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Nanovehicles for medical use: an in vitro evaluation of cytotoxicity and drug delivery efficiency
Full text linkThe recent progresses offered by nanotechnology in the manipulation of matter lead to the development of several nanoparticles (NPs) and nanodevices for medical applications. In oncology, nanosized objects are particularly attractive as drug delivery systems since it is expected that engineered nanovehicles of appropriate size and functionalised with specific ligands/antibodies will improve the efficacy and selectivity of cancer therapies by exploiting both the passive and active mechanism of tumour targeting. The use of delivery systems is particularly appealing in those therapies in which the administration of the drug in aqueous formulations leads to drug aggregation with decreased activity or scarce bioavailability and tumour selectivity. This is the case of most of the photosensitizers used in photodynamic therapy (PDT), which display hydrophobicity and poor selective accumulation in malignant tissues. In the last decades, PDT is emerging as a promising cancer treatment modality in alternative to conventional therapies, which often demonstrate systemic drug toxicity and multidrug-resistance phenomena. PDT is based on the administration of a photosensitizer (PS) that accumulates in the tumour and after activation with light of appropriate wavelengths, reacts with surrounding molecular oxygen leading to the formation of cytotoxic reactive oxygen species (ROS) with consequent cellular and vasculature damages.
In this PhD thesis, three different nanosystems, namely, liposomes, poly-(D,L-lactide-co-glycolide) nanoparticles (PLGA NPs) and ORganically Modified SILica nanoparticles (ORMOSIL NPs) were considered for the delivery of the second generation PS meta-tetra(hydroxyphenyl)chlorin (m-THPC, Temoporfin) to cancer cells in vitro. In particular, drug delivery efficiency, dark and phototoxicity of the m-THPC nanoparticle-based formulations were evaluated. To improve m-THPC bioavailability and tumour selectivity, in the design of the nanovehicles PEGylation and targeting of NPs were considered as essential strategies in order to prolong NP circulation in the bloodstream and exploit active mechanisms of tumour targeting.
For the delivery of m-THPC using unilamellar liposomes, four different PEGylated liposomal formulations (trade name Fospeg®, provided by Biolitec Research) in which the length (PEG750, PEG2000, PEG5000) and the density (2%, 8%) of PEG were varied, were tested in vitro in normal lung fibroblasts CCD-34Lu and in cancer A549 lung epithelial cells. Compared to drug delivered in the standard solvent (Foscan®, ethanol/PEG 400/water (20:30:50, by vol.)), liposomal m-THPC showed a decreased intracellular uptake in both cell lines, but the presence of the delivery system highly reduced the dark cytotoxicity of the drug. The reduction of the PS dark toxicity increased with the increasing of PEG density on liposome surface, while the length of PEG chains did not affect significantly the toxic effect of m-THPC in the dark. However, photo-toxicity measured in A549 cells was only slightly affected by the reduced uptake of m-THPC delivered by Fospeg®, and the efficiency of PDT-induced cell killing was comparable among the different liposomal formulations. Interestingly, the intracellular localization of m-THPC delivered as Fospeg® or Foscan® was the same (Golgi apparatus and endoplasmic reticulum) suggesting drug release from liposomes, especially in the presence of the serum proteins, being m-THPC only physically entrapped within liposomes. m-THPC release was confirmed by the fact that liposomes covalently labelled with rhodamine were effectively were taken up by cells but, differently from m-THPC, localized in the acidic compartments of the cells. In spite of m-THPC release from liposomes, the Fospeg® formulation was exploited to target actively cancer cells by liposome conjugation with folic acid (FA), being FA-receptors (FRs) over-expressed in several human tumours. Thus, specific uptake and photo-toxicity of FA-targeted liposomes (FA-Fospeg) with respect to liposomes of the same composition but lacking FA (un-targeted Fospeg) was evaluated in KB (FR-positive) and in A549 (FR-negative) cells. The uptake of m-THPC delivered as FA-Fospeg was twice that of un-targeted Fospeg in KB cells; however only a modest fraction (~ 15%) of the targeted vehicle was effectively internalized by FR-mediated endocytosis while nonspecific internalization remained the prevailing mechanism of liposomes uptake in both cell lines. The improved m-THPC uptake obtained with FA-Fospeg in FR over-expressing cells translated into a 1.5 higher photo-induced toxicity.
