Self-assembled peptido-nanomicelles as an engineered formulation for synergy-enhanced combinational SDT, PDT and chemotherapy to nasopharyngeal carcinoma

Zhe Liu *ab, Doudou Wang c, Jiaping Li c and Yan Jiang c
aAcademy of Medical Engineering and Translational Medicine, Tianjin University, 300072, Tianjin, China. E-mail:
bTianjin Key Laboratory of Brain Science and Neural Engineering, Tianjin University, 300072, Tianjin, China
cWenzhou Medical University, 325001, Wenzhou, Zhejiang, China

Received 16th July 2019 , Accepted 29th July 2019

First published on 31st July 2019

A formulation of self-assembled peptido-nanomicelles has been developed for a combinational treatment of SDT, PDT and chemotherapy to nasopharyngeal carcinoma. In vitro cellular tests and in vivo mice therapy proved effective for targeted tumor growth inhibition. These merits provided a novel approach to non-invasive cancer treatments.

Nasopharyngeal carcinoma (NPC) is one of the typical non-lymphatic malignant cancers which severely affects the human health quality throughout life.1–3 The incidence rate ranks top of the head-and-neck cancers. Especially in South China, the Middle East, Alaska and Canada, NPC is more common with approximately 21 cases per 100[thin space (1/6-em)]000 people.4 In the past 5 years, the overall survival rate from stage I to IV has witnessed a decline from 72% to 38%. Early-stage treatment is obviously important. However, due to insufficient specific molecular targets for efficient molecular diagnosis, current treatments against NPC are mainly confined to traditional radiation therapy, chemotherapy or surgery. Anti-cancer drugs are usually given via a vein or oral administration with limited circulation stability and unsatisfactory bioavailability, and radiation therapy or surgery is prone to cause remarkable side-effects, high metastatic risks and psychological burden to the patients in that the location of nasopharynx is in close proximity to the brain.5 As a consequence, the overall successful curing rate is always considerably low, and poor prognosis leads to even worse disease situation. To solve these bottle-neck difficulties, advanced drug formulations with more biocompatible carriers and better drug loading efficacy are expected to afford increased drug absorption at the lesion, and enhanced bioavailability and more favourable therapeutic efficiency are accordingly acquired. In the meantime, shortened body clearance and unexpected side-effects to normal tissues can be avoided. The state-of-the-art non-invasive treatments such as sonodynamic (SDT) or photodynamic (PDT) therapies with the exploitation of elaborately designed sono- or photo-sensitizers render easy handling and intuitively visualized convenience for disease therapy.6–10 The reactive oxygen species (ROS) can be localized and generated, and cancerous tissues may be completely eliminated. Thus, a synergistic enhancement combining these modalities might be realized to broaden their emerging biomedical uses for curing a series of deadly human diseases.

Rose Bengal (RB), an analogue of fluorescein prepared in 1882, is nowadays used in photochemistry, biological staining and skin disease treatments.11–15 RB-loaded nanoparticles have been reported for their anti-bacterial activity and application to cancer therapies.16–18 Multifunctional polypeptides are a great pool of bioactive and pharmaceutical substances which play a critical role in biomarker recognition, receptor binding, trans-membrane penetration and signal transduction.19–22 In particular, amphiphilic peptides of C18GR7RGDS are capable of forming stable self-assembled peptido-nanomicelles encapsulating the therapeutic payloads. In response to the relatively acidic micro-environment at the tumor, the pH-sensitive peptido-nanomicelles release the loaded RB for SDT, PDT and chemotherapy. Therefore, by loading RB into the multifunctional polypeptide, cancerous lesion-targeting, receptor-mediated drug delivery and consecutive in situ treatments can be achieved with high precision. In this context, self-assembled RB-loaded peptido-nanomicelles (RBNs) have been developed, and they were used as both engineered molecular probes and a novel nano-formulation for synergy-enhanced NPC treatment by SDT, PDT and chemotherapy (Fig. 1A).

image file: c9cc05463d-f1.tif
Fig. 1 A schematic illustration of elaborately fabricated RB-loaded peptido-nanomicelles (RBNs) for synergy-enhanced therapy of SDT, PDT and chemotherapy (CT) (i.v.: intravenous injections; i.t.: intra-tumoral injections) (A), the size and zeta characterizations (B) and cytotoxicity to CNE-2Z cells comparing free RB and the as-fabricated nano-formulation of RBNs (C).

