Microwave-assisted synthesis of TEMPO-labeled hydrogels traceable with MRI.

Polymer functionalization strategies have recently attracted considerable attention for several applications in biomaterials science. In particular, technological advancements in medical imaging have focused on the design of polymeric matrices to improve non-invasive approaches and diagnostic accuracy. In this scenario, the use of microwave irradiation of aqueous solutions containing appropriate combinations of polymers is gaining increasing interest in the synthesis of sterile hydrogels without using monomers, eliminating the need to remove unreacted species. In this study, we developed a method for the in situ fabrication of TEMPO-labeled hydrogels based on a one-pot microwave reaction that can then be tracked by magnetic resonance imaging (MRI) without using toxic compounds that could be hostile for the target tissue. Click chemistry was used to link TEMPO to the polymeric scaffold. In an in vivo model, the system was able to preserve its TEMPO paramagnetic activity up to 1 month after hydrogel injection, showing a clear detectable signal on T1-weighted MRI with a longitudinal relaxivity value of 0.29 mM s-1, comparable to a value of 0.31 mM s-1 characteristic of TEMPO application. The uncleavable conjugation between the contrast agent and the polymeric scaffold is a leading point to record these results: the use of TEMPO only physically entrapped in the polymeric scaffold did not show MRI traceability even after few hours. Moreover, the use of TEMPO-labeled hydrogels can also help to reduce the number of animals sacrificed being a longitudinal non-invasive technique.

On the parallelism between the mechanisms behind chromatography and drug delivery: the role of interactions with a stationary phase.

A huge number of studies and work in the drug delivery literature are focused on understanding and modeling transport phenomena, the pivotal point for a good device design. The rationalization of all phenomena involved is fundamental, but several concerns arise leaving many issues unsolved. In order to change the point of view we decided to focus our attention on the parallelisms between two fields that seem to be very far from each other: chromatography and drug release. Taking advantages of the studies conducted by many researchers using chromatographic columns we decided to explain all the phenomena involved in drug delivery considering sodium ibuprofen (IP) molecules as analytes and hydrogel as a stationary phase. In particular, we considered not only diffusion, but also drug-polymer interactions as adsorption on the stationary phase and drug-drug interactions as aggregation of analytes. The hydrogel investigated is a promising formulation made of agarose and carbomer 974p (AC) loaded with IP, a non-steroidal common anti-inflammatory drug. The self-diffusion coefficient of IP in AC formulations was measured by using an innovative method based on a magic angle spinning NMR spectroscopic technique to produce high resolution (liquid-like) spectra. This method (HR-MAS NMR) is used in combination with pulsed field gradient spin echo (PGSE) liquid-state techniques. The model predictions satisfactorily match with the experimental data obtained in water and the gel environment, indicating that the model presented here, despite its simplicity, is able to describe the key phenomena governing the device behavior and could be used to rationalize the experimental activity.

Drug-Polymer Interactions in Hydrogel-based Drug-Delivery Systems: An Experimental and Theoretical Study.

In drug-delivery systems, drug transport is a key step, but the interpretation of the transport mechanism is still controversial. Here, we investigated a promising hydrogel library loaded with the anticonvulsant drug ethosuximide (ESM). The self-diffusion coefficient of ESM was measured using two methods: a direct and advanced measurement with a pulsed field gradient spin-echo (PFGSE) method, using an NMR spectrometer equipped with high-resolution magic angle spinning (HR-MAS) probe, and an indirect one based on fitting in vitro drug-delivery data. Starting from the experimental data a mathematical model without fitted parameters was developed and all the phenomena involved, that is, adsorption and diffusion, were considered. At low drug concentrations, adsorption prevails and consequently the diffusivity in the gels is lower than that in water. At high drug concentrations, where all adsorption sites are saturated, the diffusion in the gels is similar to that in a water solution. This study may pave the way for better device design.

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.

The ß-arrestin1/endothelin axis bolsters ovarian fibroblast-dependent invadosome activity and cancer cell metastatic potential.

Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix (ECM) remodeling, invasion, and metastasis, highlighting the need to investigate the molecular mechanisms driving CAF function. Endothelin-1 (ET-1) regulates the communication between cancer and stroma and facilitates the progression of serous ovarian cancer (SOC). By binding to Endothelin A (ETA) and B (ETB) receptors, ET-1 enables the recruitment of ß-arrestin1 (ß-arr1) and the formation of signaling complexes that coordinate tumor progression. However, how ET-1 receptors might "educate" human ovarian fibroblasts (HOFs) to produce altered ECM and promote metastasis remains to be elucidated. This study identifies ET-1 as a pivotal factor in the activation of CAFs capable of proteolytic ECM remodeling and the generation of heterotypic spheroids containing cancer cells with a propensity to metastasize. An autocrine/paracrine ET-1/ETA/BR/ß-arr1 loop enhances HOF proliferation, upregulates CAF marker expression, secretes pro-inflammatory cytokines, and increases collagen contractility, and cell motility. Furthermore, ET-1 facilitates ECM remodeling by promoting the lytic activity of invadosome and activation of integrin ß1. In addition, ET-1 signaling supports the formation of heterotypic HOF/SOC spheroids with enhanced ability to migrate through the mesothelial monolayer, and invade, representing metastatic units. The blockade of ETA/BR or ß-arr1 silencing prevents CAF activation, invadosome function, mesothelial clearance, and the invasive ability of heterotypic spheroids. In vivo, therapeutic inhibition of ETA/BR using bosentan (BOS) significantly reduces the metastatic potential of combined HOFs/SOC cells, associated with enhanced apoptotic effects on tumor cells and stromal components. These findings support a model in which ET-1/ß-arr1 reinforces tumor/stroma interaction through CAF activation and fosters the survival and metastatic properties of SOC cells, which could be counteracted by ETA/BR antagonists.

Modulation of Methacrylated Hyaluronic Acid Hydrogels Enables Their Use as 3D Cultured Model.

Bioengineered hydrogels represent physiologically relevant platforms for cell behaviour studies in the tissue engineering and regenerative medicine fields, as well as in in vitro disease models. Hyaluronic acid (HA) is an ideal platform since it is a natural biocompatible polymer that is widely used to study cellular crosstalk, cell adhesion and cell proliferation, and is one of the major components of the extracellular matrix (ECM). We synthesised chemically modified HA with photo-crosslinkable methacrylated groups (HA-MA) in aqueous solutions and in strictly monitored pH and temperature conditions to obtain hydrogels with controlled bulk properties. The physical and chemical properties of the different HA-MA hydrogels were investigated via rheological studies, mechanical testing and scanning electron microscopy (SEM) imaging, which allowed us to determine the optimal biomechanical properties and develop a biocompatible scaffold. The morphological evolution processes and proliferation rates of glioblastoma cells (U251-MG) cultured on HA-MA surfaces were evaluated by comparing 2D structures with 3D structures, showing that the change in dimensionality impacted cell functions and interactions. The cell viability assays and evaluation of mitochondrial metabolism showed that the hydrogels did not interfere with cell survival. In addition, morphological studies provided evidence of cell-matrix interactions that promoted cell budding from the spheroids and the invasiveness in the surrounding environment.

Editorial on the Special Issue "Advances in Nanogels".

In recent decades, the rise of nanotechnology has led to the design of innovative nano-biomaterials which are used to improve pharmacological therapies and assist with disease diagnosis [...].

Functionalization strategies of polymeric nanoparticles for drug delivery in Alzheimer's disease: Current trends and future perspectives.

Alzheimer's disease (AD), the most common form of dementia, is a progressive and multifactorial neurodegenerative disorder whose primary causes are mostly unknown. Due to the increase in life expectancy of world population, including developing countries, AD, whose incidence rises dramatically with age, is at the forefront among neurodegenerative diseases. Moreover, a definitive cure is not yet within reach, imposing substantial medical and public health burdens at every latitude. Therefore, the effort to devise novel and effective therapeutic strategies is still of paramount importance. Genetic, functional, structural and biochemical studies all indicate that new and efficacious drug delivery strategies interfere at different levels with various cellular and molecular targets. Over the last few decades, therapeutic development of nanomedicine at preclinical stage has shown to progress at a fast pace, thus paving the way for its potential impact on human health in improving prevention, diagnosis, and treatment of age-related neurodegenerative disorders, including AD. Clinical translation of nano-based therapeutics, despite current limitations, may present important advantages and innovation to be exploited in the neuroscience field as well. In this state-of-the-art review article, we present the most promising applications of polymeric nanoparticle-mediated drug delivery for bypassing the blood-brain barrier of AD preclinical models and boost pharmacological safety and efficacy. In particular, novel strategic chemical functionalization of polymeric nanocarriers that could be successfully employed for treating AD are thoroughly described. Emphasis is also placed on nanotheranostics as both potential therapeutic and diagnostic tool for targeted treatments. Our review highlights the emerging role of nanomedicine in the management of AD, providing the readers with an overview of the nanostrategies currently available to develop future therapeutic applications against this chronic neurodegenerative disease.

