Theranostic Niosomes as a Promising Tool for Combined Therapy and

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Theranostic Niosomes as Promising Tool for the Combined Therapy and Diagnosis: ‘All in One Approach’ Bilal Demir, Firat Baris Barlas, Z. Pinar Gumus, Perihan Unak, and Suna Timur ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00468 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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ACS Applied Nano Materials

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Theranostic Niosomes as Promising Tool for the

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Combined Therapy and Diagnosis: ‘All in One

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Approach’

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Bilal Demir *,†, F. Baris Barlas †, Z. Pinar Gumus ‡, Perihan Unak §, Suna Timur *,†,‡

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†

‡

Ege University Faculty of Science Biochemistry Department 35100 Bornova, Izmir/Turkey

Ege University, Central Research Testing and Analysis Laboratory Research and Application

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Center, 35100 Bornova, Izmir/Turkey §

Ege University, Institute of Nuclear Sciences, 35100 Bornova, Izmir/Turkey

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*Corresponding Authors: Prof. Suna Timur ([email protected]) and Dr. Bilal Demir

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([email protected])

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KEYWORDS: Theranostics, niosomes, multimodal nanoparticles, cell imaging, targeted therapy

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ACS Paragon Plus Environment

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ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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Since the great achievement and progress made for the generation of novel nanostructures,

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theranostic nano-platforms have been the trend topic due to their intensive capability of therapy

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and diagnosis. Hence, theranostics have also been a generic strategy for the personalized

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medicine, recently. Moreover, traditional therapy modalities limit the use of chemotherapeutic

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agents for every patient and this requires more effective drug carrier systems by designing the

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formulation of drug in a specified way. Herein, we performed a generic theranostic platform in

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"all-in-one" concept by the combination of two therapy modalities with active targeting

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approach. To achieve this, 10 nm gold nanoparticles (AuNPs) and protoporphyrine IX (PpIX)

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were encapsulated into folic acid (FA) tagged niosome vesicles. Resulted AuNP-PpIX-FA

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niosomes were characterized and their particle size was 93±17 nm with a high surface charge

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and encapsulation efficiency (around 85%). In the case of bio-applications for AuNP-PpIX-FA

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niosomes, folate receptor positive (FR(+)) human cervical cancer cell line (HeLa) and FR

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negative (FR(-)) human alveolar type-II (A549) like cell line were examined with the relative

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control groups of theranostic vesicles. By testing the toxicity of vesicles, non-toxic

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concentrations were introduced to cell with the combined treatment of radiotherapy and

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photodynamic therapy, successfully. On the other hand, cellular uptake of niosomes were also

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showed its great potential for FR (+) HeLa cells as the theranostic platform with all-in-one

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approach.

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INTRODUCTION

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Synthesis of theranostic nano-structured materials such as nanoparticles (NPs) and nanovesicles

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is the promising matter of nanotechnology and gained a great attraction. Besides

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biocompatibility, theranostic nanoplatforms are needed to have various functionalities such as

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imaging, therapy and drug delivery abilities.1,2 Also, these materials can be adapted to targeting

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strategies or designed for passive processes.3

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Drug targeting approaches have been divided into categories as “Passive” and “Active”.

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“Passive targeting” is based on drug accumulation to around the tumors with leaky vasculature;

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which is referred to as the enhanced permeation and retention (EPR) effect. On the other hand,

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“Active targeting” denotes to selective ligand–receptor type interaction after nanomaterials reach

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to the target cells and requires an efficient interaction between the drug carrier and the target

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cells.4

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Niosomes are vesicular drug carriers exhibiting a bilayer structure similar with the liposomes

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and are in most cases formed by self-association of non-ionic surfactants and cholesterol in

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aqueous media. Various therapeutics with a wide range of solubility could be entrapped into the

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aqueous core or in between membrane bilayer of these structures.5,6 Therefore, these nanocarriers

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could act as multifunctional platforms which allow loading various molecules such as imaging

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and therapy agents. The efficiency of niosomal systems can be improved by active targeting

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process by using a proper ligand attached to the surface of niosomes. Recently, polyethylene

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glycolated niosomes were used to encapsulate both doxorubicin and curcumin and then,

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successfully targeted to glioblastoma cells by using tumor homing and penetrating peptide.7 In

