Simonetta Papa

Selective Nanovector Mediated Treatment of Activated Proinflammatory Microglia/Macrophages in Spinal Cord Injury

Much evidence shows that acute and chronic inflammation in spinal cord injury (SCI), characterized by immune cell infiltration and release of inflammatory mediators, is implicated in development of the secondary injury phase that occurs after spinal cord trauma and in the worsening of damage. Activation of microglia/macrophages and the associated inflammatory response appears to be a self-propelling mechanism that leads to progressive neurodegeneration and development of persisting pain state. Recent advances in polymer science have provided a huge amount of innovations leading to increased interest for polymeric nanoparticles (NPs) as drug delivery tools to treat SCI. In this study, we tested and evaluated in vitro and in vivo a new drug delivery nanocarrier: minocycline loaded in NPs composed by a polymer based on poly-ε-caprolactone and polyethylene glycol. These NPs are able to selectively target and modulate, specifically, the activated proinflammatory microglia/macrophages in subacute progression of the secondary injury in SCI mouse model. After minocycline-NPs treatment, we demonstrate a reduced activation and proliferation of microglia/macrophages around the lesion site and a reduction of cells with round shape phagocytic-like phenotype in favor of a more arborized resting-like phenotype with low CD68 staining. Treatment here proposed limits, up to 15 days tested, the proinflammatory stimulus associated with microglia/macrophage activation. This was demonstrated by reduced expression of proinflammatory cytokine IL-6 and persistent reduced expression of CD68 in traumatized site. The nanocarrier drug delivery tool developed here shows potential advantages over the conventionally administered anti-inflammatory therapy, maximizing therapeutic efficiency and reducing side effects.

Multiple drug delivery hydrogel system for spinal cord injury repair strategies

The multifactorial pathological progress of spinal cord injury (SCI) is probably the main reason behind the absence of efficient therapeutic approaches. Hence, very recent highlights suggest the use of new multidrug delivery systems capable of local controlled release of therapeutic agents. In this work, a biocompatible hydrogel-based system was developed as multiple drug delivery tool, specifically designed for SCI repair strategies. Multiple release profiles were achieved by loading gel with a combination of low and high steric hindrance molecules. In vitro, in vivo and ex vivo release studies showed an independent combination of fast diffusion-controlled kinetics for smaller molecules together with slow diffusion-controlled kinetics for bigger ones. A preserved functionality of loaded substances was always achieved, confirming the absence of any chemical stable interactions between gel matrix and loaded molecules. Moreover, the relevant effect of the cerebrospinal fluid flux dynamics on the drug diffusion in the spinal cord tissue was here revealed for the first time: an oriented delivery of the released molecules in the spinal cord tract caudally to the gel site is demonstrated, thus suggesting a more efficient gel positioning rostrally to the lesion.

Polymeric nanoparticle system to target activated microglia/macrophages in spinal cord injury

The possibility to control the fate of the cells responsible for secondary mechanisms following spinal cord injury (SCI) is one of the most relevant challenges to reduce the post traumatic degeneration of the spinal cord. In particular, microglia/macrophages associated inflammation appears to be a self-propelling mechanism which leads to progressive neurodegeneration and development of persisting pain state. In this study we analyzed the interactions between poly(methyl methacrylate) nanoparticles (PMMA-NPs) and microglia/macrophages in vitro and in vivo, characterizing the features that influence their internalization and ability to deliver drugs. The uptake mechanisms of PMMA-NPs were in-depth investigated, together with their possible toxic effects on microglia/macrophages. In addition, the possibility to deliver a mimetic drug within microglia/macrophages was characterized in vitro and in vivo. Drug-loaded polymeric NPs resulted to be a promising tool for the selective administration of pharmacological compounds in activated microglia/macrophages and thus potentially able to counteract relevant secondary inflammatory events in SCI.

A new three dimensional biomimetic hydrogel to deliver factors secreted by human mesenchymal stem cells in spinal cord injury.