Droplet-based microfluidic synthesis of nanogels for controlled drug delivery: tailoring nanomaterial properties via pneumatically actuated flow-focusing junction.

Conventional batch syntheses of polymer-based nanoparticles show considerable shortcomings in terms of scarce control over nanomaterials morphology and limited lot-to-lot reproducibility. Droplet-based microfluidics represents a valuable strategy to overcome these constraints, exploiting the formation of nanoparticles within discrete microdroplets. In this work, we synthesized nanogels (NGs) composed of hyaluronic acid and polyethyleneimine using a microfluidic flow-focusing device endowed with a pressure-driven micro-actuator. The actuator achieves real-time modulation of the junction orifice width, thereby regulating the microdroplet diameter and, as a result, the NG size. Acting on process parameters, NG hydrodynamic diameter could be tuned in the range 92-190 nm while preserving an extremely low polydispersity (0.015); those values are hardly achievable in batch syntheses and underline the strength of our toolbox for the continuous in-flow synthesis of nanocarriers. Furthermore, NGs were validated in vitro as a drug delivery system in a representative case study still lacking an effective therapeutic treatment: ovarian cancer. Using doxorubicin as a chemotherapeutic agent, we show that NG-mediated release of the drug results in an enhanced antiblastic effect vs. the non-encapsulated administration route even at sublethal dosages, highlighting the wide applicability of our microfluidics-enabled nanomaterials in healthcare scenarios.

Synthesis of Nanogels: Current Trends and Future Outlook.

Nanogels represent an innovative platform for tunable drug release and targeted therapy in several biomedical applications, ranging from cancer to neurological disorders. The design of these nanocarriers is a pivotal topic investigated by the researchers over the years, with the aim to optimize the procedures and provide advanced nanomaterials. Chemical reactions, physical interactions and the developments of engineered devices are the three main areas explored to overcome the shortcomings of the traditional nanofabrication approaches. This review proposes a focus on the current techniques used in nanogel design, highlighting the upgrades in physico-chemical methodologies, microfluidics and 3D printing. Polymers and biomolecules can be combined to produce ad hoc nanonetworks according to the final curative aims, preserving the criteria of biocompatibility and biodegradability. Controlled polymerization, interfacial reactions, sol-gel transition, manipulation of the fluids at the nanoscale, lab-on-a-chip technology and 3D printing are the leading strategies to lean on in the next future and offer new solutions to the critical healthcare scenarios.

Graphene-laden hydrogels: A strategy for thermally triggered drug delivery.

The synthesis of graphene-based materials has attracted considerable attention in drug delivery strategies. Indeed, the conductivity and mechanical stability of graphene have been investigated for controlled and tunable drug release via electric or mechanical stimuli. However, the design of a thermo-sensitive scaffold using pristine graphene (without distortions related to the oxidation processes) has not been deeply investigated yet, although it may represent a promising approach for several therapeutic treatments. Here, few-layer graphene was used as a nanofiller in a hydrogel system with a thermally tunable drug release profile. In particular, varying the temperature (25 °C, 37 °C and 44 °C), responsive drug releases were noticed and hypothesized depending on the formation and perturbation of π-π interactions involving graphene, the polymeric matrix and the model drug (diclofenac). As a result, these hybrid hydrogels show a potential application as thermally triggered drug release systems in several healthcare scenarios.

Nano-encapsulation of hydroxytyrosol into formulated nanogels improves therapeutic effects against hepatic steatosis: An in vitro study.

Nanomaterials hold promise as a straightforward approach for enhancing the performance of bioactive compounds in several healthcare scenarios. Indeed, nanoencapsulation represents a valuable strategy to preserve the bioactives, maximizing their bioavailability. Here, a nanoencapsulation strategy for the treatment of nonalcoholic fatty liver disease (NAFLD) is presented. NAFLD represents the most common chronic liver disease in Western societies, and still lacks an effective therapy. Hydroxytyrosol (HT), a naturally occurring polyphenol, has been shown to protect against hepatic steatosis through its lipid-lowering, antioxidant and anti-inflammatory activities. However, the efficient delivery of HT to hepatocytes remains a crucial aspect: the design of smart nanogels appears as a promising tool to promote its intracellular uptake. In this paper, we disclose the synthesis of nanogel systems based on polyethylene glycol and polyethyleneimine which have been tested in an in vitro model of hepatic steatosis at two different concentrations (0.1 mg/mL and 0.5 mg/mL), taking advantage of high-content analysis tools. The proposed HT-loaded nanoscaffolds are non-toxic to cells, and their administration showed a significant decrease in the intracellular triglyceride levels, restoring cell viability and outperforming the results achievable with HT in its non-encapsulated form. Moreover, nanogels do not induce oxidative stress, thus demonstrating their biosafety. Overall, the formulated nanogel system achieves superior performance compared to conventional drug administration routes and hence represents a promising strategy for the management of NAFLD.

