A. Guerrini

Mesenchymal stem cells as delivery vehicle of porphyrin loaded nanoparticles: Effective photoinduced in vitro killing of osteosarcoma

Mesenchymal stem cells (MSC) have the unique ability to home and engraft in tumor stroma. These features render them potentially a very useful tool as targeted delivery vehicles which can deliver therapeutic drugs to the tumor stroma. In the present study, we investigate whether fluorescent core-shell PMMA nanoparticles (FNPs) post-loaded with a photosensitizer, namely meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS) and uploaded by MSC could trigger osteosarcoma (OS) cell death in vitro upon specific photoactivation. In co-culture studies we demonstrate using laser confocal microscopy and time lapse imaging, that only after laser irradiation MSC loaded with photosensitizer-coated fluorescent NPs (TPPS@FNPs) undergo cell death and release reactive oxygen species (ROS) which are sufficient to trigger cell death of all OS cells in the culture. These results encourage further studies aimed at proving the efficacy of this novel tri-component system for PDT applications.

Chlorin e6 keratin nanoparticles for photodynamic anticancer therapy

This work describes the preparation of keratin and chlorin e6-conjugated keratin nanoparticles, KNPs and KNPs@Ce6 respectively, by comparing self-assembling and desolvation methodologies. Nanoparticles were characterized in terms of yield, size, morphology, Ce6 loading ratio and ability to produce reactive oxygen species (ROS) upon irradiation with white light. Overall, both methods provided nanoparticles of comparable dimensions, morphology and Ce6 loading ratio. KNPs@Ce6 obtained by a self-assembling procedure were able to produce ROS in a concentration and irradiation-time dependent manner, while displaying compelling evidence of their photostability. In vitro internalization and photo-toxicity studies were performed on osteosarcoma (U2OS) and glioblastoma (U87) cells lines to assess the ability of KNPs@Ce6 nanoparticles to act as delivery systems for photodynamic therapy of cancer. Importantly, at all the Ce6 considered concentrations, e.g. 0.5, 2.5 and 5.0 μg mL−1, no dark toxicity was detected while the amount of Ce6 inside the cells, significantly increased when loaded onto KNPs. The irradiation of tumor cells loaded with KNPs@Ce6 resulted in a greater cell death percentage (approximately 90%) as compared to free Ce6 in both cell types and at all the considered concentrations, thus showing KNPs as effective and promising delivery vehicles for photodynamic therapy applications.

Thiophene-based compounds as fluorescent tags to study mesenchymal stem cell uptake and release of taxanes.

Human mesenchymal stem cells (hMSC) are multipotent cells that display the unique ability to home and engraft in tumor stroma. This remarkable tumor tropic property has generated a great deal of interest in many clinical settings. Recently, we showed that hMSC represent an excellent base for cell-mediated anticancer therapy since they are able to internalize paclitaxel (PTX) and to release it in an amount sufficient to inhibit tumor cell proliferation. In order to shed light on the dynamics of drug uptake and release, in the present paper we describe the synthesis of two novel thiophene-based fluorophore-paclitaxel conjugates, namely PTX-F32 and PTX-F35, as tools for in vitro drug tracking. We aimed to study the ability of these novel derivatives to be efficiently internalized by hMSC and, in a properly engineered coculture assay, to be released in the medium and taken up by tumor cells. In order to ensure better stability of the conjugates toward enzymatic hydrolysis, the selected oligothiophenes were connected to the taxol core at the C7 position through a carbamate linkage between PTX and the diamino linker. Antiproliferative experiments on both tumor cells and stromal cells clearly indicate that, in good correlation with the parent compound, cells are sensitive to nanomolar concentrations of the fluorescent conjugates. Moreover, in the coculture assay we were able to monitor, by fluorescence microscopy, PTX-F32 trafficking from hMSC toward glioblastoma U87 tumor cells. Our work paves the way for novel possibilities to perform extensive and high quality fluorescence-based analysis in order to better understand the cellular mechanisms involved in drug trafficking, such as microvescicle/exosome mediated release, in hMSC vehicle cells.

Intracellular delivery of molecular beacons by PMMA nanoparticles and carbon nanotubes for mRNA sensing

We describe here the use of carbon nanotubes (CNTs) and polymeric nanoparticles made of a core of polymethylmethacrylate (PMMA) surrounded by a shell bearing cationic groups, as intracellular delivery tools of molecular beacons (MBs), particular fluorescent DNA probes, for the detection and localization of a specific mRNA. Survivin mRNA targeting MBs have been used with Atto647N and Blackberry 650 as fluorophore/quencher pair. The MB was anchored to the surface of CNTs and PMMA nanoparticles via a commercial sulfhydryl-reactive heterobifunctional crosslinker and the achieved nanomaterials were then characterized in vitro.