Stem cell therapy with human mesenchymal stem cells (hMSCs) represents a promising strategy in spinal cord injury (SCI). However, both systemic and parenchymal hMSCs administrations show significant drawbacks as a limited number and viability of stem cells in situ. Biomaterials able to encapsulate and sustain hMSCs represent a viable approach to overcome these limitations potentially improving the stem cell therapy. In this study, we evaluate a new agarose/carbomer based hydrogel which combines different strategies to optimize hMSCs viability, density and delivery of paracrine factors. Specifically, we evaluate a new loading procedure on a lyophilized scaffold (soaked up effect) that reduces mechanical stress in encapsulating hMSCs into the hydrogel. In addition, we combine arginine-glycine-aspartic acid (RGD) tripeptide and 3D extracellular matrix deposition to increase the capacity to attach and maintain healthy hMSCs within the hydrogel over time. Furthermore, the fluidic diffusion from the hydrogel toward the injury site is improved by using a cling film that oriented efficaciously the delivery of paracrine factors in vivo. Finally, we demonstrate that an improved combination as here proposed of hMSCs and biomimetic hydrogel is able to immunomodulate significantly the pro-inflammatory environment in a SCI mouse model, increasing M2 macrophagic population and promoting a pro-regenerative environment in situ.

Current options for drug delivery to the spinal cord

Introduction: Spinal cord disorders (SCDs) are among the most devastating neurological diseases, due to their acute and long-term health consequences, the reduced quality of life and the high economic impact on society. Here, drug administration is severely limited by the blood–spinal cord barrier (BSCB) that impedes to reach the cord from the bloodstream. So, developing a suitable delivery route is mandatory to increase medical chances. Areas covered: This review provides an overview of drug delivery systems used to overcome the inaccessibility of the cord. On one side, intrathecal administration, either with catheters or with biomaterials, represents the main route to administer drugs to the spinal cord; on the other side, more recent strategies involve chemical or electromagnetic disruption of the barrier and synthesis of novel functionalized compounds as nanoparticles and liposomes able to cross BSCB. Expert opinion: Both the multifactorial pathological progression and the restricted access of therapeutic drugs to the spine are probably the main reasons behind the absence of efficient therapeutic approaches for SCDs. Hence, very recent highlights suggest the use of original strategies to overcome the BSCB, and new multidrug delivery systems capable of local controlled release of therapeutic agents have been developed. These issues can be addressed by using nanoparticles technology and smart hydrogel drug delivery systems, providing an increased therapeutic compound delivery in the spinal cord environment and multiple administrations able to synergize treatment efficacy.

Non-invasive in vitro and in vivo monitoring of degradation of fluorescently labeled hyaluronan hydrogels for tissue engineering applications

UNLABELLED Tracking of degradation of hydrogels-based biomaterials in vivo is very important for rational design of tissue engineering scaffolds that act as delivery carriers for bioactive factors. During the process of tissue development, an ideal scaffold should remodel at a rate matching with scaffold degradation. To reduce amount of animals sacrificed, non-invasive in vivo imaging of biomaterials is required which relies on using of biocompatible and in situ gel forming compounds carrying suitable imaging agents. In this study we developed a method of in situ fabrication of fluorescently labeled and injectable hyaluronan (HA) hydrogel based on one pot sequential use of Michael addition and thiol-disulfide exchange reactions for the macromolecules labeling and cross-linking respectively. Hydrogels with different content of HA were prepared and their enzymatic degradation was followed in vitro and in vivo using fluorescence multispectral imaging. First, we confirmed that the absorbance of the matrix-linked near-IR fluorescent IRDye® 800CW agent released due to the matrix enzymatic degradation in vitro matched the amount of the degraded hydrogel measured by classical gravimetric method. Secondly, the rate of degradation was inversely proportional to the hydrogel concentration and this structure-degradation relationship was similar for both in vitro and in vivo studies. It implies that the degradation of this disulfide cross-linked hyaluronan hydrogel in vivo can be predicted basing on the results of its in vitro degradation studies. The compliance of in vitro and in vivo methods is also promising for the future development of predictive in vitro tissue engineering models. STATEMENT OF SIGNIFICANCE The need for engineered hydrogel scaffolds that deliver bioactive factors to endogenous progenitor cells in vivo via gradual matrix resorption and thus facilitate tissue regeneration is increasing with the aging population. Importantly, scaffold should degrade at a modest rate that will not be too fast to support tissue growth nor too slow to provide space for tissue development. The present work is devoted to longitudinal tracking of a hydrogel material in vivo from the time of its implantation to the time of complete resorption without sacrificing animals. The method demonstrates correlation of resorption rates in vivo and in vitro for hydrogels with varied structural parameters. It opens the possibility to develop predictive in vitro models for tissue engineered scaffolds and reduce animal studies.

Early modulation of pro-inflammatory microglia by minocycline loaded nanoparticles confers long lasting protection after spinal cord injury.

