Laura Pandolfi

PLGA-Mesoporous Silicon Microspheres for the in Vivo Controlled Temporospatial Delivery of Proteins.

In regenerative medicine, the temporospatially controlled delivery of growth factors (GFs) is crucial to trigger the desired healing mechanisms in the target tissues. The uncontrolled release of GFs has been demonstrated to cause severe side effects in the surrounding tissues. The aim of this study was to optimize a translational approach for the fine temporal and spatial control over the release of proteins, in vivo. Hence, we proposed a newly developed multiscale composite microsphere based on a core consisting of the nanostructured silicon multistage vector (MSV) and a poly(dl-lactide-co-glycolide) acid (PLGA) outer shell. Both of the two components of the resulting composite microspheres (PLGA-MSV) can be independently tailored to achieve multiple release kinetics contributing to the control of the release profile of a reporter protein in vitro. The influence of MSV shape (hemispherical or discoidal) and size (1, 3, or 7 μm) on PLGA-MSVs morphology and size distribution was investigated. Second, the copolymer ratio of the PLGA used to fabricate the outer shell of PLGA-MSV was varied. The composites were fully characterized by optical microscopy, scanning electron microscopy, ζ potential, Fourier transform infrared spectroscopy, and thermogravimetric analysis-differential scanning calorimetry, and their release kinetics over 30 days. PLGA-MSVs biocompatibility was assessed in vitro with J774 macrophages. Finally, the formulation of PLGA-MSV was selected, which concurrently provided the most consistent microsphere size and allowed for a zero-order release kinetic. The selected PLGA-MSVs were injected in a subcutaneous model in mice, and the in vivo release of the reporter protein was followed over 2 weeks by intravital microscopy, to assess if the zero-order release was preserved. PLGA-MSV was able to retain the payload over 2 weeks, avoiding the initial burst release typical of most drug delivery systems. Finally, histological evaluation assessed the biocompatibility of the platform in vivo.

Enabling cytoplasmic delivery and organelle targeting by surface modification of nanocarriers

Nanocarriers are designed to specifically accumulate in diseased tissues. In this context, targeting of intracellular compartments was shown to enhance the efficacy of many drugs and to offer new and more effective therapeutic approaches. This is especially true for therapies based on biologicals that must be encapsulated to favor cell internalization, and to avoid intracellular endosomal sequestration and degradation of the payload. In this review, we discuss specific surface modifications designed to achieve cell cytoplasm delivery and to improve targeting of major organelles; we also discuss the therapeutic applications of these approaches. Last, we describe some integrated strategies designed to sequentially overcome the biological barriers that separate the site of administration from the cell cytoplasm, which is the drugs site of action.

Composite microsphere-functionalized scaffold for the controlled release of small molecules in tissue engineering

Current tissue engineering strategies focus on restoring damaged tissue architectures using biologically active scaffolds. The ideal scaffold would mimic the extracellular matrix of any tissue of interest, promoting cell proliferation and de novo extracellular matrix deposition. A plethora of techniques have been evaluated to engineer scaffolds for the controlled and targeted release of bioactive molecules to provide a functional structure for tissue growth and remodeling, as well as enhance recruitment and proliferation of autologous cells within the implant. Recently, novel approaches using small molecules, instead of growth factors, have been exploited to regulate tissue regeneration. The use of small synthetic molecules could be very advantageous because of their stability, tunability, and low cost. Herein, we propose a chitosan–gelatin scaffold functionalized with composite microspheres consisting of mesoporous silicon microparticles and poly(dl-lactic-co-glycolic acid) for the controlled release of sphingosine-1-phospate, a small molecule of interest. We characterized the platform with scanning electron microscopy, Fourier transform infrared spectroscopy, and confocal microscopy. Finally, the biocompatibility of this multiscale system was analyzed by culturing human mesenchymal stem cells onto the scaffold. The presented strategy establishes the basis of a versatile scaffold for the controlled release of small molecules and for culturing mesenchymal stem cells for regenerative medicine applications.

