Engineering and biological assessment of double core nanoplatform for co-delivery of hybrid fluorophores to human melanoma

https://doi.org/10.1016/j.jinorgbio.2020.111088Get rights and content

Highlights

  • TOPO-stabilized NaYF4:Er3+,Yb3+ NPs and Rose Bengal were co-encapsulated by double emulsion method

  • PLGA, Span 80 and Cremophor A25 act as the obtained nanocontainers stabilizers

  • The co-encapsulation process retained the optical properties of the hybrid cargo

  • The in vitro biological studies shown the protective effect of the nanocarriers upon normal human keratinocytes

  • The hybrid cargo supported by up-converting NPs activation showed an improved photodynamic effect toward human melanoma cells.

Abstract

We investigated new development in photodynamic therapy (PDT), aiming at enhanced tumor selectivity and biocompatibility, which included application of a third-generation photosensitizing agent, i.e. xanthene-origin Rose Bengal (RB) co-encapsulated with up-converting NaYF4 nanoparticles (NPs) co-doped with lanthanide ions: Er3+ (2%) and Yb3+ (20%). The hybrid fluorophores were applied as components of double core nanocarriers (NCs) obtained by double (multiple) emulsion solvent evaporation process. Next, to improve the biocompatibility and photodynamic activity, biodegradable polymer: poly(lactide-co-glycolide) PLGA and non-ionic surfactants with different hydrophobicity: Span 80 and Cremophor A25, were used. After the engineering process, controlled by dynamic light scattering (DLS) measurements, ζ-potential evaluation, transmission electron and atomic force microscopy (TEM and AFM) imaging, as well as optical analysis provided by measurements of the up-conversion emission spectra and luminescence kinetics for encapsulated only NaYF4:Er3+,Yb3+ NPs and co-encapsulated RB + NaYF4:Er3+,Yb3+ molecules, spherical polyester NCs with average size <200 nm, were tested on human melanoma (Me-45 and MeWo) cells and a control human keratinocyte (HaCaT) cell line. The photodynamic action of the investigated NCs was assessed by oxidoreductive potential measurements with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, that corresponds to percentage of the viable cells. Immunofluorescence and the NCs internalization studies were visualized by confocal laser scanning microscopy (CLSM studies). Our results indicated effective photosensitizer delivery into the cancer cells and significant photodynamic efficiency enhanced by the near infrared (NIR)-activation of the encapsulated hybrid cargo in the skin melanoma cells.

Graphical abstract

The trioctylphosphine oxide-stabilized NaYF4:Er3+,Yb3+ nanoparticles and Rose Bengal were co-encapsulated by double-emulsion evaporation method, recognized as an effective nanoemulsion structural design. The physicochemical properties of the hybrid cargo were investigated in detail. The photodynamic activity upon 980 nm irradiation was checked in vitro against human melanoma (Me45 and MeWo) cells.

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Introduction

The possibility of challenging skin melanoma efficient inactivation has recently attracted significant interest, mainly due to the improvement of novel antitumor treatments protocols [1,2]. Among the well-known approaches, photodynamic therapy (PDT) is recognized as an effective anticancer tool, having many advantageous in comparison with conventional treatments such as chemo- or radio-therapy. Although, some side effects of PDT may occur after irradiation, e.g. light-triggered photosensitive reactions and swelling in the treated area, causing pain and difficulties in swallowing or breathing. Additionally, insufficient therapeutic outcome of PDT is frequently associated with poor efficiency of applied photosensitizing agents, and rising drug resistance taking place after repeated application of the same treatment procedures [2,3]. Thus, alternative tools for enhanced photosensitizer delivery and activation are still required to improve the PDT method outcome. The latest research indicates that nanoscopic drug delivery systems [4,5], novel inorganic fluorescent markers [6,7], and their combinations [8,9] can significantly boost the photosensitizer active delivery, and thus increase the therapeutic effect. Innovative developments in the field of nanotechnology and nanoencapsulation, regarding the drug delivery in PDT, show considerable progress in the design of novel nanoemulsions and their structural features via layering, embedding and clustering processes [9,10]. These approaches enable creation of polymeric or lipid nanocarriers (oil-core nanocapsules, nanospheres, polymeric micelles, solid lipid nanoparticles, lyotropic liquid crystals) for encapsulation/entrapment/loading of organic or inorganic (or both) compounds, which may increase their solubility, bioavailability and photosensitivity of applied PDT protocols [[10], [11], [12]].