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the other study, encapsulation of gadolinium nanoparticles and protoporphyrin IX (PpIX) into


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niosomes were carried out and these structures were applied to cancer cells via passive targeting

2

process.8

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encapsulate gold nanoparticles (AuNP) and PpIX together. These nanovesicles could be

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promising candidates for photodynamic therapy (PDT) and radiotherapy (RT) as well as

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combined therapy (PDT+RT) and cell imaging applications. All through the experiments, FR

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targeted nanocarriers were applied to FR positive and negative cell lines (HeLa and A549 cells)

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as the model cancer cells, by means of active targeting. After characterization steps, efficiency of

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targeted niosomes were tested as imaging (fluorescence) and RT, PDT and combined therapy

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(RT plus PDT) systems.

Herein, we described folate receptor (FR) targeted niosomes which are used to

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PDT has emerged as a promising alternative for the treatment of malignant diseases. PDT

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involves the administration of photosensitizers (PSs) followed by illumination of the tumor with

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a localized energy source to activate the specific PS. Due to the presence of those components,

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cells are exposed to reactive oxygen species (ROS) such as singlet oxygens, which are generated

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by the excitation of PSs to T1 state from a ground state with light. Numerous clinical trials of

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PDT have been conducted and PDT is used with increasing frequency in a variety of cancers,

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such as skin, lung, and cervix.9 Recently, PSs used in clinical purposes are mainly originated

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from porphyrins, chlorophylls, and dyes.10 Porphyrins are extensively applied for clinical uses;

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for instance, ‘Photofrin’ has been approved by FDA. It also has a good potential for

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radiosensitivity under ionizing irradiation by increasing energy uptake in cancer cells in

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comparison with healthy cells. On the other hand, RT has traditionally been one of the most

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common and efficient treatments of cancer and other diseases with ionizing radiation. RT causes

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lethal damage to disease cells by damaging the cellular DNA. Undifferentiated tumor cells are


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considered more susceptible to RT as they have a diminished ability to repair sub-lethal DNA

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damage.9,11

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In addition to the use of NPs in nanomedicine,12-15 promising demonstrations of the

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radiosensitizing potential of NPs in the last decade, both in vitro and in vivo, now mean that

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significant research efforts focus on NPs for improved dose localization for radiotherapy.

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Besides, novel sensitizers, such as NPs, have shown to locally increase the damaging effect of

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both photon and ion radiation, when both are applied to the tumor area. As NP systems, AuNP

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have become particularly popular due to several advantages such as biocompatibility, well-

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controlled methods for synthesis in a wide range of sizes, and the possibility of functionalization

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of their surface with various molecules to provide partial control of, for example, surface charge

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or interaction with serum proteins.16

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By creating an "all-in-one" approach, we engineered a novel theranostic platform with niosome

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vesicles as the main nanocarrier materials. RT effect of AuNPs and PDT effect of

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protoporphyrine IX (PpIX) under proper irradiation conditions were utilized after their

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encapsulation into folic acid tagged niosome vesicles. The following characterization related to

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physcochemical parameters such as size and surface charge and morphology, bio-investigations

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of the final AuNP-PpIX-FA theranostically engineered particles were examined with the folate

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receptor positive (FR(+)) human cervical cancer cell line (HeLa) and FR negative (FR(-)) human

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alveolar type-II (A549) like cell line. Within RT and PDT, combinatorial therapy modality

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studies and cellular uptake of AuNP-PpIX-FA vesicles were tested in the comparison of their

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relative control groups, accordingly.

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2. EXPERIMENTAL SECTION

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Materials. Tween 80, folic acid (FA), 1,1'-Carbonyldiimidazole (CDI), cholesterol (Chol),

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protoporhyrin IX (PpIX), dimethylsulfoxide (DMSO), chloroform, methanol, sodium dodecyl

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sulfate (SDS) and 3-(4,5-dimethylthiazolyl-2)- 2,5-diphenyltetrazolium bromide (MTT) and 4',6-

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diamino-2-phenylindol (DAPI) were purchased from Sigma Aldrich (St. Louis, USA). 10 nm

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citrate-stabilized gold nanoparticles (AuNPs) were purchased from BBI Solutions (Cardiff, UK).

