Diogo Silva Pellosi

Polymeric Nanoparticles for Cancer Photodynamic Therapy.

In chemotherapy a fine balance between therapeutic and toxic effects needs to be found for each patient, adapting standard combination protocols each time. Nanotherapeutics has been introduced into clinical practice for treating tumors with the aim of improving the therapeutic outcome of conventional therapies and of alleviating their toxicity and overcoming multidrug resistance. Photodynamic therapy (PDT) is a clinically approved, minimally invasive procedure emerging in cancer treatment. It involves the administration of a photosensitizer (PS) which, under light irradiation and in the presence of molecular oxygen, produces cytotoxic species. Unfortunately, most PSs lack specificity for tumor cells and are poorly soluble in aqueous media, where they can form aggregates with low photoactivity. Nanotechnological approaches in PDT (nanoPDT) can offer a valid option to deliver PSs in the body and to solve at least some of these issues. Currently, polymeric nanoparticles (NPs) are emerging as nanoPDT system because their features (size, surface properties, and release rate) can be readily manipulated by selecting appropriate materials in a vast range of possible candidates commercially available and by synthesizing novel tailor-made materials. Delivery of PSs through NPs offers a great opportunity to overcome PDT drawbacks based on the concept that a nanocarrier can drive therapeutic concentrations of PS to the tumor cells without generating any harmful effect in non-target tissues. Furthermore, carriers for nanoPDT can surmount solubility issues and the tendency of PS to aggregate, which can severely affect photophysical, chemical, and biological properties. Finally, multimodal NPs carrying different drugs/bioactive species with complementary mechanisms of cancer cell killing and incorporating an imaging agent can be developed. In the following, we describe the principles of PDT use in cancer and the pillars of rational design of nanoPDT carriers dictated by tumor and PS features. Then we illustrate the main nanoPDT systems demonstrating potential in preclinical models together with emerging concepts for their advanced design.

Biotin-targeted Pluronic® P123/F127 mixed micelles delivering niclosamide: A repositioning strategy to treat drug-resistant lung cancer cells

With the aim to develop alternative therapeutic tools for the treatment of resistant cancers, here we propose targeted Pluronic(®) P123/F127 mixed micelles (PMM) delivering niclosamide (NCL) as a repositioning strategy to treat multidrug resistant non-small lung cancer cell lines. To build multifunctional PMM for targeting and imaging, Pluronic(®) F127 was conjugated with biotin, while Pluronic(®) P123 was fluorescently tagged with rhodamine B, in both cases at one of the two hydroxyl end groups. This design intended to avoid any interference of rhodamine B on biotin exposition on PMM surface, which is a key fundamental for cell trafficking studies. Biotin-decorated PMM were internalized more efficiently than non-targeted PMM in A549 lung cancer cells, while very low internalization was found in NHI3T3 normal fibroblasts. Biotin-decorated PMM entrapped NCL with good efficiency, displayed sustained drug release in protein-rich media and improved cytotoxicity in A549 cells as compared to free NCL (P<0.01). To go in depth into the actual therapeutic potential of NCL-loaded PMM, a cisplatin-resistant A549 lung cancer cell line (CPr-A549) was developed and its multidrug resistance tested against common chemotherapeutics. Free NCL was able to overcome chemoresistance showing cytotoxic effects in this cell line ascribable to nucleolar stress, which was associated to a significant increase of the ribosomal protein rpL3 and consequent up-regulation of p21. It is noteworthy that biotin-decorated PMM carrying NCL at low doses demonstrated a significantly higher cytotoxicity than free NCL in CPr-A549. These results point at NCL-based regimen with targeted PMM as a possible second-line chemotherapy for lung cancer showing cisplatin or multidrug resistance.

Pluronic ® P123/F127 mixed micelles delivering sorafenib and its combination with verteporfin in cancer cells

Here, we developed Pluronic® P123/F127 (poloxamer) mixed micelles for the intravenous delivery of the anticancer drug sorafenib (SRB) or its combination with verteporfin (VP), a photosensitizer for photodynamic therapy that should complement well the cytotoxicity profile of the chemotherapeutic. SRB loading inside the core of micelles was governed by the drug:poloxamer weight ratio, while in the case of the SRB–VP combination, a mutual interference between the two drugs occurred and only specific ratios could ensure maximum loading efficiency. Coentrapment of SRB did not alter the photophysical properties of VP, confirming that SRB did not participate in any bimolecular process with the photosensitizer. Fluorescence resonance energy-transfer measurement of micelles in serum protein-containing cell-culture medium demonstrated the excellent stability of the system in physiologically relevant conditions. These results were in line with the results of the release study showing a release rate of both drugs in the presence of proteins slower than in phosphate buffer. SRB release was sustained, while VP remained substantially entrapped in the micelle core. Cytotoxicity studies in MDA-MB231 cells revealed that at 24 hours, SRB-loaded micelles were more active than free SRB only at very low SRB concentrations, while at 24+24 hours a prolonged cytotoxic effect of SRB-loaded micelles was observed, very likely mediated by the block in the S phase of the cell cycle. The combination of SRB with VP under light exposure was less cytotoxic than both the free combination and VP-loaded micelles + SRB-loaded micelles combination. This behavior was clearly explainable in terms of micelle uptake and intracellular localization. Besides the clear advantage of delivering SRB in poloxamer micelles, our results provide a clear example that each photochemotherapeutic combination needs detailed investigations on their particular interaction, and no generalization on enhanced cytotoxic effects should be derived a priori.

