/therapeutic effects in vitro. We also show that in vitro, PMMP-NP uptake is not affected by hypoxia. Interestingly, after in vivo brain ischaemia and reperfusion injury achieved by transient middle cerebral artery occlusion (tMCAo) in mice, iv hAMSC treatment resulted in significant improvement in cognitive function compared to PBS-treated tMCAo mice. Our study provides evidence that tracing placental MSC with PMMP-NPs does not alter their in vitro and in vivo functions. These observations are grounds for the use of PMMP-NPs as tools to investigate the therapeutic mechanisms of hAMSC and hCV-MSC in preclinical models of inflammatory-driven diseases.

PEGylated Nanoparticles Obtained through Emulsion Polymerization as Paclitaxel Carriers.

Polymer nanoparticles (NPs) represent a promising way to deliver poorly water-soluble anticancer drugs without the use of unwanted excipients, whose presence can be the cause of severe side effects. In this work, a Cremophor-free formulation for paclitaxel (PTX) has been developed by employing PEGylated polymer nanoparticles (NPs) as drug delivery carriers based on modified poly(ε-caprolactone) macromonomers and synthesized through free radical emulsion polymerization. Paclitaxel was loaded in the NPs in a postsynthesis process which allowed to obtain a drug concentration suitable for in vivo use. In vivo experiments on drug biodistribution and therapeutic efficacy show comparable behavior between the NPs and the Cremophor formulation, also showing good tolerability of the new formulation proposed.

Fate of PLA and PCL-Based Polymeric Nanocarriers in Cellular and Animal Models of Triple-Negative Breast Cancer.

An integrated platform to assess the interaction between nanocarriers and biological matrices has been developed by our group using poly methyl-methacrylate nanoparticles. In this study, we exploited this platform to evaluate the behavior of two biodegradable formulations, poly-ε-caprolactone (PCL3) and poly lactic-acid (PLA8), respectively, in cellular and animal models of triple-negative breast cancer (TNBC). Both NPs shared the main physicochemical parameters (size, shape, ζ-potential) and exclusively differentiated on the material on which they are composed. Our results showed that (1) PLA8 NPs, systemically injected in mice, underwent rapid degradation without penetration into tumors; (2) PLA8 NPs were not internalized in the human TNBC cell line (MDA-MB-231); (3) PCL3 NPs had a longer bioavailability, reached the tumor parenchyma, and efficiently penetrated in MDA-MB-231 cells. Our data highlight the relevance of the material selection to both improve bioavailability and target tropism, and make PCL3 NPs an interesting tool for the development of nanodrugs against TNBC.

Small interfering RNA delivery through positively charged polymer nanoparticles.

Small interfering RNA (siRNA) is receiving increasing attention with regard to the treatment of many genetic diseases, both acquired and hereditary, such as cancer and diabetes. Being a high molecular weight (MW) polyanion, siRNA is not able to cross a cell membrane, and in addition it is unstable in physiological conditions. Accordingly, a biocompatible nanocarrier able to deliver siRNA into cells is needed. In this work, we synthesized biocompatible positively charged nanoparticles (NPs) following a two-step process that involves ring opening polymerization (ROP) and emulsion free radical polymerization (EFRP). Firstly, we proved the possibility of fine tuning the NPs' characteristics (e.g. size and surface charge) by changing the synthetic process parameters. Then the capability in loading and delivering undamaged siRNA into a cancer cell cytoplasm has been shown. This latter process occurs through the biodegradation of the polymer constituting the NPs, whose kinetics can be tuned by adjusting the polymer's MW. Finally, the ability of NPs to carry siRNA inside the cells in order to inhibit their target gene has been demonstrated using green flourescent protein positive cells.

Polymer hydrogel functionalized with biodegradable nanoparticles as composite system for controlled drug delivery.

The possibility to direct pharmacological treatments targeting specific cell lines using polymer nanoparticles is one of the main novelties and perspectives in nanomedicine. However, sometimes, the ability to maintain NPs localized at the site of the injection that work as a drug reservoir can represent a good and complementary option. In this direction we built a composite material made of polymeric hydrogel functionalized with polymer NPs. ϵ-caprolactone and polyethylene glycol have been copolymerized in a two-step synthesis of PEGylated NPs, while hydrogel was synthesized through polycondensation between NPs, agarose and branched polyacrylic acid. NP functionalization was verified with Fourier transform infrared spectroscopy (FTIR), high resolution magic angle spinning-nuclear magnetic resonance (HRMAS-NMR) spectroscopy and release kinetics from a hydrogel matrix and compared with NPs only physically entrapped into a hydrogel matrix. The characteristics of the resulting composite hydrogel-NPs system were studied both in terms of rheological properties and in its ability to sustain the release of To-Pro3, used as a drug mimetic compound to represent a promising drug delivery device.

Synthesis of hetero-nanoclusters: the case of polymer-magnetite systems.