The RBNs were fabricated by a one-pot sonication-promoted self-assembly protocol. The amphiphilic peptide with both hydrophobic and hydrophilic terminals at spacer-bridged ends was mildly dissolved in water, and a RB aqueous solution was added along with external sonication. The amphiphilic peptide underwent a sonication-promoted self-assembly, and the thermodynamically stabilized peptido-nanomicelles were generated simultaneously with RB encapsulation into the core. The resulting RBNs displayed an average size of 17.28 ± 0.88 nm in diameter and a mean zeta potential of 26.2 ± 0.81 mV (Fig. 1B). It was also observed that a vast majority of RBNs in volume distribution were smaller than 50 nm, a suitable particle size for penetrating the endothelial gaps and permeating into the tumor via the EPR effect. The RB encapsulation efficiency was determined in the range of 43–58%, which was much higher than those of some reported nano-formulations. The long-term detection of the size variation and RB encapsulation verified their prominent stability from RB leakiness and nanomicelle disintegration.23 In order to evaluate the cytotoxicity of RBNs compared to free RB, CNE-2Z cells as a representative of the NPC cell line were co-incubated with them respectively. As shown in Fig. 1C, it was reasonable that the concentrated RBNs afforded increased cytotoxicity, and 20, 60 and 100 μg mL−1 of RBNs gave dramatically decreased cell survivals of 53.8%, 22.3% and 7.8%. Whereas, free RB showed negligible toxicity even after 24 h incubation. This remarkable difference can be explained that the elaborately fabricated RBNs have an appropriate spacer length and the RGDS residue, an αvβ3 integrin-targeted ligand on the outer shell, which provides them specific binding affinity to the CNE-2Z cells. The RBNs may be internalized via RGDS-mediated endocytosis, and the loaded RB can be transported, which resulted in significant cytotoxicity.

In order to utilize the RBNs for SDT and PDT, sono- and photo-activation of RBNs to ROS generation was assessed, and their in vitro cell killing capability has also been verified. Herein, a fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCF-DA) was used as a water-soluble redox ROS indicator.24,25 As displayed in Fig. 2A, the RBNs were internalized into the cytoplasm, and under ultrasound (US) exposure ROS were produced by sono-activation of RBNs, which exerted strong sono-induced CNE-2Z cell death or necrosis. Meanwhile, the self-assembly structure of RBNs collapsed upon tumor micro-environmental acidity, and the polypeptide and loaded RB were gradually released from the lysosomes and decomposed in vitro. This could be affirmed by confocal laser scanning microscopy (CLSM) (Fig. 2B). It was visualized that RBNs + US exhibited the strongest ROS production (FITC channel) along with a maximal RBN collapse (TRITC channel). In contrast, ROS generation in the RBN group was moderate, and most RBNs remained intact without US exposure. The RB, RB + US, US groups or the control showed no ROS generation, which confirmed that only the combination of RBNs and US contributed to ROS production and ultimately sono-induced CNE-2Z cell death or necrosis. Similarly, the photo-activation of RBNs for ROS generation and the cell killing behaviour were studied, and it was also concluded that a combination of RBNs and laser irradiation facilitated abundant ROS production along with photo-induced CNE-2Z cell elimination (Fig. 2C and D).

image file: c9cc05463d-f2.tif
Fig. 2 (A) The proposed mechanism of RBNs internalization into CNE-2Z cells treated with SDT; (B) ROS generation by RBNs + US compared to other groups; (C) the proposed mechanism of RBNs internalization into CNE-2Z cells treated with PDT; and (D) ROS generation by RBNs + laser compared to other groups (scale bar: 100 μm).