A novel formulation of bare and PEGylated PLGA NPs in which m-THPC was physically entrapped were synthesized (Prof J. Kos, University of Ljubljana) and evaluated in vitro and in vivo for phototherapy and fluorescence-based tumour imaging applications. In vitro studies carried out on A549, MCF10A neo T (breast cancer cells) and U937 (lymphoma derived pro-monocytic cells) cell lines, showed reduced uptake of PEGylated NPs with respect to non PEGylated NPs. As for Fospeg®, the use of the delivery system led to a significant reduction of m-THPC dark toxicity.. As expected for PEGylated NPs, the efficiency of cell internalization of m-THPC entrapped in PEG PLGA was reduced by 50% with respect to that in the standard solvent, but surprisingly cytotoxicity induced in irradiated A549 cells was quite comparable. At 24 h post-injection in vivo biodistribution of bare and PEGylated PLGA NPs compared to Foscan® was assessed in mice, showing very similar drug accumulation in the major organs but reduced skin uptake for both NP formulations. Thus, even if m-THPC release in the presence of serum proteins was measured in vitro, PEGylated PLGA NPs appeared potentially useful as stealth and biodegradable PS delivery systems.
The premature release of the PS from the delivery system was completely avoided with the covalent link of m-THPC to the silane matrix of highly PEGylated ORMOSIL NPs (Prof. F. Mancin, University of Padova). This type of NPs exhibited a very low extent of cell internalization in vitro due to their high degree of PEGylation, making NP targeting an essential prerequisite to enhance intracellular drug delivery. In addition to FA, the RGD peptide and the antibody Cetuximab, which bind respectively the integrin α5ß3 receptor and epidermal growth factor receptor (EGFR), were exploited as targeting agents for ORMOSIL NPs and the specific uptake and photo-toxicity of m-THPC delivered by conjugated NPs were evaluated in vitro. The study revealed how the characteristics of the targeting agents are of crucial importance in determining the performances of targeted PEGylated nanosystems. In fact, the hydrophobic FA was very likely buried in the PEG layer and was unable to drive the selective uptake of ORMOSIL NPs while RGD peptide and Cetuximab antibody displayed some selectivity toward cells over-expressing their receptors (HUVEC cells over-expressing integrin α5ß3 receptors and A431 cells over-expressing EGFR). Unfortunately, the enhanced and selective uptake of m-THPC obtained by the two latter targeted ORMOSIL NPs was not accompanied by efficient and selective photo-induced cytotoxicity; it appeared that the selectivity of NP uptake was achieved in scarce drug cell loading conditions, determining only low PDT efficacy.
The assessment of the biocompatibility of NPs is of fundamental importance for their safe use in nanomedicine. Since ORMOSIL NPs are not well characterised from this point of view, a toxicological characterization of empty ORMOSIL NPs were carried out in vitro in normal (CCD-34Lu) and cancer (A549, NCIH-2347) lung cells. The study included traditional cell viability and cytotoxicity tests (MTS test, LDH release assay, ROS production, cell membrane permeabilization measurements and electron microscopy analyses) in combination with a genome-wide analysis of gene expression profiles of cells exposed to NPs. The results pointed out that different types of cells respond quite differently to NPs and PEGylation of NPs highly affected the cytotoxicity profiles. PEGylation of ORMOSIL NPs completely abolished the toxicity of the nanosystem in CCD-34Lu and NCIH-2347 cells. On the contrary PEG ORMOSIL NPs induced necrotic cell death of A549 by increasing the permeability of the plasma membrane. At sub-lethal concentrations alteration of gene expression and inflammation were measured in A549 cells exposed to. The different response to PEG NPs is very likely explained considering the peculiarity of the cell type and the particular interaction of NPs with cell and internalization mechanisms. In fact, it was shown clearly that NPs internalized in A549 cells localized in and affected the morphology and the functioning of pulmonary surfactant containing lamellar bodies, peculiar of alveolar type II cells of which A459 cells represents an in vitro models
Folate-targeted PEGylated liposomes improve the selectivity of PDT with meta-tetra(hydroxyphenyl)chlorin (m-THPC)
No full textThe folate receptor (FR) is over-expressed in many human tumours and is being intensively studied also
in the field of nanomedicine as a target to enhance the selectivity of drug delivery to cancer cells by
using nanocarriers bearing folic acid (FA) on their surface. In this study we report the encapsulation of