Multifunctional theranostic Pluronic mixed micelles improve targeted photoactivity of Verteporfin in cancer cells

Nanotechnology development provides new strategies to treat cancer by integration of different treatment modalities in a single multifunctional nanoparticle. In this scenario, we applied the multifunctional Pluronic P123/F127 mixed micelles for Verteporfin-mediated photodynamic therapy in PC3 and MCF-7 cancer cells. Micelles functionalization aimed the targeted delivery by the insertion of biotin moiety on micelle surface and fluorescence image-based through rhodamine-B dye conjugation in the polymer chains. Multifunctional Pluronics formed spherical nanoparticulated micelles that efficiently encapsulated the photosensitizer Verteporfin maintaining its favorable photophysical properties. Lyophilized formulations were stable at least for 6months and readily reconstituted in aqueous media. The multifunctional micelles were stable in protein-rich media due to the dual Pluronic mixed micelles characteristic: high drug loading capacity provided by its micellar core and high kinetic stability due its biocompatible shell. Biotin surface functionalized micelles showed higher internalization rates due biotin-mediated endocytosis, as demonstrated by competitive cellular uptake studies. Rhodamine B-tagged micelles allowed monitoring cellular uptake and intracellular distribution of the formulations. Confocal microscopy studies demonstrated a larger intracellular distribution of the formulation and photosensitizer, which could drive Verteporfin to act on multiple cell sites. Formulations were not toxic in the dark condition, but showed high Verteporfin-induced phototoxicity against both cancer cell lines at low drug and light doses. These results point Verteporfin-loaded multifunctional micelles as a promising tool to further developments in photodynamic therapy of cancer.

β-Cyclodextrin Nanosponges as Multifunctional Ingredient in Water-Containing Semisolid Formulations for Skin Delivery

A β-cyclodextrin nanosponge cross-linked with pyromellitic dianhydride (βNS-PYRO) is reported for the first time as multifunctional ingredient in semisolid formulations for drug delivery to the skin. The role of βNS-PYRO on solubilization and stabilization of the photosensitizer benzoporphyrin-derivative monoacid ring A (BPDMA) and all-trans retinoic acid (atRA) as well as its effect on skin permeation of diclofenac (DIC) was investigated. Aqueous solutions, gels, and cream-gels were prepared from mixtures of βNS-PYRO with a conventional gelling agent at specific ratios. The incorporation of BPDMA in βNS-PYRO water solutions prevented its aggregation and gave kinetically stable complexes with high photostability and singlet oxygen generation upon irradiation. atRA incorporated in the βNS-PYRO-containing gel demonstrated a remarkable stability as compared with the formulation without βNS-PYRO, resulting in an eightfold increase of its lifetime. Skin permeation studies highlighted that βNS-PYRO in gels and cream-gels containing DIC significantly decreased the amount of drug permeated through the skin while increasing its amount in stratum corneum and viable epidermis. Overall, swellable βNS-PYRO turns to be a multifunctional coingredient with potential in topical monophasic and biphasic formulations to stabilize light-sensitive drugs and to localize the action of highly penetrating drugs in the external layers of skin.

Singlet oxygen production by combining erythrosine and halogen light for photodynamic inactivation of Streptococcus mutans

BACKGROUNDnPhotodynamic inactivation of microorganisms is based on a photosensitizing substance which, in the presence of light and molecular oxygen, produces singlet oxygen, a toxic agent to microorganisms and tumor cells. This study aimed to evaluate singlet oxygen quantum yield of erythrosine solutions illuminated with a halogen light source in comparison to a LED array (control), and the photodynamic effect of erythrosine dye in association with the halogen light source on Streptococcus mutans.nnnMETHODSnSinglet oxygen quantum yield of erythrosine solutions was quantified using uric acid as a chemical-probe in an aqueous solution. The in vitro effect of the photodynamic antimicrobial activity of erythrosine in association with the halogen photopolimerizing light on Streptococcus mutans (UA 159) was assessed during one minute. Bacterial cultures treated with erythrosine alone served as negative control.nnnRESULTSnSinglet oxygen with 24% and 2.8% degradation of uric acid in one minute and a quantum yield of 0.59 and 0.63 was obtained for the erythrosine samples illuminated with the halogen light and the LED array, respectively. The bacterial cultures with erythrosine illuminated with the halogen light presented a decreased number of CFU mL(-1) in comparison with the negative control, with minimal inhibitory concentrations between 0.312 and 0.156mgmL(-1).nnnCONCLUSIONSnThe photodynamic response of erythrosine induced by the halogen light was capable of killing S. mutans. Clinical trials should be conducted to better ascertain the use of erythrosine in association with halogen light source for the treatment of dental caries.