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Penicillin/streptomycin (10 000 UI/mL), Dulbecco's Modified Eagle's Medium (DMEM), L-

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glutamine (200 mM), phosphate buffered saline (PBS) and trypsin/ethylenediaminetetraacetic

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acid (EDTA) (0.05% trypsin in 0.2 g/L EDTA) and phoshate buffer saline (PBS) used in cell

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culture experiments were purchased from Lonza (Basel, Switzerland). Fetal bovine serum (FBS)

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was purchased from Biowest (Nuaillé, France). Phosphate buffer saline (PBS) was prepared with

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8.0 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4 and 0.24 g/L KH2PO4 at pH 7.4 for synthesis and

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characterization studies. Water was purified using a Milli-Q system (Millipore, Molsheim,

14

France).

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Synthesis of Tween 80-FA conjugate. Prior to the preparation of niosomes, Tween 80-FA

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conjugate was synthesized according to a previous protocol by Chen et al.17 Briefly, 1.66 g

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Tween 80 and 0.66 g CDI were dissolved in 5.0 mL DMSO and incubated for 2 h at 40 oC with

18

stirring. Diethylether was added onto the Tween 80:CDI mixture thoroughly to separate the

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excess CDI from the activated Tween 80. After separation by using the funnel, activated Tween

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80 was kept in oven (55 oC) for 2 h to concentrate the intermediate. Tween 80 which has

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activated -OH groups was introduced into 30 mL Na-Carbonate buffer (10 mM, pH 9.0)

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containing 0.36 g FA for 4 h at room temperature under shaking. Following the reaction, dialysis

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(MW: 1000 Da) was applied to purify the final Tween 80-FA conjugate. In the final step, water


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soluble conjugate was lyophilized for 3 days with a benchtop freeze dryer (Labconco, Missouri,

2

USA) and kept at 4 oC for the further characterization and use in preparation of theranostic

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niosomes. The synthesis of the conjugate is demonstrated in Scheme 1.

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Scheme 1. Synthesis of Tween 80-FA conjugate with carbonyldiimidazole (CDI) activation of -

6

OH groups.

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Preparation of theranostic niosomes. Theranostically designed niosome formulations were

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prepared via traditional thin film hydration method followed by a sonication step. Initially, 0.1

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mM Tween 80: Chol (1:1 molar ratio) were dissolved in chloroform: methanol (3:1 v/v) mixture

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in a 50 mL round-bottom flask for the further rotary evaporation to obtain a surfactant film by a

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Buchi-RII Rotavapor model evaporator (BUCHI Labortechnik AG, Flawil, Switzerland)

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equipped with a vacuum pump. Surfactant/Chol film was hydrated with 10 mL PBS (pH 7.4)

13

which contains 500 µL, 10 nm AuNP solution (taken directly from commercial stock) and 100

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µM PPIX. The addition of Tween 80-FA conjugate solution (which was dissolved in DMSO:

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PBS (1:9) for 0.0387 mg/mL as the final concentration in niosome solution) into niosome

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bilayers was applied via insertion during hydration process. Following the 2 h of incubation at 50

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o

C, latter vigorous vortexing was applied until the complete detaching of film layer from the


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flask surface. In order to get small unilamellar vesicle distribution, niosome suspension was

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sonicated for 30 min at room temperature. In the final step, the sonicated AuNP-PpIX-FA

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niosomes were dialyzed against distilled water overnight by a dialysis membrane (MW: 12000 –

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14 000 Da).

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To create control groups for cellular bio-applications which can demonstrate the strategic

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differences, AuNP-PpIX and PpIX-FA containing niosomes were prepared for the comparison,

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separately. The constructed niosomal samples were stored at +4 oC by protecting from light.

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Characterization. To illuminate the conjugation of Tween80-FA after lyophilization, Fourier

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transform infrared (FTIR) spectra of Tween 80-FA was obtained by using a Pyris 1 FTIR

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Spectrometer (Perkin–Elmer Instruments, Massachusettes, USA) on KBr plates. Moreover, high

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performance liquid chromatography (HPLC) was applied to the sample to verify the conjugation

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efficiency of FA to CDI activated Tween80. The chromatographic analysis of FA was conducted

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by using HPLC (Agilent) with DAD detector (Santa Clara, CA, USA) and the elution of the

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peaks in the chromatogram was performed with an Eclipse XDB-C18 column (5.0 µm particle