Functionalized nanogel for treating activated astrocytes in spinal cord injury.

Astrogliosis has a unique reaction during spinal cord damage, with helpful or adverse impacts on recovery. There is consequently a pressing need for treatment to target activated astrocytes and their unsafe response after injury to ensure some preservative effect during the progressive damage. We specifically developed and characterized a functionalized nanogel-based nanovector in vitro and in vivo, demonstrating its selectivity towards astrocytes, and limited uptake by macrophages when functionalized with both NH2 and Cy5 groups. In vitro experiments showed that the internalization was mediated by a clathrin-dependent endocytic pathway. After internalization into the cytoplasm of astrocytes, nanogels undergo lysosomal degradation and release compounds with potential therapeutic efficacy.

Ester coupling of ibuprofen in hydrogel matrix: A facile one-step strategy for controlled anti-inflammatory drug release.

Ibuprofen (IBU) is a non-steroidal anti-inflammatory drug (NSAID) commonly used in the treatment of pain, fever and inflammation. However, the administration of IBU in its free carboxylic acid form is strongly dependent on its limited solubility in aqueous solution. This mandates for an increased drug concentration to reach the therapeutic window, and promotes the alternative use of IBU sodium salt, even if this latter form poses significant constraints in terms of tunable release due to its uncontrolled and rapid diffusion. A potential solution is represented by oral administration through physical encapsulation of ibuprofen in designed carriers, despite this route limits the application of this therapeutic agent. In this work, we propose the covalent tethering of ibuprofen to a hydrogel matrix via esterification reaction. Exploiting the cleavability of the ester bond under physiological conditions, we propose a controlled drug delivery system where the whole drug payload can be released, thus overcoming the questioned aspects of over-dosage and solubility-dependent administration. In particular, we tested the biological activity of cleaved ibuprofen in terms of cyclooxygenase inhibition, reporting that chemical tethering did not alter the efficiency of the NSAID. Moreover, due to the sol-gel transition of the hydrogel matrix, these ibuprofen-functionalized hydrogels could be used as injectable tools in several clinical scenarios, performing a localized drug release and opening advanced avenues for in situ treatments.

Effects of primary amine-based coatings on microglia internalization of nanogels.

Nanogels represent a pivotal class of biomaterials in the therapeutic intracellular treatment of many diseases, especially those involving the central nervous system (CNS). Their biocompatibility and synergy with the biological environment encourage their cellular uptake, releasing the curative cargo in the desired area. As a main drawback, microglia are generally able to phagocytize any foreign element overcoming the blood brain barrier (BBB), including these materials, drastically limiting their bioavailability for the target cells. In this work, we investigated the opportunity to tune and therefore reduce nanogel internalization in microglia cultures, exploiting the orthogonal chemical functionalization with primary amine groups, as a surface coating strategy. Nanogels are designed by following two methods: the direct grafting of aliphatic primary amines and the linkage of -NH2 modified PEG on the nanogel surface. The latter synthesis was proposed to evaluate the combination of PEGylation with the basic nitrogen atom. The achieved results indicate the possibility of effectively modulating the uptake of nanogels, in particular limiting their internalization using the PEG-NH2 coating. This outcome could be considered a promising strategy for the development of carriers for drugs or gene delivery that could overcome microglia scavenging.

Selective Modulation of A1 Astrocytes by Drug-Loaded Nano-Structured Gel in Spinal Cord Injury.

Astrogliosis has a very dynamic response during the progression of spinal cord injury, with beneficial or detrimental effects on recovery. It is therefore important to develop strategies to target activated astrocytes and their harmful molecular mechanisms so as to promote a protective environment to counteract the progression of the secondary injury. The challenge is to formulate an effective therapy with maximum protective effects, but reduced side effects. In this study, a functionalized nanogel-based nanovector was selectively internalized in activated mouse or human astrocytes. Rolipram, an anti-inflammatory drug, when administered by these nanovectors limited the inflammatory response in A1 astrocytes, reducing iNOS and Lcn2, which in turn reverses the toxic effect of proinflammatory astrocytes on motor neurons in vitro, showing advantages over conventionally administered anti-inflammatory therapy. When tested acutely in a spinal cord injury mouse model, it improved motor performance, but only in the early stage after injury, reducing the astrocytosis and preserving neuronal cells.