Light source is critical to induce glioblastoma cell death by photodynamic therapy using chloro-aluminiumphtalocyanine albumin-based nanoparticles.

Selection of an efficient light source is fundamental in the development of photodynamic therapy (PDT) protocols. However, few studies provide a comparison of different light sources with regard to phototoxic effects. Here, we compared the cell death induced by photoactivation of chloro-aluminiumphtalocyanine (AlClPc)-loaded human serum albumin nanoparticles under irradiation with different light sources: continuous laser (CL), pulsed laser (PL), and light-emitting diode (LED). Cells were exposed to three different AlClPc concentrations (1, 3, and 5μM) and three different light doses (200, 500, and 700mJ/cm2) for each light source. Cell death and differentiation of apoptosis and necrosis pathway were measured by flow cytometry. CL was the best light source for improving the photodynamic action of AlClPc-loaded albumin nanoparticles in glioblastoma cells and avoiding undesirable side effects, especially at low photosensitizer doses (200mJ/cm2). In addition, apoptosis was the main cell death pathway in all evaluated cases (70% for CL, and greater than 50% for PL and LED). In conclusion, the search for optimal light sources and light/photosensitizer doses is a crucial step in improving PDT outcomes and enhancing the clinical translation of PDT.

Photodegradation in Micellar Aqueous Solutions of Erythrosin Esters Derivatives

Strong light absorption and high levels of singlet oxygen production indicate erythrosin B as a viable candidate as a photosensitizer in photodynamic therapy or photodynamic inactivation of microorganisms. Under light irradiation, erythrosin B undergoes a photobleaching process that can decrease the production of singlet oxygen. In this paper, we use thermal lens spectroscopy to investigate photobleaching in micellar solutions of erythrosin ester derivatives: methyl, butyl, and decyl esters in low concentrations of non-ionic micellar aqueous solutions. Using a previously developed thermal lens model, it was possible to determine the photobleaching rate and fluorescence quantum efficiency for dye-micelle solutions. The results suggest that photobleaching is related to the intensity of the dye-micelle interaction and demonstrate that the thermal lens technique can be used as a sensitive tool for quantitative measurement of photochemical properties in very diluted solutions.

Spotlight on the delivery of photosensitizers: different approaches for photodynamic-based therapies

ABSTRACT Introduction: Nanomedicine development allowed the discovery of new photosensitizers (PS) and drug delivery systems (DDS) to overcome current issues on phototherapy. Nano-engineered materials have the potential to improve the solubility of PS, control drug pharmacokinetics, decreasing side effects, increasing bioavailability, and overcoming multidrug resistance. A recent approach is the co-delivery of PS with other therapeutic agents in a multimodal platform for synergic and improved results. Areas covered: This paper discusses the delivery of PS-nanostructured platforms for conventional, photothermal, and antimicrobial photodynamic therapies, as well as in a recent therapeutic modality for photobiomodulation, covering applications of cancer diagnosis, targeting to skin pathogens, photoregeneration and wound healing. The focus of the present review is to describe the use of different DDS to enhance the therapeutic outcomes triggered by the combination of delivered PS, light, and oxygen. Expert opinion: Nanotechnology allowed the development of site-specific delivery of PS molecules, expanding possibilities poorly explored before to enhance photodynamic efficacy and extrapolate the concept to other treatment protocols. Research in this area embraces potential and pitfalls of PS delivery, allowing new clinical phase outcomes and long-term issues to be established, which will impact on several biomedical applications.

Magneto low-density nanoemulsion (MLDE): A potential vehicle for combined hyperthermia and photodynamic therapy to treat cancer selectively

In this paper, we introduce a new drug delivery system (DDS) called magneto low-density nanoemulsion (MLDE), which can carry maghemite nanoparticles and Chlorin e6 as an active photosensitizer drug. This design can enhance tumor damage after minor heat dissipation and/or minimum visible light photosensitization doses by classical magnetic hyperthermia (MHT) and photodynamic therapy (PDT), respectively. We establish protocols to prepare the MLDE and to load the drug combination onto it. The MLDE prepared herein is nanometric (<200u202fnm), has high encapsulation efficiency, and is stable for at least 12u202fmonths in water dispersions. Flow cytometry results demonstrated that MLDE presents targeted selectivity toward the MCF-7 breast cancer cell line but not in NHI-3T3 mouse fibroblast cell lines, because the MCF-7 cancer cell surface contains overexpressed low density lipoprotein (LDL) receptors. Despite this targeted effect, MHT or PDT alone does not prompt significant antiproliferative outcomes. On the other hand, MHT and PDT in combination induce a strong and synergic action on MCF-7 cells and reduce the cell viability. In conclusion, the developed MLDE deserves further investigation because it is biocompatible, displays good encapsulation efficiency, and is highly stable. Moreover, it is selectively taken up by cancer cell surfaces with receptor recognition based on LDL receptor overexpression, which potentiates the action of combined MHT and PDT.