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size, 4.6x150 mm). For the FA analysis, the following procedure was applied. The mobile phase

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consisted of a mixture of (A) 1.0 % (v/v) aqueous phosphoric acid and (B) acetonitrile (90:10,

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v/v). Flow rate was adjusted to 1.2 mL/min and the detector wavelength was set at 283 nm. The

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injection volume was 20 µL and the column temperature was maintained at 25 °C. The stock

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solutions of FA (10-500 ppb) were prepared in methanol and conjugation efficiency of Tween80-

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FA was determined via the obtained calibration curve from HPLC with the equation of y =

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0.032x + 0.083 (R2 = 0.999). Limit of detection and limit of quantitation values are 1.58 ng/mL

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and 5.28 ng/mL, respectively. Similar conditions were also carried out to exhibit the Tween80-

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FA insertion efficiency into niosomes.


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After the synthesis of theranostically designed AuNP-PpIX-FA and control formulations, initial

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step for the first characterization related to those theranostic vesicles was to determine the

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hydrodynamic particle size distributions and surface charges. Prior to the measurements of

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surface and hydrodynamic characteristics, each niosome sample was diluted as 20 times in water.

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The measurements were carried out as via a Zetasizer Nano ZS (Malvern Instruments Ltd., U.K.)

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at a scattering angle of 90o using a wavelength of 633 nm and at room temperature. Zeta

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potential analysis was performed by the same device according to Smoluchowski equation.

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Surface charge and size measurements were repeated as three times with the samples prepared in

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different days.

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Encapsulation efficiency (EE) of developed niosomes was also calculated by using PpIX as the

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model photosensitizer in this work. For that, freshly dialyzed niosome stocks were used. The

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decomposition of vesicle bilayer was performed by vortexing and sonication for 10 min in

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methanol. The prepared standard solutions of PpIX in PBS with varying concentrations between

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1.0-200 µM were used for the calibration curve of PpIX. Afterwards, decomposed PpIX

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niosomes were diluted with PBS and EE% of each niosome formulation was calculated

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according the following equation:

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Entrapment efficiency (EE%) = (Encapsulated PpIX Amount/Initial PpIX Amount) x 100

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Inductively coupled plasma mass spectrometer (ICP-MS; 7500ce octopole reaction system,

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Agilent, California, USA) experimentation was carried out to detect total Au concentration after

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the decomposition of niosomes as well. The linear calibration curve for Au concentration is

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between 0-1000 ppm.


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To investigate the morphological structures of developed surfactant vesicles, atomic force

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microscopy (AFM) was introduced. Prior to the sample deposition over indium tin oxide (ITO)

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glasses, a cleaning procedure was performed with sonication successively for 15 min, in

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detergent solution, de-ionized water, acetone and 2-propanol, sequentially. The measurements

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were carried out under ambient conditions by using an NT-MDT NTEGRA SOLARIS. To obtain

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the topographic images, the non-contact mode (tapping mode) was selected. A 10 mm scanner

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equipped with silicon tips with 10 nm tip curvature was used for measurements. After drying in

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nitrogen stream, niosome solutions (diluted as 10 times in water) were immediately spin coated

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on the ITO substrates at 20 oC and then directly measured via AFM.

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Transmission electron microscopy (TEM) was performed to visualize the AuNPs inside vesicles.

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Firstly, main stock of AuNP-PpIX-FA sample was diluted 50 times (20 µL sample + 1980 µL

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water) and dispersed for 5 min in a bath sonicator. 20 µL of this dilution was covered onto grid

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and after drying process, images were captured by a JEM-2100F (JEOL, Japan).

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Cellular Investigation of Theranostic Niosomes

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Human cervical cancer cell line (HeLa) and human alveolar type-II (ATII)-like cell line (A549)

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were sub-cultured in DMEM supplemented with 10% (v/v) FBS, 2.0 mM glutamine, 100 µg/mL

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penicillin/streptomycin, in 75 cm2 flasks at 37 °C, 5.0% CO2 and 100% humidity, until reaching

18

80% confluency. 0.25% (w/v) trypsin/EDTA in PBS was used for the cell passage as two times

19

per week to maintain the cultivation during cell culture studies.