Intracellular Nanosensing and Nanodelivery by PMMA Nanoparticles

The delivery of fluorescent agents or probes to cells and tissues by using nanoparticles is currently receiving a growing interest because such nanodimensional structures can conveniently allow the preparation of small tools to spy at cellular mechanisms without interferences. We describe here the synthesis and characterization of fluorescently labeled cationic core–shell nanoparticles (NPs), made up of a core of polymethylmethacrylate (PMMA), surrounded by a shell bearing cationic groups, modified for nanosensing purposes. In particular, the NPs have been characterized as potential intracellular nanocarriers of molecular beacons (MB) for tumor mRNA sensing. Survivin targeting MBs have been used with Atto647N and Blackberry 650 as fluorophore/quencher pair. The MB was anchored to the surface of the PMMA nanoparticles via a commercial sulfhydryl-reactive heterobifunctional crosslinker and the achieved nanomaterials have been then characterized in vitro.

Oligonucleotide switches and nanomaterials for intracellular mRNA sensing

We describe here the conjugation of polymethylmethacrylate nanoparticles to particular oligonucleotide switches, termed molecular beacons (MBs), as potential intracellular nanosensors. Survivin mRNA targeting MBs have been used with Atto647N and Blackberry 650 as fluorophore/quencher pair. The nanosensors have been characterized in vitro by investigating the analytical performances of the chosen molecular beacon and its functionalities after conjugation to the nanoparticles.

PO-435 Photoactivation of nanoparticles delivered by mesenchymal stem cells induces osteosarcoma cell death in in vitro 3D co-culture models

Introduction Osteosarcoma (OS) is a rare and aggressive tumour that mainly affects long bones of adolescents. Currently, OS patients are treated with a combination of multi-agent chemotherapy and surgery. However, 30% of patients do not respond to standard treatment. Therefore, innovative therapeutic agents are needed. Mesenchymal stem cells (MSCs) display a specific tumour-tropism and have been previously used in successful preclinical studies to deliver several therapeutic agents. Furthermore, the safety of genetically engineered MSCs was demonstrasted in ongoing clinical trial. The goal of the present study was to test in vitro whether MSCs could uptake photoactivable nanoparticles (NPs) and induce cell death of OS cells upon photoactivation. Material and methods Ptl@PMMA NPs were produced by adding tetrasulfonate aluminium phthalocyanine (Ptl) to an aqueous solution of positively charged poly-methylmethacrylate (PMMA) nanoparticles. The photosensitizer Ptl is activated in near-infrared light allowing a deep tissue penetration. Human MSC lines, isolated from the bone marrow of multiple donors, were loaded with Ptl@PMMA NPs. The MSCs’ ability to internalise and retain NPs, along with their migratory properties, were tested. Cell death upon photoactivation (PDT) was evaluated in vitro, on a monolayer co-culture of MSCs and OS cells and in 3D multicellular spheroids, generated via cell suspension in ultralow attachment plates Results and discussions MSCs showed an internalisation rate of Plt@PMMA>95%, which did not alter cell viability and migratory capacity. When Ptl@PMMA-MSCs were co-cultured with a human OS cell line (SaOS-2) in monolayers, they efficiently triggered cell death upon PDT. In particular, AnnexinV/PI and CalceinAM/EthD staining showed 70% of cell death in the co-culture system. These results were also validated by a metabolic assay. Interestingly, in a 3D co-culture of the OS cell line MG63 and Ptl@PMMA-MSCs, we observed a marked reduction of the viability (<5%) measured by the ATP content 24 hours after PDT. A massive cell necrosis induced by the photoactivation of the Ptl in the whole spheroid mass was confirmed by CalceinAM/EthD staining and TEM imaging. Conclusion For the first time, we demonstrated that photoactivation of MSCs loaded with Ptl@PMMA NPs can successfully induce OS cell death in a three-dimensional OS model. These results encourage further in vivo evaluation to demonstrate the specific targeting of Plt@PMMA loaded MSCs to the tumour stroma and the efficacy of PDT treatment

Raman spectroscopic characterisation of photo-active keratin doped with Methylene Blue for wound dressings and tissue engineering

BACKGROUND: The design of wound dressings with extraordinary functionalities that fully address the problem of wound healing is an ambitious challenge in biomedical field. Keratin is a protein most abundant in nature, being the major component of wool, feather, hair, etc., with promising applications in biomedical and regenerative medicine fields. A high level of antibacterial functionality is another desirable property for applications in biomedical field in response to the increasing resistance of bacteria to antibiotics. One of the emerging methods of disinfection and sterilization is the antimicrobial photodynamic therapy (APDT), which uses light combined to a photosensitizer and oxygen to produce phototoxic species. OBJECTIVE: Biomatrices (photo-active keratin) made of wool keratin functionalized with methylene blue, a powerful photosensitizer, have been developed and tested as systems that combine the bioactive properties with the antimicrobial photodynamic functionality. METHODS: The biomatrix resistance to photo-degradation and the formation of reactive oxygen species were evaluated by spectroscopic methods, whereas the antibacterial properties were tested towards gram-positive bacteria. RESULTS: The Raman analysis revealed that specific damages occur at sensitive amino acid sites, selectively, rather than indiscriminately. However, keratin resulted to be a suitable biomaterial for APDT, since it has enough resistance to photodegradation and the radical-induced oxidation is not able to induce strong structural changes in the protein. CONCLUSIONS: The results clearly indicate the potential use of these novel photo-active keratin biomatrices in wound dressing and tissue engineering.