Nanoclusters (NCs) composed of nanoparticles (NPs) with different functionalities and having final size in the sub-micrometer range are of great interest for biomedical imaging, drug delivery, sensors, etc. Because some of the functionalities cannot be incorporated into a single NP, e.g., high drug loading combined with strong magnetic properties, here, we present a proof of the concept using an alternative way to combine these properties using different NPs. In particular, starting from polymer and magnetite nanoparticles (MNPs), we produce NCs made out of a statistical distribution of the two components through a process based on aggregation and breakup. The effect of all involved operating parameters, i.e., primary NP size and composition, surfactant type and concentration, and applied hydrodynamic stress on the NC size and internal structure, was systematically investigated using dynamic light scattering (DLS), static light scattering (SLS), and transmission electron microscopy (TEM) analyses. It was found that, by properly tuning the balance between attractive and steric repulsive forces on one side and hydrodynamic stress on the other, NCs as small as 100 nm can be produced. In all cases, the produced NCs have a very compact internal structure characterized by fractal dimension around 2.6. The proposed production strategy to synthesize hetero-NCs composed of mixtures of various primary particles is suitable for the production of multifunctional devices of nanometer size (i.e., approximately 100 nm) for material and biomedical applications.

Integrated multiplatform method for in vitro quantitative assessment of cellular uptake for fluorescent polymer nanoparticles.

Studies of cellular internalization of nanoparticles (NPs) play a paramount role for the design of efficient drug delivery systems, but so far they lack a robust experimental technique able to quantify the NP uptake in terms of number of NPs internalized in each cell. In this work we propose a novel method which provides a quantitative evaluation of fluorescent NP uptake by combining flow cytometry and plate fluorimetry with measurements of number of cells. Single cell fluorescence signals measured by flow cytometry were associated with the number of internalized NPs, exploiting the observed linearity between average flow cytometric fluorescence and overall plate fluorimeter measures, and previous calibration of the microplate reader with serial dilutions of NPs. This precise calibration has been made possible by using biocompatible fluorescent NPs in the range of 20-300 nm with a narrow particle size distribution, functionalized with a covalently bonded dye, Rhodamine B, and synthesized via emulsion free-radical polymerization. We report the absolute number of NPs internalized in mouse mammary tumor cells (4T1) as a function of time for different NP dimensions and surface charges and at several exposure concentrations. The obtained results indicate that 4T1 cells incorporated 10(3)-10(4) polymer NPs in a short time, reaching an intracellular concentration 15 times higher than the external one.

Quantum mechanical investigation on bimolecular hydrogen abstractions in butyl acrylate-based free radical polymerization processes.

The present computational study focuses on the investigation of bimolecular hydrogen abstractions that can occur during free radical polymerization (FRP) processes. In particular, several hydrogen abstractions from four monomers (butyl acrylate, BA; styrene, ST; butyl methacrylate, BMA; vinyl acetate, VA) and three different backbone chains (poly-BA, poly-BA-co-VA, and poly-BA-co-ST) have been studied. The aim is to provide an overview of the kinetics for all possible intermolecular hydrogen abstraction reactions from all chemical species present in a bulk FRP as well as to support the understanding of the influence of chemical environment on hydrogen abstractions. All simulations were performed using density functional theory (DFT) with quantum tunneling factors estimated using the Eckart model. This study provides proof that the presence of an electron donating group in the chemical environment of the abstracted hydrogen atoms can lead to lower activation energies and higher rate coefficients for abstraction whereas the presence of an electron withdrawing group leads to opposite effects.

Quantum chemistry investigation of fluorinated polymer systems of industrial interest.

In this work, the free-radical polymerization (FRP) of widely used fluorinated monomers was investigated. Computational studies were conducted to assess the FRP kinetics of each binary copolymerization between vinylidene fluoride (VDF), hexafluoropropylene (HFP), and tetrafluoroethylene (TFE). More specifically, all calculations were performed using density functional theory (DFT), and the B3LYP level of theory was used to optimize structures and determine absolute minimum energy geometries, whereas the electronic energies were estimated using B3LYP/6-31G(d,p) as well as a higher level of theory, MPWB1K/6-31G(d,p). Transition state theory was employed to determine kinetic parameters according to the terminal model of copolymerization. The homopolymerization of VDF and all of its corresponding copolymerizations were investigated by taking into account every possible propagation reaction (head to head, head to tail, tail to tail, head to monomer, tail to monomer, etc.) to estimate the Arrhenius parameters for each system. This study provides the estimation of a large set of rate coefficients, which gives detailed pictures of the specific copolymerization systems examined and is highly valuable to generate a comprehensive overview of the polymerization kinetics of relevant fluorinated monomers.

Zwitterionic nanoparticles for thermally activated drug delivery in hyperthermia cancer treatment.