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Cell viability. A conventional MTT set up was established to investigate the cell viability

21

responses of developed vesicles in a dose-dependent way by using the standard protocol in our

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previous studies.18,19 In brief, 8.0x103 cells were inoculated into 96 well-plate and incubated for


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48 h in standard cell culture conditions. Subsequently, the culture medium was replaced with the

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treatment medium including different concentrations of PpIX containing niosomes for 24 h

3

incubation. In the end of incubation with samples, cells were treated with 110 µL/ well MTT

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solution (10%, 5.0 mg/mL in sterilized PBS, pH 7.4) in medium for 4 h. Following the MTT

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treatment, 100 µL SDS (1.0 g SDS in 10 mL of 0.01 M HCl) was then added to the wells to

6

dissolve the purple-colored formazan crystals which were produced in cells. In the final step of

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24 h incubation, the optical densities of each well were analyzed with a spectrophotometric plate

8

reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) at 570 nm and 630 nm as well. As

9

designed in characterization part, same control samples including AuNP-PpIX and PpIX-FA

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niosomes were applied to the cells for the comparison.

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Radiotherapy (RT). To exhibit the possible therapeutic efficiency of AuNP-PpIX-FA niosomes,

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samples were treated with cells under irradiation. For that, we carried out an established protocol

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from our previous study related to AuNP conjugates for cancer therapy.20 Briefly, 4.0x103 cells

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were seeded to 96-well plates and incubated overnight. Then, medium was replaced with the

15

maximum nontoxic concentrations of samples after 3 times washing with PBS. Afterwards, cells

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were irradiated for 2 h with 2.5 Gray (Gy) of radiation which was delivered by a 6 MV linear

17

accelerator system (LINAC, Siemens Primus, Germany). As the next step, cells were incubated

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for 72 h in ideal cell culture conditions (37 oC, 5.0% CO2 with 100 % humidity) and the standard

19

MTT assay was conducted to observe the cytotoxic effect as described in Cell Viability part.

20

Photodynamic therapy (PDT). In PDT experiment, a previous protocol by Morimoto and co-

21

workers was carried out with a small re-modification.21 Briefly, 1.5x104 cells were seeded in 24-

22

well plates and cultivated for 48 under ideal cell culture conditions. Afterwards, HeLa and A549

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cells were pre-treated with the AuNP-PpIX-FA, AuNP-PpIX and PpIX-FA samples, accordingly.


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Following 2 h, a homemade LED lamp (5.1 J/cm2) was used to deliver light for 5 min exposure

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between 400 and 700 nm wavelength ranges. Cell viability was measured by MTT method after

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24 h incubation at the ideal conditions as described above.

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Combined therapy assay. This novel assay which was early optimized in our lab was carried

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out to investigate the combined therapy effect of PDT (5 min light with 5.1 J/cm2) and RT (2.5

6

Gy) on the cell viability.8 Within this combination, a similar treatment procedure was also

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conducted for AuNP-PpIX-FA and the control groups at their maximum nontoxic concentrations.

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In short, PDT for 5 min with 5.1 J/cm2 and a subsequent 2 h irradiation with 2.5 Gy by LINAC

9

system were applied to cells and after 72 h cell viabilities were detected by MTT method as well.

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Cell imaging. In order to monitor the intralocalizations of FR targeted AuNP-PpIX-FA

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theranostic vesicles and also control groups HeLa and A549 cells, 100 µL of maximum nontoxic

12

concentration of samples were introduced into the cells grown in a chamber slide for 2 h after 2

13

day-cultivation under ideal cell culture conditions. Following the treatment for 2 h at 37 °C in

14

CO2 incubator, the cells were washed twice with PBS. The cell images were taken by a

15

fluorescence microscope (Olympus BX53F) equipped with a CCD camera (Olympus DP72).

16

Cell images were overlapped with the red fluorescence from PpIX and blue fluorescence form

17

DAPI in order to monitor nucleus via Image J software.

18

Statistical analysis. All the experiments placed in this study were repeated at least 3 times and

19

data were expressed as average ±SD (standard deviation) unless particularly outlined. A one-way

20

analysis of variance (ANOVA) was conducted with a post-test of Tukey's multiple comparison

21

test for the statistical evaluation. The difference between two groups was considered to be


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significant when the ‘P’ value was less than 0.05 and highly significant when the ‘P’ value was

2

less than 0.01 or 0.001.