the photosensitizer (PS) meta-tetra(hydroxyphenyl)chlorin (m-THPC) in FA-targeted PEGylated liposomes
used as a novel drug delivery system for photodynamic therapy (PDT) of cancer. Our in vitro investigations
revealed that only a modest fraction of targeted liposomes were internalized by specific endocytosis in
FR-positive KB cells. However, FA-liposomes doubled the uptake of the entrapped m-THPC with respect
to un-targeted liposomes and enhanced the photo-induced cytotoxicity in KB cells. In contrast, in FRnegative
A549 cells FA-targeted or un-targeted liposomes exhibited a very similar extent of internalization
and as a consequence the same photo-killing efficiency
Conjugation of photosensitisers to antimicrobial peptides increases the efficiency of photodynamic therapy in cancer cells
No full textStrategies for optimizing the delivery to tumors of macrocyclic photosensitizers used in photodynamic therapy (PDT)
No full textThis review briefly summaries the principles and mechanisms of action of photodynamic therapy (PDT) as concerns its application in the oncological field, highlighting its drawbacks and some of the strategies that have been or are being explored to overcome them. The major aim is to increase the efficiency and selectivity of the photosensitizer (PS) uptake in the cancer cells for optimizing the PDT effects on tumors while sparing normal cells. Some attempts to achieve this are based on the conjugation of the PS to biomolecules (small ligands, peptides) functioning as carriers with the ability to efficiently penetrate cells and/or specifically recognize and bind proteins/receptors overexpressed on the surface of cancer cells. Alternatively, the PS can be entrapped in nanocarriers derived from various types of materials that can target the tumor by exploiting the enhanced permeability and retention (EPR) effects. The use of nanocarriers is particularly attractive because it allows the simultaneous delivery of more than one drug with the possibility of combining PDT with other therapeutic modalities.</jats:p
Cationic antimicrobial peptides as carriers of photosensitizers for photodynamic therapy of tumours
No full textIn the last decades, several applications of photodynamic therapy (PDT) have been proposed, including the treatment of cancerous and non-cancerous diseases as well as antimicrobial infections. The therapy is based on the administration of a photosensitizing agent (PS) which, after activation with visible light, exerts cytotoxicity via production of highly reactive oxygen species (ROS) that cause photo-oxidative damages in the cellular sites of PS accumulation and as a consequence cell death. We studied porphyrins and porphyrin-derivatives to explore their potential usefulness for cancer and antimicrobial PDT; in particular we explored strategies for improving the efficiency and the selectivity of PDT in killing cancer cells and bacteria. In this study we evaluate the delivery to cancer cells of PSs conjugated to cationic antimicrobial peptides (CAMPs). CAMPs are known to exert antibacterial activity against selected bacteria but they also exhibit several features typical of cell-penetrating peptides (CPPs) largely studied as carriers of anticancer drugs. Some of the CAMPs appear also to exert selective anticancer activity. Based on this, we are studying CAMPs as new class of carriers for the delivery of PSs for tumour PDT. We are evaluating, in cancer cells in vitro, the selectivity of uptake and the efficiency of the delivery of the PS 5(4’-carboxyphenyl)-10,15,20-triphenylporphyrin (cTPP) conjugated to selected CAMPs (Buforin II, Magainin II, Apidaecin). Preliminary experiments in A549 lung epithelial cancer cells revealed that the intracellular delivery of cTPP conjugated to Buforin II or Magainin II was increased about tenfold and thirtyfold, respectively, with respect to the un-conjugated form of the PS, after 5 h of cell incubation. Therefore, when cells were irradiated with 1.5 J cm-2 of blue light, comparable photo-killing efficiencies were measured with the cTPP-conjugates using concentrations at least 10 times lower than those used for the un-conjugated porphyrin. Notwithstanding the greater improvement of PDT outcomes measured in A549 cells using CAMPs-porphyrin conjugates, further investigations are needed in order to determine the interactions of the drug conjugates with non-cancerous cells and to assess that the mechanisms of action of these CPPs in driving the delivery of the PS increases the efficiency and selectivity of the PDT treatment
Uptake and photo-toxicity of Foscan®, Foslip® and Fospeg® in multicellular tumor spheroids
