Ligand-targeted Theranostic Liposomes combining methylene blue attached upconversion nanoparticles for NIR activated bioimaging and photodynamic therapy against HER-2 positive breast cancer

Highlights

• Anti-HER2 Theranostic Liposome for Photodynamic therapy (PDT) and selective bioimaging.

• Enhancing PDT efficiency of methylene Blue by capping with upconverting nanoparticle v/s free methylene Blue.

• Liposome loaded with upconversion nanoparticle (UCNPs) and methylene blue.

• Enhanced Reactive oxygen species (ROS) generation after NIR-light exposure.

Abstract

Methylene Blue (MB) based Photodynamic therapy (PDT) has gained much interest recently due to its FDA approval for treating various diseased conditions in human. Herein, we report for the first time the systematic evaluation of NIR-controlled PDT efficiency of free MB and MB attached on NaYF₄:Yb, Er nanoparticles (UCNPs) i.e., MB@UCNPs encapsulated within Liposomes (LPs). The UCNPs act as an upconverting energy source for the photosensitizers dye MB for enhanced ROS generation and utilizing the remnant energy for selective tracking or bioimaging. The specificity of our nano LPs system was achieved by conjugating our newly discovered anti-HER2 peptides by phage display technique and demonstrated its specificity by confocal imaging of HER-2 overexpressing in vitro model. A significant decline in the cell viability by 83% and 64% was achieved by the LPs containing MB@UCNPs and free MB with UCNPs, respectively after 975 nm NIR laser exposure (0.60 W/cm⁻²) for 5 min continuous exposure. Also, the flowcytometry assay confirmed that enhanced ROS generation capability of LPs with MB@UCNPs was higher than the free MB with UCNPs, which states the relevance of the minimal distance between MB and UCNPs for higher anti-cancer efficiency.

Introduction

Breast cancer is one of the leading causes of death with very poor prognosis [1,2]. The molecular carcinogenesis of breast cancer relies on the presence and absence of many receptors such as estrogen, progesterone, and HER2 receptor [3]. Among all, it has been reported that the HER2 receptor accounts for 30% of total breast cancer with a very poor prognosis [4]. In consideration of this it is vital to develop next-generation treatment modalities which include the theranostics for efficient breast cancer management. The word “theranostics” was coined recently by Fankhouser in 2002, where he termed it as a combination of the therapy and diagnostics [[5], [6], [7]]. The development of multi-modal theranostics platform with the potential of increasing the treatment efficiency along with the utilization of ultra-low concentration could be an important factor. Such a multi-modal theranostics platform includes the combination of photodynamic therapy (PDT), photothermal therapy (PTT), chemotherapy, bioimaging etc. [8,9].

PDT could be one of the promising tools for treating breast cancer due to its high efficiency, non-invasive property, and controlled therapeutic outcome [10]. Under light irradiation, the photosensitizer (PS) generates reactive oxygen species (ROS) which can induce apoptosis and activate the immune responses [11]. It has been also reported that the PDT can be combined with chemotherapy [12,13], and PTT [14,15] to enhance the treatment outcome. The PDT mainly depends on the key elements such as PS, ROS, and light source. In turn, when we excite the PS with a specific wavelength of monochromatic light can transfer the absorbed photon to the neighboring oxygen molecules to generate the ROS capable of anti-cancer effect [12,16]. The main drawbacks associated with the PDT is a light to outreach deeper sited tumor tissues to photoexcite the PS [17]. The source of light from UV to visible region has shown to have low penetration deep when compared with the NIR light within the biological window [18]. Also, considering the fact that NIR light has low absorption and scattering by the biomolecules, tissues, and body fluids [18]. However, the main drawback of utilizing the NIR light is that most of the readily available PS are not photoexcitable at this wavelength of light [19,20]. One such strategy for improving the application of PDT will be combining such modality with upconverting nanoparticles (UCNPs) and leveraging their emission to photoexcite the PS [[21], [22], [23], [24], [25], [26]]. It has been reported that the anti-stokes and sharp emission property in high energy photon after excitation with low energy photon makes them a possible candidate for cancer theranostics [27]. Furthermore, such visible emission can be tuned by the appropriate combination of lanthanide ions enhancing the resonance with PS. There have been many reports by many groups including our own for improving cancer based theranostics using the UCNPs for PDT [12,16,[28], [29], [30]], and PTT [31] applications.

The methylene blue (MB) approval by the FDA for direct clinical based translations makes it an ideal candidate for PDT applications [32,33]. MB is a phenothiazine derivative with good solubility in the aqueous phase. There has been some recent development of combining the graphene oxide [34] and encapsulating only MB directly within liposomes (LPs) [35,36]. However, the wavelength of light used for PDT at ~655 nm shows relatively poor penetration power in the deep sited tumors and could affect its anti-cancer efficiency. The main factor impeding the MB application for PDT is the stability, inactivation, and bioaccumulation in endothelial and erythrocyte cells [37,38]. These issues could be resolved by fabricating the nanocarrier i.e., LPs, which can protect and carry the MB within for enhanced accumulation in the tumor site for enhanced PDT. The LPs are highly efficient because of its controlled nanosized, membrane modifications capabilities and are recognized as the most suitable system to carry the nanoparticles or cargo loads within them due to their capability to amend the pharmacokinetics and reduction of adverse side effects by adjusting the adsorption, distribution, excretion, and metabolism processes (i.e., ADME) [39].

Recently our group reported the development of a nano Liposome system capable of chemo-drug delivery along with NIR controlled PDT using MB. Since there we describe the fabrication of nanoliposome system and PDT evaluation against HER2 positive breast cancer cells, but such reports do not summarize the efficiency of the MB in the free form along with UCNPs. Herein, we report the systematic evaluation of the PDT efficiency of MB attached UCNPs (i.e., MB@UCNPs) and its free form within the LPs using deep-penetrating NIR excitation sources, in the in vitro model. The efficiency of such a PDT system to selectively tack the HER-2 positive breast cancer cell was done using our own newly discovered anti-HER2 peptide which showed selective tracking and high cell penetration capabilities. The production of ROS after 975 nm laser excitation was demonstrated using ROS sensitive probe molecule, flow cytometry assay, and by in vitro fluorogenic marker. To the best of our knowledge, it's the first report of demonstrating the ROS generation capabilities, depending on the distance of free MB along with UCNPs and MB@UCNPs by using the ligand targeted LPs as a nanocarrier for enhanced PDT-based HER2 positive breast cancer management.