3

3. RESULTS AND DISCUSSION

4

The increasing trend to find novel and more effective therapeutic approaches in the war of cancer

5

revealed many opportunities resulting in required multidisciplinary way. To keep up with this

6

trend, researchers have been studied on multifunctional nanocarriers which enable multimodal

7

therapy and diagnosis as well or their combinations. In this work, we mainly focused on the

8

design and bio-application of a novel FR targeted niosomal formulation. To create a more

9

effective platform in one carrier, theranostic engineering was conducted to niosome vesicles by

10

including PpIX and AuNPs to the structures as PDT agent and RT agent, respectively.

11

Concomitantly, niosomes as the surfactant vesicles have gained great attraction as a nanocarrier

12

platform thanks to their advantageous properties over lipid-based vesicles. Although it is known

13

that liposomes are more easy-to-modify and open for post-functionalization to create specificity,

14

these vesicles are more fragile due to rapid oxidation under atmosphere and light for long

15

durations. In order to overcome these drawbacks, niosomes were prepared as FR targeted with

16

the addition of FA conjugated Tween80.

17

Synthesis of Tween 80-FA conjugate. Prior to the preparation of niosomes, FA conjugation was

18

enabled to Tween80 thanks to its structure bearing pendant -OH groups via CDI coupling. After

19

the activation of each -OH group at Tween80, -NH2 group of each FA was successfully

20

conjugated and purified as mentioned in experimental part. In order to analyze the conjugation

21

efficiency, HPLC analysis was accomplished with the standard curve and equation for FA in ppb

22

level accuracy. During the synthesis, 0.36 g, FA in 30 mL Na-carbonate buffer was used for

23

conjugation (12000 ppm FA as the initial concentration). After purification, it was found that 237


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Page 14 of 31

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ppm FA couldn't bind to Tween 80. Therefore, 98% of FA could bind to Tween80, successfully.

2

Within that result we can admit that (2-3) FA molecules might be linked to each Tween80

3

molecule. Moreover, this high conjugation efficiency was supported by FTIR analysis as well.

4

As illustrated in Figure 1, Tween80-FA was compared with plain Tween80 and FA to observe

5

the structural changes via specific peaks which have exhibited in FTIR spectra.

6 7

Figure 1. FTIR spectra of Tween80, FA and Tween80-FA conjugate.

8

FA is formed from a pteridine ring, p-amino benzoic acid and glutamic acid. In the FTIR

9

spectrum of free FA, the most common characteristic peaks at 1610 and 1700 cm-1 were

10

generally attributed to -COOH, C=O and aromatic C=C residues on pteridine and phenyl rings,

11

vividly.22 There are some characteristic absorption bands which could confirm the presence of

12

Tween80 in the conjugation. The bands centered at 2932 and 2873 cm-1 are associated with the

13

asymmetric (vas) and symmetric (vs) stretching vibrations of methylene (-CH2), respectively.23


14

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1

The band at 1730 cm-1 originates from the C=O stretching of the ester group. The strong band

2

between 3400-3500 cm-1 can be attributed to the O-H stretching vibrations as the most common

3

peak for primary alcohol groups. In the spectrum of Tween80-FA conjugation, the characteristic

4

peaks from both structures can be clearly observed due to the similar peak absorptions around

5

3450 cm-1 for FA and 3448 cm-1 for conjugate which are related to N-H stretching beside the

6

presence of -CH2 in conjugate. Moreover, the peak absorption which also presents the

7

conjugation efficiency might be attributed to C-N (amide bond). As a result, it is clearly

8

demonstrated that the first step to construct the FR targeted theranostic platforms was

9

successfully completed for the rest of the study.

10

Design of theranostic niosomes and characterization. In the concept of theranostic particles,

11

niosomes which carry both PpIX and AuNPs were synthesized with the FA tagging over the

12

surface of bilayer nonionic surfactant membrane. Following the synthesis, hydrodynamic particle

13

sizes of those multifunctional carriers were estimated via DLS method. The final theranostic

14

vesicle which contains both PpIX and AuNPs with the FA tagging presents a size of 93±17 nm

15

as show in Table 1. Likewise, control vesicles which prepared for the further bio-investigations

16

enabled similar particle sizes after sonication to reduce particle size and to achieve unilamellar

17

vesicle formation. Besides, AuNPs were also measured within other samples and their size was

18

verified as 8.0±2.0 nm as enabled in the manufacturer's instructions.