Enhancing Vascularization through the Controlled Release of Platelet-Derived Growth Factor-BB

Using delivery systems to control the in vivo release of growth factors (GFs) for tissue engineering applications is extremely desirable as the clinical use of GFs is limited by their fast in vivo turnover. Hence, the development of effective platforms that are able to finely control the release of GFs in vivo remains a challenge. Herein, we investigated the ability of multiscale microspheres, composed by a nanostructured silicon multistage vector (MSV) core and a poly(dl-lactide-co-glycolide) acid (PLGA) forming outer shell (PLGA-MSV), to release functional platelet-derived growth factor-BB (PDGF-BB) to induce in vivo localized neovascularization. The in vitro release of PDGF-BB was assessed by enzyme-linked immunosorbent assay (ELISA) over 2 weeks and showed a sustained, zero-order release kinetics. The ability to promote in vivo localized neovascularization was investigated in a subcutaneous injection model in BALB/c mice and followed by intravital microscopy up to 2 weeks. Fully functional newly formed vessels were found within the area where PLGA-MSVs were localized and covered 3.0 ± 0.9 and 19 ± 5.1% at 7 and 14 days, respectively, showing a 6-fold increase in 1 week. The distribution of CD31+ and α-SMA+ cells was detected by immunofluorescence on harvested tissues. CD31 was significantly more expressed (4-fold increase) compared to the untreated control. Finally, the level of up-regulation of angiogenesis-associated genes (Vegfa, Vwf, and Col3a1) was assessed by q-PCR, resulting in a significantly higher expression where PLGA-MSVs were localized (Vegfa: 2.32 ± 0.50 at 7 days and 4.37 ± 0.75 at 14 days; Vwf: 4.13 ± 0.82 and 7.74 ± 0.91; Col3a1: 5.43 ± 0.37 and 6.66 ± 0.89). Altogether, our data supported the conclusion that the localized delivery of PDGF-BB from PLGA-MSVs induced the localized de novo formation of fully functional vessels in vivo. With this study, we demonstrated that PLGA-MSV holds promise for accomplishing the controlled localized in vivo release of GFs for the design of innovative tissue engineering strategies.

Nanocomposite Hydrogels as Platform for Cells Growth, Proliferation, and Chemotaxis

The challenge of mimicking the extracellular matrix with artificial scaffolds that are able to reduce immunoresponse is still unmet. Recent findings have shown that mesenchymal stem cells (MSC) infiltrating into the implanted scaffold have effects on the implant integration by improving the healing process. Toward this aim, a novel polyamidoamine-based nanocomposite hydrogel is synthesized, cross-linked with porous nanomaterials (i.e., mesoporous silica nanoparticles), able to release chemokine proteins. A comprehensive viscoelasticity study confirms that the hydrogel provides optimal structural support for MSC infiltration and proliferation. The efficiency of this hydrogel, containing the chemoattractant stromal cell-derived factor 1α (SDF-1α), in promoting MSC migration in vitro is demonstrated. Finally, subcutaneous implantation of SDF-1α-releasing hydrogels in mice results in a modulation of the inflammatory reaction. Overall, the proposed SDF-1α-nanocomposite hydrogel proves to have potential for applications in tissue engineering.

Patterning Biomaterials for the Spatiotemporal Delivery of Bioactive Molecules

The aim of tissue engineering is to promote the repair of functional tissues. For decades, the combined use of biomaterials, growth factors (GFs), and stem cells has been the base of several regeneration strategies. Among these, biomimicry emerged as a robust strategy to efficiently address this clinical challenge. Biomimetic materials, able to recapitulate the composition and architecture of the extracellular matrix, are the materials of choice, for their biocompatibility and higher rate of efficacy. In addition, it has become increasingly clear that restoring the complex biochemical environment of the target tissue is crucial for its regeneration. Toward this aim, the combination of scaffolds and GFs is required. The advent of nanotechnology significantly impacted the field of tissue engineering by providing new ways to reproduce the complex spatial and temporal biochemical patterns of tissues. This review will present the most recent approaches to finely control the spatiotemporal release of bioactive molecules for various tissue engineering applications.

A nanofibrous electrospun patch to maintain human mesenchymal cell stemness

AbstractMesenchymal stem cells (MSCs) have been extensively investigated in regenerative medicine because of their crucial role in tissue healing. For these properties, they are widely tested in clinical trials, usually injected in cell suspension or in combination with tridimensional scaffolds. However, scaffolds can largely affect the fates of MSCs, inducing a progressive loss of functionality overtime. The ideal scaffold must delay MSCs differentiation until paracrine signals from the host induce their change. Herein, we proposed a nanostructured electrospun gelatin patch as an appropriate environment where human MSCs (hMSCs) can adhere, proliferate, and maintain their stemness. This patch exhibited characteristics of a non-linear elastic material and withstood degradation up to 4 weeks. As compared to culture and expansion in 2D, hMSCs on the patch showed a similar degree of proliferation and better maintained their progenitor properties, as assessed by their superior differentiation capacity towards typical mesenchymal lineages (i.e. osteogenic and chondrogenic). Furthermore, immunohistochemical analysis and longitudinal non-invasive imaging of inflammatory response revealed no sign of foreign body reaction for 3 weeks. In summary, our results demonstrated that our biocompatible patch favored the maintenance of undifferentiated hMSCs for up to 21 days and is an ideal candidate for tridimensional delivery of hMSCs.Graphical Abstract The present work reports a nanostructured patch gelatin-based able to maintain in vitro hMSCs stemness features. Moreover, hMSCs were able to differentiate toward osteo- and chondrogenic lineages once induces by differentiative media, confirming the ability of this patch to support stem cells for a potential in vivo application. These attractive properties together with the low inflammatory response in vivo make this patch a promising platform in regenerative medicine.