Many efforts have been performed in order to understand the role of recruited macrophages in the progression of spinal cord injury (SCI). Different studies revealed a pleiotropic effect played by these cells associated to distinct phenotypes (M1 and M2), showing a predictable spatial and temporal distribution in the injured site after SCI. Differently, the role of activated microglia in injury progression has been poorly investigated, mainly because of the challenges to target and selectively modulate them in situ. A delivery nanovector tool (poly-ε-caprolactone-based nanoparticles) able to selectively treat/target microglia has been developed and used here to clarify the temporal and spatial involvement of the pro-inflammatory response associated to microglial cells in SCI. We show that a treatment with nanoparticles loaded with minocycline, the latter a well-known anti-inflammatory drug, when administered acutely in a SCI mouse model is able to efficiently modulate the resident microglial cells reducing the pro-inflammatory response, maintaining a pro-regenerative milieu and ameliorating the behavioral outcome up to 63 days post injury. Furthermore, by using this selective delivery tool we demonstrate a mechanistic link between early microglia activation and M1 macrophages recruitment to the injured site via CCL2 chemokine, revealing a detrimental contribution of pro-inflammatory macrophages to injury progression after SCI.

Differences in protein quality control correlate with phenotype variability in 2 mouse models of familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a disease of variable severity in terms of speed of progression of the disease course. We found a similar variability in disease onset and progression of 2 familial ALS mouse strains, despite the fact that they carry the same transgene copy number and express the same amount of mutant SOD1G93A messenger RNA and protein in the central nervous system. Comparative analysis of 2 SOD1G93A mouse strains highlights differences associated with the disease severity that are unrelated to the degree of motor neuron loss but that appear to promote early dysfunction of these cells linked to protein aggregation. Features of fast progressing phenotype are (1) abundant protein aggregates containing mutant SOD1 and multiple chaperones; (2) low basal expression of the chaperone alpha-B-crystallin (CRYAB) and β5 subunits of proteasome; and (3) downregulation of proteasome subunit expression at disease onset. In contrast, high levels of functional chaperones such as cyclophillin-A and CRYAB, combined with delayed alteration of expression of proteasome subunits and the sequestration of TDP43 into aggregates, are features associated with a more slowly progressing pathology. These data support the hypothesis that impairment of protein homeostasis caused by low-soluble chaperone levels, together with malfunction of the proteasome degradation machinery, contributes to accelerate motor neuron dysfunction and progression of disease symptoms. Therefore, modulating the activity of these systems could represent a rational therapeutic strategy for slowing down disease progression in SOD1-related ALS.

Tunable Hydrogel - Nanoparticles Release System for Sustained Combination Therapies in the Spinal Cord

Poly(methyl methacrylate) (PMMA) nanoparticles (NPs) were prepared by emulsion free radical polymerization. NPs with controlled dimension, as monitored by dynamic light scattering (DLS) and transmission electron microscopy (TEM), were produced by changing experimental parameters, such as the amount of emulsifier and the monomer feeding mode (batch or semi-batch). Then, different sized NPs (60, 80 and 130nm) were loaded in polysaccharide-polyacrylic acid based hydrogels, cross-linked by covalent ester bonds between polyacrylic acid (PAA) and agarose chains, with different pore sizes (30, 60, 90nm). The characteristics of the resulting composite hydrogel-NPs system were firstly studied in terms of rheological properties and ability to release Rhodamine B that presents steric hindrance similar to many neuroprotective agents used in spinal cord injury (SCI) repair. Then, diffusion-controlled release of different sized NPs from different entangled hydrogels was investigated in vitro and correlated with NPs diameter and hydrogel mean mesh size, showing different hindrances to the diffusion pathways. Release experiments and diffusion studies, rationalized by mathematical modeling and verified in vivo, allowed to build a material library able to satisfy different medical drug delivery needs.

Sustained Delivery of Chondroitinase ABC from Hydrogel System.

In the injured spinal cord, chondroitin sulfate proteoglycans (CSPGs) are the principal responsible of axon growth inhibition and they contribute to regenerative failure, promoting glial scar formation. Chondroitinase ABC (chABC) is known for being able to digest proteoglycans, thus degrading glial scar and favoring axonal regrowth. However, its classic administration is invasive, infection-prone and clinically problematic. An agarose-carbomer (AC1) hydrogel, already used in SCI repair strategies, was here investigated as a delivery system capable of an effective chABC administration: the material ability to include chABC within its pores and the possibility to be injected into the target tissue were firstly proved. Subsequently, release kinetic and the maintenance of enzymatic activity were positively assessed: AC1 hydrogel was thus confirmed to be a feasible tool for chABC delivery and a promising device for spinal cord injury topic repair strategies.