Design of polymer-based antimicrobial hydrogels through physico-chemical transition.

The antimicrobial activity represents a cornerstone in the development of biomaterials: it is a leading request in many areas, including biology, medicine, environment and industry. Over the years, different polymeric scaffolds are proposed as solutions, based on the encapsulation of metal ions/particles, antibacterial agents or antibiotics. However, the compliance with the biocompatibility criteria and the concentration of the active principles to avoid under- and over-dosing are being debated. In this work, we propose the synthesis of a versatile hydrogel using branched polyacrylic acid (carbomer 974P) and aliphatic polyetherdiamine (elastamine®) through physico-chemical transition, able to show its ability to counteract the bacterial growth and infections thanks to the polymers used, that are not subjected to further chemical modifications. In particular, the antimicrobial activity is clearly demonstrated against Staphyloccoccus aureus and Candida albicans, two well-known opportunistic pathogens. Moreover, we discuss the hydrogel use as drug carrier to design a unique device able to combine the antibacterial/antimicrobial properties to the controlled drug delivery, as a promising tool for a wide range of biomedical applications.

Evaluation of RGD functionalization in hybrid hydrogels as 3D neural stem cell culture systems.

The use of neural stem cells (NSCs) in cell therapy has become a powerful tool used for the treatment of central nervous system diseases, including traumatic brain and spinal cord injuries. However, a significant drawback is related to the limited viability after transplantation in situ. The design of three-dimensional (3D) scaffolds that are capable of resembling the architecture and physico-chemical features of an extracellular environment could be a suitable approach to improve cell survival and preserve their cellular active phase over time. In this study, we investigated NSC adhesion and proliferation in hydrogel systems. In particular, we evaluated the effect of RGD binding domains on cell fate within the polymeric scaffold. The introduction of a tripeptide via hydrogel chemical functionalization improved the percentage of proliferating cells until 8 days after seeding when compared to the unmodified scaffold. The beneficial effects of this 3D culture system was further evident when compared to a NSC monolayer (2D) culture, resulting in an approximately 40% increase in cells in the active phases at 4 and 8 days, and maintained a difference of 25% until 21 days after seeding.

Hydrogel-Nanoparticles Composite System for Controlled Drug Delivery.

Biodegradable poly(ethylene glycol)-block-poly(-lactic acid) (PEG-b-PLA) nanoparticles (NPs) were prepared by nanoprecipitation with controlled dimension and with different electric charges, as monitored by dynamic light scattering (DLS). Then NPs were loaded within hydrogels (HG) developed for biomedical applications in the central nervous system, with different pore sizes (30 and 90 nm). The characteristics of the resulting composite hydrogel-NPs system were firstly studied in terms of ability to control the release of small steric hindrance drug mimetic. Then, diffusion-controlled release of different charged NPs from different entangled hydrogels was studied in vitro and correlated with NPs electric charges and hydrogel mean mesh size. These studies showed different trends, that depend on NPs superficial charge and HG mesh size. Release experiments and diffusion studies, then rationalized by mathematical modeling, allowed us to build different drug delivery devices that can satisfy different medical needs.

Novel functionalization strategies to improve drug delivery from polymers.

INTRODUCTION: The utility of controlled and sustained release of drugs from polymeric systems, both bulk (hydrogels) and colloids (nanoparticles), is a key point that should be addressed. Unfortunately, classic delivery systems are essentially driven by diffusion, which is very quick due to the high concentration gradient present in the body. Area covered: This review provides an overview of functionalization strategies which have been used to reduce release rates by taking advantage of post-polymerization functionalization of polymers. This paradigm is extremely useful in the pharmacological treatment of several diseases, particularly multifactorial diseases, which may require a variety of release kinetics for different drugs from a single device. Expert opinion: Polymer chains can be functionalized with several post-polymerization strategies in order to link, with a cleavable bond, drug molecules to reactive points of the polymeric network. Following this strategy, the main mechanism related to drug release is the breakage of the link that could be opportunely chosen depending on the medical needs: the weaker the bond, the higher the release rate.