Immune tuning scaffold for the local induction of a pro-regenerative environment

In mammals, tissue regeneration is accomplished through a well-regulated, complex cascade of events. The disruption of the cellular and molecular processes involved in tissue healing might lead to scar formation. Most tissue engineering approaches have tried to improve the regenerative outcome following an injury, through the combination of biocompatible materials, stem cells and bioactive factors. However, implanted materials can cause further healing impairments due to the persistent inflammatory stimuli that trigger the onset of chronic inflammation. Here, it is described at the molecular, cellular and tissue level, the body response to a functionalized biomimetic collagen scaffold. The grafting of chondroitin sulfate on the surface of the scaffold is able to induce a pro-regenerative environment at the site of a subcutaneous implant. The early in situ recruitment, and sustained local retention of anti-inflammatory macrophages significantly reduced the pro-inflammatory environment and triggered a different healing cascade, ultimately leading to collagen fibril re-organization, blood vessel formation, and scaffold integration with the surrounding native tissue.

Heparan Sulfate: A Potential Candidate for the Development of Biomimetic Immunomodulatory Membranes

Clinical trials have demonstrated that heparan sulfate (HS) could be used as a therapeutic agent for the treatment of inflammatory diseases. Its anti-inflammatory effect makes it suitable for the development of biomimetic innovative strategies aiming at modulating stem cells behavior toward a pro-regenerative phenotype in case of injury or inflammation. Here, we propose collagen type I meshes fabricated by solvent casting and further crosslinked with HS (HS-Col) to create a biomimetic environment resembling the extracellular matrix of soft tissue. HS-Col meshes were tested for their capability to provide physical support to stem cells’ growth, maintain their phenotypes and immunosuppressive potential following inflammation. HS-Col effect on stem cells was investigated in standard conditions as well as in an inflammatory environment recapitulated in vitro through a mix of pro-inflammatory cytokines (tumor necrosis factor-α and interferon-gamma; 20 ng/ml). A significant increase in the production of molecules associated with immunosuppression was demonstrated in response to the material and when cells were grown in presence of pro-inflammatory stimuli, compared to bare collagen membranes (Col), leading to a greater inhibitory potential when mesenchymal stem cells were exposed to stimulated peripheral blood mononuclear cells. Our data suggest that the presence of HS is able to activate the molecular machinery responsible for the release of anti-inflammatory cytokines, potentially leading to a faster resolution of inflammation.

Controlled Release of Small Molecules for Cardiac Differentiation of Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have been shown to differentiate to functional cardiomyocytes (CM) with high efficiency through temporally controlled inhibition of the GSK3/Wnt signaling pathways. In this study, we investigated the ability of temporally controlled release of GSK3/Wnt small-molecule inhibitors to drive cardiac differentiation of iPSC without manual intervention. Porous silica particles were loaded with GSK3 inhibitor CHIR99021 or Wnt inhibitor IWP2, and the particles containing IWP2 were coated with 5 wt% poly(lactic-co-glycolic acid) 50:50 to delay release by ∼72 h. iPSCs reprogrammed through mRNA transfection were cultured with these particles up to 30 days. High-performance liquid chromatography suggests a burst release of CHIR99021 within the first 24 h and a delayed release of IWP2 after 72 h. Annexin V/propidium iodide staining did not show a significant effect on apoptosis or necrosis rates. Cultured cells upregulated both early (Nkx 2.5, Isl-1) and late (cTnT, MHC, Cx43) cardiac markers, assayed with a quantitative real-time polymerase chain reaction, and began spontaneous contraction at 3.0 ± 0.6 Hz at 15-21 days after the start of differentiation. CM had clear sarcomeric striations when stained for β-myosin heavy chain, and showed expression and punctate membrane localization of gap junction protein Connexin43. Calcium and voltage-sensitive imaging showed both action potential and calcium transients typical of immature CM. This study showed that the cardiac differentiation of pluripotent stem cells can be directed by porous silica vectors with temporally controlled release of small-molecule inhibitors. These results suggest methods for automating and eliminating variability in manual maintenance of inhibitor concentrations in the differentiation of pluripotent stem cells to CM.