The sono- and photo-induced cell elimination capability was also evaluated by live/dead staining (Fig. 3). RBNs + Laser + US exhibited the highest cell death, which implied a PDT-SDT synergistic effect in addition to the intrinsic RB chemotherapy. Furthermore, single PDT- or SDT-activation approaches, i.e., RBNs + laser and RBNs + US, showed relatively reduced cell killing, and other groups without the exploitation of RBNs displayed nearly no cell elimination performances.

image file: c9cc05463d-f3.tif
Fig. 3 Live/dead staining and fluorescence microscopy of CNE-2Z cells to evaluate the sono- and photo-induced cell elimination capability by RBNs. Viable cells were stained green with calcein-AM, and dead cells were stained red with PI (scale bar: 200 μm).

To quantitatively compare the therapeutic contributions of SDT and PDT, the cell viability under varied conditions was analyzed (Fig. 4). It was interesting that all groups upon US exposure showed a certain extent of cell killing. Even US alone contributed to 15.3% cell death, and with the utilization of RBNs, 64.7% cell death was achieved under US exposure (Fig. 4A). In contrast, only laser irradiation could not result in apparent cell killing, and a decreased viability of 29.6% was found under laser irradiation (Fig. 4B). The strongest cell killing was observed in association with RBNs, Laser and US, and cell viability (16.0%) was acquired. On the other hand, the time duration for SDT and PDT also plays a critical role. 1–5 min of US exposure and laser irradiation were respectively applied to the RBNs, and the corresponding cell viability has been obtained (Fig. 4C and D). Co-incubation of RBNs with CNE-2Z cells without US or laser irradiation contributed to a moderate cytotoxicity (40% viability) which was in good accordance with the results in Fig. 2. Prolonged US or laser exposure from 1 to 5 min could not lead to an increased cell elimination. Nevertheless, the SDT-PDT combination at the same time duration of 3 min provided the lowest viability and an optimal cell elimination. All these findings concluded that SDT-PDT proved an eminent therapy modality in combination with RBNs as a chemotherapeutic formulation, and a synergy enhancement between the tri-modalities facilitated efficacious cell elimination.

image file: c9cc05463d-f4.tif
Fig. 4 Quantitative analysis for individual contributions of SDT and PDT (A and B), and the influences of US and laser irradiation time (C and D) on the CNE-2Z cell viability.

Encouraged by these findings and in order to validate the synergy effect, the RBNs were administered into the CNE-2Z-xenografted nude mice and a proof-of-principle in vivo therapy study was carried out (Fig. 5). All experiments were performed in accordance with the institutional guidelines and regulations made by the Administrative Panel of Wenzhou Medical University, and approved by the ethics committee at Wenzhou Medical University. Study participants were fully informed regarding the purposes of the study and consent was obtained. A 10 day treatment program was carried out. RBNs were intravenously injected, and this program was repeated 3 times followed by intra-tumoral administration till day 10 (Fig. 5A). The tumor size, variation and mice weight were measured and recorded. As shown in Fig. 5B and C, the combination therapy of RBNs + Laser + US demonstrated an overwhelming tumor inhibition tendency. It was noticeable that the efficiency of intra-tumoral administration of RBNs starting from day 6 was as good as that by intravenous administration. The NPC lesions were remarkably damaged by SDT, PDT and chemotherapy, which is attributed to RBN injection, and the tumor size became smaller and erased to a large extent (Fig. S1, ESI). The body weight did not significantly change through the 10 day treatment (Fig. 5D). With regard to other experimental groups, the tumor growth was not efficiently controlled and the tumor size constantly proliferated. Hence, the RBNs for the first time proved their full availability and prominent anti-tumor efficacy as a nano-formulation for synergy-enhanced combinational therapy by means of SDT, PDT and chemotherapy.

image file: c9cc05463d-f5.tif
Fig. 5 A proof-of-principle in vivo study on CNE-2Z tumor-xenografted nude mice for synergy-enhanced combinational therapy of SDT, PDT and chemotherapy by means of RBNs. A treatment program including a repeated intravenous injection (orange grids), a repeated intra-tumoral injection (green grids) and a final sacrifice (dark grid) (A); and variations of tumor sizes (B and C) and mice body weight (D) during the 10 day treatment (n = 4).