No full textPORPHYRIN-TYPE PHOTOSENSITISERS FOR KILLING BACTERIA AND CANCER CELLS WITH PHOTODYNAMIC THERAPY
No full textThe photosensitising properties displayed by many porphyrin-type molecules have been widely exploited for therapeutic purposes in the so called photodynamic therapy (PDT). In PDT, cells and tissues are first loaded with a photosensitizing drug which is then activated by illumination with suitable wavelengths of visible light to generate reactive oxygen species, mainly singlet oxygen, that kill cells through oxidative damages. Both prokaryotic and eukaryotic cells can be killed by PDT by selecting photosensitizers with physico-chemical properties favoring interaction/internalization with the particular cell type. We have been studying porphyrins and some derivatives to explore their potential usefulness for photodynamic therapy of tumours as well as for antimicrobial photodynamic therapy. In the last years we are exploring strategies that are able to improve efficiency and selectivity of PDT in killing cancer cells as well as bacteria. In the attempt of increasing the efficiency in killing bacteria, porphyrins have been conjugated to cationic antimicrobial peptides that are known to exhibit selectivity against bacterial cells. When compared to the corresponding free porphyrins, the conjugates showed very different outcomes, in terms of efficiency of bacteria photokilling, that were determined by the type of bacteria (Gram-positive or Gram-negative), the hydro/lipo-philicity and/or electric charge of the porphyrin that affect the strength of interaction of the conjugates with the bacterial cells. Our results showed that an anionic and hydrophobic porphyrin, devoid of photoactivity against the Gram-negative E. coli, acquired the ability to efficiently kill this bacterium when conjugated to the peptides. The effect of conjugation to cationic antimicrobial peptides of a tri-cationic porphyrin was less obvious very likely because, as other positively charged PS, this porphyrin exhibited by itself some photoactivity against both Gram-positive and –negative bacteria, which is preserved or even reduced by the conjugates [1]. In spite of the selectivity displayed by antimicrobial cationic peptides against bacterial cells, the porphyrin-peptide conjugates resulted very toxic by photoactivation also against eukaryotic cells. We found that normal human fibroblasts were completely killed by illumination with blue light after incubation with 5 and 3 μM of the free anionic or cation porphyrin respectively. The same effect was found with the porpyrin-peptides conjugates at concentrations 2-3 orders of magnitude lower than the free porphyrins. Therefore it appears that cationic antimicrobial peptides may represent useful carriers for improving internalization of drugs in mammalian cells, as already found for cell penetrating peptides. Based on this we are exploring the potential of the peptides to improve the delivery of PSs also to cancer cells.
[1] Dosselli R., Tampieri C., Ruiz-Gonzalez R., De Munari S., Ragàs X., Sánchez-García D., Agut M., Nonell S., Reddi E., Gobbo M. J. .Med. Chem. 2013, 56, 1052
Overview of Nanoparticle-Based Approaches for the Combination of Photodynamic Therapy (PDT) and Chemotherapy at the Preclinical Stage
Full text linkThe widespread diffusion of photodynamic therapy (PDT) as a clinical treatment for solid tumors is mainly limited by the patient’s adverse reaction (skin photosensivity), insufficient light penetration in deeply seated neoplastic lesions, unfavorable photosensitizers (PSs) biodistribution, and photokilling efficiency due to PS aggregation in biological environments. Despite this, recent preclinical studies reported on successful combinatorial regimes of PSs with chemotherapeutics obtained through the drugs encapsulation in multifunctional nanometric delivery systems. The aim of the present review deals with the punctual description of several nanosystems designed not only with the objective of co-transporting a PS and a chemodrug for combination therapy, but also with the goal of improving the therapeutic efficacy by facing the main critical issues of both therapies (side effects, scarce tumor oxygenation and light penetration, premature drug clearance, unspecific biodistribution, etc.). Therefore, particular attention is paid to the description of bio-responsive drugs and nanoparticles (NPs), targeted nanosystems, biomimetic approaches, and upconverting NPs, including analyzing the therapeutic efficacy of the proposed photo-chemotherapeutic regimens in in vitro and in vivo cancer models
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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