Hyperthermia is considered a promising strategy to boost the curative outcome of traditional chemotherapeutic treatments. However, this thermally mediated drug delivery is still affected by important limitations. First, the poor accumulation of the conventional anticancer formulations in the target site limits the bioavailability of the active ingredient and induces off-site effects. In addition, some tumoral scenarios, such as ovarian carcinoma, are characterized by cell thermotolerance, which induces tumoral cells to activate self-protecting mechanisms against high temperatures. To overcome these constraints, we developed thermoresponsive nanoparticles (NPs) with an upper critical solution temperature (UCST) to intracellularly deliver a therapeutic payload and release it on demand through hyperthermia stimulation. These NPs were synthesized via reversible addition-fragmentation chain transfer (RAFT) emulsion polymerization and combine polyzwitterionic stabilizing segments and an oligoester-based biodegradable core. By leveraging the pseudo-living nature of RAFT polymerization, important physicochemical properties of the NPs were controlled and optimized, including their cloud point (Tcp) and size. We have tuned the Tcp of NPs to match the therapeutic needs of hyperthermia treatments at 43 °C and tested the nanocarriers in the controlled delivery of paclitaxel, a common anticancer drug. The NPs released almost entirely the encapsulated drug only following 1 h incubation at 43 °C, whereas they retained more than 95% of the payload in the physiological environment (37 °C), thus demonstrating their efficacy as on-demand drug delivery systems. The administration of drug-loaded NPs to ovarian cancer cells led to therapeutic effects outperforming the conventional administration of non-encapsulated paclitaxel, which highlights the potential of the zwitterionic UCST-type NPs as an innovative hyperthermia-responsive drug delivery system.

Degradable Alternating Copolymers by Radical Copolymerization of 2-Methylen-1,3-dioxepane and Crotonate Esters.

Producing backbone degradable copolymers via free-radical copolymerization is a promising, yet challenging method to develop more sustainable materials for many applications. In this work, we present the copolymerization of 2-methylen-1,3-dioxepane (MDO) with crotonic acid derivative esters. MDO can copolymerize by radical ring-opening polymerization incorporating degradable ester moieties in the polymer backbone, although this can often be difficult due to the very unfavorable reactivity ratios. Crotonic acid derivatives, on the other hand, can be easily produced completely from biomass but are typically very difficult to (co)polymerize due to low propagation rates and very unfavorable reactivity ratios. Herein, we present the surprisingly easy copolymerization between MDO and butyl crotonate (BCr), which shows the ability to form alternating copolymers. The alternating nature of the copolymer was characterized by MALDI-TOF and supported by the reactivity ratios calculated experimentally (rMDO = 0.105 and rBCr = 0.017). The alternating nature of the copolymers favored the degradability that could be achieved under basic conditions (in 2 h, all chains have molar masses smaller than 2 kg/mol). Last, the work was expanded to other crotonate monomers to expand the portfolio and show the potential of this copolymer family.

3D-printed boluses for radiotherapy: influence of geometrical and printing parameters on dosimetric characterization and air gap evaluation.

The work investigates the implementation of personalized radiotherapy boluses by means of additive manufacturing technologies. Boluses materials that are currently used need an excessive amount of human intervention which leads to reduced repeatability in terms of dosimetry. Additive manufacturing can solve this problem by eliminating the human factor in the process of fabrication. Planar boluses with fixed geometry and personalized boluses printed starting from a computed tomography scan of a radiotherapy phantom were produced. First, a dosimetric characterization study on planar bolus designs to quantify the effects of print parameters such as infill density and geometry on the radiation beam was made. Secondly, a volumetric quantification of air gap between the bolus and the skin of the patient as well as dosimetric analyses were performed. The optimization process according to the obtained dosimetric and airgap results allowed us to find a combination of parameters to have the 3D-printed bolus performing similarly to that in conventional use. These preliminary results confirm those in the relevant literature, with 3D-printed boluses showing a dosimetric performance similar to conventional boluses with the additional advantage of being perfectly conformed to the patient geometry.

Biocompatible fluorescent nanoparticles for in vivo stem cell tracking.

Efficient application of stem cells to the treatment of neurodegenerative diseases requires safe cell tracking to follow stem cell fate over time in the host environment after transplantation. In this work, for the first time, fluorescent and biocompatible methyl methacrylate (MMA)-based nanoparticles (fluoNPs) were synthesized through a free-radical co-polymerization process with a fluorescent macromonomer obtained by linking Rhodamine B and hydroxyethyl methacrylate. We demonstrate that the fluoNPs produced by polymerization of MMA-Rhodamine complexes (1) were efficient for the labeling and tracking of multipotent human amniotic fluid cells (hAFCs); (2) did not alter the main biological features of hAFCs (such as viability, cell growth and metabolic activity); (3) enabled us to determine the longitudinal bio-distribution of hAFCs in different brain areas after graft in the brain ventricles of healthy mice by a direct fluorescence-based technique. The reliability of our approach was furthermore confirmed by magnetic resonance imaging analyses, carried out by incubating hAFCs with both superparamagnetic iron oxide nanoparticles and fluoNPs. Our data suggest that these finely tunable and biocompatible fluoNPs can be exploited for the longitudinal tracking of stem cells.