In addition, the nude mice were sacrificed for a routine blood examination. It was observed that most blood indexes were in the standard range of healthy nude mice, which proved the minimized side-effects and relative safety to the blood and circulation for SDT, PDT and chemotherapy. The white blood cell (WBC) values of both therapy and control groups demonstrated a slight decline, which indicated an immune activation attributed to the existence of the tumor (Fig. 6). Analysis of two cytokines (IL-6 and TNF-α) after the treatment supports the conclusion that the combination therapy of RBNs + laser + US came into effect via tumor angiogenesis inhibition, and tumor vasculature injury has been induced to the final lesion necrosis.26 Furthermore, a favorable prognosis and minimized side-effects to the blood were assured by IL-6.27 The H&E-TUNEL histological staining assay of CNE-2Z tumor tissues was also performed, and the NPC tumor and normal organs including the heart, liver, spleen, lungs, and kidneys were sliced to estimate the molecular specificity of RBNs and their potential side-effects to normal organs (Fig. 7). It was evidenced that no significant organ damage occurred, and the tumor lesion treated with RBNs + laser + US demonstrated a large-scale coagulative necrosis with a typical karyolysis (Fig. 7A). According to the TUNEL assay, RBNs + laser + US led to a large area of CNE-2Z cell apoptosis, and this combinational therapy provides both tumor specificity and RBN-mediated synergy enhancement resulting in successful tumor lesion inhibition (Fig. 7B). These advantages by means of SDT, PDT and chemotherapy using RBNs as an engineered nano-formulation have provided a promising combinational modality and potential medical translations to clinical NPC treatments in a non-invasive and precise manner.

image file: c9cc05463d-f6.tif
Fig. 6 Routine blood examination indexes of the nude mice after the treatment, and cytokines analysis of IL-6 and TNF-α indicating a favorable prognosis and a tumor angiogenesis inhibition, respectively.

image file: c9cc05463d-f7.tif
Fig. 7 The H&E-TUNEL histological staining assay of representative normal organs and CNE-2Z tumors after RBN-mediated SDT, PDT and chemotherapy (scale bar: 50 μm).

In summary, an engineered nano-formulation of self-assembled RB-loaded peptido-nanomicelles was developed for synergy-enhanced combinational therapy using SDT, PDT and chemotherapy against nasopharyngeal carcinoma. In vitro cellular tests and in vivo nude mice therapy verified RBNs as effective payloads for NPC-targeted cell killing. It was disclosed that the RBNs + laser + US therapy demonstrated an overwhelming tumor inhibition and lesion elimination capability due to the synergy-enhanced treatment. Meanwhile, tumor angiogenesis inhibition and vasculature injury with minimized side-effects to normal organs have been elucidated. Thus, the RBN-mediated combination of SDT, PDT and chemotherapy provided a promising strategy and unprecedented translational opportunities to clinical NPC treatments in a non-invasive, efficacious and precise manner.

We are grateful for the financial support from the National Natural Science Foundation of China (21575106), the Scientific Research Foundation for Returned Scholars, the Ministry of Education of China and Beijing National Laboratory for Molecular Sciences (BNLMS).

Conflicts of interest

There are no conflicts to declare.

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Electronic supplementary information (ESI) available: Experimental details on fabrication of RBNs, in vitro cellular tests and in vivo mice therapy. See DOI: 10.1039/c9cc05463d

This journal is © The Royal Society of Chemistry 2019