Our recent studies involving encapsulation of inorganic, up-converting NaYF4 nanoparticles (NPs) [9,13,14] inside various lipid-polymeric nanostructures encouraged us to research up-conversion-supported near infrared (NIR)-light triggered PDT of human melanoma - cancer type which is highly resistant to standard chemotherapy and radiotherapy [15], in the present contribution. By using the double emulsion embedding technique we were able to create polymeric nanocarriers (NCs) based on a poly(lactide-co-glycolide) (PLGA) copolymer stabilized by non-ionic surfactants (Span 80 and Cremophor A25) containing loaded: (i) second generation photosensitizer – Rose Bengal (RB) and (ii) trioctylphosphine oxide (TOPO)-stabilized NaYF4:2%Er3+,20%Yb3+ NPs. This approach allowed us to fabricate the biocompatible NCs with hydrophilic (organic) and hydrophobic (inorganic) cargo showing high colloidal stability and loading capacity, as well as resistance to chemical degradation. Latest data indicate RB as an effective photosensitizer for human cancer therapy, including promising treatment of melanoma [16]. The role of up-converting NaYF4:2%Er3+,20%Yb3+ NPs relies on both, the possibility to use PDT triggering by the 980 nm NIR light, which falls into the so called therapeutic window of cells and tissues, simultaneously avoiding the spectrum overlapping with the main biological dyes resulting in background auto-fluorescence suppression. Additionally, the possible static or dynamic energy transfer between the up-converting NPs and the xanthene-origin photosensitizer assures improvement of the photodynamic activity of the obtained NCs [13]. Furthermore, selected for this study lanthanide doped NPs showed stable in time up-conversion luminescence with minimal photo-bleaching and suppressed influence of the surroundings, thus being versatile luminescent probes for any other bio-related applications [17].

In our latest contributions significant effort was made to evaluate the obtained systems, in order to choose the most promising and optimized nanocontainer capable to co-encapsulate hybrid fluorophores with different hydrophobicity [9,13,18]. We also provided pilot biological experiments with free and loaded NaYF4 NPs, performed on cancer (SKOV-3) and normal (HUVEC) cells, which indicated that only encapsulated NPs show low toxicity in appropriate concentrations [19]. Thus, in the present studies we decided to verify the photoactive performance of RB in the encapsulated and co-encapsulated with the up-converting NPs form, which in fact revealed more toxic properties in cancer cells after irradiation, simultaneously protecting normal cells. The combination of organic (hydrophilic) photosensitizer (RB) and inorganic (poorly water soluble) up-converting NPs (NaYF4:2%Er3+,20%Yb3+) co-loaded in the NCs dual compartment structure (double core), for simultaneous deliver of the hybrid cargo and in vitro PDT, arises as efficient and safe approach for the novel anti-melanoma protocols. This biological evaluation was performed using two cell line models - human melanoma: wild type of melanoma granular fibroblasts (MeWo) and malignant cells derived from a lymph node metastasis of skin melanoma (Me-45), as well as - normal skin cells, i.e. spontaneously immortalized human cutaneous keratinocyte line (HaCaT) applied as control. Our data have showed that the applied combination of organic and inorganic agents, as well as encapsulation technology and NIR light 980 nm optical activation enhances the dual platform transport to the target skin melanoma cells and improves the effectiveness of the PDT treatment. We strongly believe that these well-defined theranostic NCs can be valuable, not only to anticancer protocols, but also to multi-modal imaging applications, targeted molecular diagnosis of melanoma and phototherapy of cancers.

Section snippets

Materials

The following reagents applied for the synthesis of NaYF4:2%Er3+,20%Yb3+ NPs: lanthanide oxides (Y2O3, Yb2O3, Er2O3), sodium trifluoroacetate, trifluoroacetic acid, octadecane and trioctylphosphine oxide as well as Rose Bengal (RB) were purchased from Sigma Aldrich and used as received. Non-ionic surfactants with different hydrophobicity: Cremophor A25 and Span 80 were purchased from BASF Care Creations and Sigma Aldrich, respectively. Ester endcap poly(lactide-co-glycolide) (PLGA,

Evaluation of the co-loaded NC engineering.

As the main scientific purpose of the present contribution, we applied effective co-encapsulation approach, i.e. double emulsion evaporation technique – described as one of the most favorable method of nanoemulsion structural design [20]. The main reason for selecting this method was to enhance the solubility and antitumor photodynamic activity of hybrid fluorophores, i.e. a hydrophilic xanthene-origin photosensitizing agent of second generation –RB and up-converting NaYF4:Er3+,Yb3+NPs, which

Conclusions

The results presented in this work may support designing and developing a new generation of bioinspired drug delivery nanocarriers for potential application in conventional (green light activated) or NIR-induced (980 nm) photodynamic therapy of melanoma skin cancer. We demonstrated the usefulness of the co-encapsulated hybrid cargo (RB photosensitizer and TOPO-stabilized NaYF4:Er3+,Yb3+NPs) for effective tumor destruction via photoactivation. The polyester-derived nanocarriers with double-core

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work has been supported by National Science Centre (Poland) within a framework of SONATA 8 programme (No. 2014/15/D/ST4/00808) and by Wroclaw University of Science and Technology.

References (29)

  • T. Datea et al.

    Lipid-polymer hybrid nanocarriers for delivering cancer therapeutics

    J. Control. Release

    (2018)
  • M. Buchner et al.

    Photosensitiser functionalised luminescent upconverting nanoparticles for efficient photodynamic therapy of breast cancer cells

    Photochem. Photobiol. Sci.

    (2019)
  • D. Wawrzyńczyk et al.

    Ligand-dependent luminescence of ultra-small Eu3+-doped NaYF4 nanoparticles

    J. Nanopart. Res.

    (2013)
  • K.M. Vargas et al.

    Hybrid lipid–nanoparticle complexes for biomedical applications

    J. Mater. Chem. B

    (2019)
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