19

polydispersity index (PDI) illustrates the homogeneity of particles in measurement solution.

20

According to the obtained results, final theranostic particles AuNP-PpIX-FA have the smaller

21

PDI value among three niosome formulations. In the case of zeta potential of developed

22

niosomes, all of the formulations demonstrated similar characteristics as -46 and -50 mV which

23

indicates all the formulations are stabile.

Concomitantly,


15


1

Page 16 of 31

Table 1. Psychochemical characteristics of developed niosome vesicles. Particle size PDI±S.D. (nm) ±S.D.a

a

Zeta

potential

(mV) ±S.D.a

Encapsulation Efficiency

PpIX (%)±S.D.a

AuNP

8±2

0.14±0.01

-42±7

AuNP-PpIX

69±10

0.64±0.02

-48.5±8

84.16±1.08

PpIX-FA

70±13

0.54±0.1

-46±8

86.31±0.96

AuNP-PpIX-FA

93±17

0.41±0.04

-50±9

86.57±4.26

a

of

±SD values obtained from free-independent measurements of freshly prepared samples.

2

Moreover, those three nanovesicles were also tested for their 30 days short-term stability via

3

particle size monitoring. After 30 days, the particles sizes of niosomes were found as 71 nm for

4

AuNP-PpIX, 77 nm for PpIX-FA and 89 nm for AuNP-PpIX-FA nanovesicles, respectively.

5

This data is also in accordance with the high negative surface charges which prove the high

6

stability for one month duration.

7

As the next step for the characterization, encapsulation efficiencies (EE) of PpIX and AuNP

8

particles into niosome vesicles were investigated. EE% of PpIX was determined by

9

spectrophotometric method resulting a standard curve with linearity between 1.0- 200 µM PpIX

10

in PBS. To calculate the EE%, the equation of y = 0.028x + 0.102 (R² = 0.997) was used. Three

11

formulations which contain PpIX have ̴ 85% PpIX amount inside the total niosomes for each

12

formulation (Table 1). This obtained result shows the efficiency of the formulation with

13

Tween80 and Chol, vividly by giving a high-rate encapsulation for drug molecules like PpIX. On

14

the other hand, the presence of AuNPs was investigated for the formulations of AuNP-PpIX and


16

Page 17 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

AuNP-PpIX-FA. In order to estimate the total Au concentration, ICP-MS system was used and

2

results were expressed as mg/L. According to that, 8.1 mg/L (36% encapsulation) for AuNP-

3

PpIX-FA and 25.3 mg/L (87% encapsulation) for AuNP-PpIX were found. The dramatic

4

difference for the Au concentration inside the vesicles might reveal from the fact that AuNP-

5

PpIX vesicles have more polydispersity than final theranostic particles, thus bigger particles

6

could contain more AuNPs. In addition, the FA presence in the bilayer membrane might create

7

an electrostatic repulsion between free -COOH groups of FA and citrate coated AuNPs by

8

resulting less AuNP entrance to the vesicles.

9

Within the successful EE of both PpIX and AuNPs, another important parameter for the FA

10

tagged vesicles is the insertion efficiency of Tween 80-FA conjugates. This was also calculated

11

by HPLC method and insertion efficiencies were found as 97.54% for PpIX-FA and 98.27% for

12

AuNP-PpIX-FA vesicles which strongly supports our theory of lesser AuNP amount in AuNP-

13

PpIX-FA vesicles.

14

Following the physicochemical and encapsulation characteristics, AFM technique was applied in

15

order to monitor the morphology of niosome vesicles by spin-coating the samples on a ITO glass

16

slide. Figure 2 demonstrates the morphology of three vesicle formulations as well as their

17

dispersity over ITO slide by height images. The comparison of three images reveals the spherical

18

shapes of each vesicle sample, accordingly. In addition to AFM, transmission electron

19

microscopy (TEM) was used to monitor the vesicle structure and presence of AuNPs. Figure S1

20

illustrates the one vesicle of AuNP-PpIX-FA sample with a particle size of 88 nm which is very

21

close to the DLS result. Moreover, the black intensity inside the vesicles is the proof of AuNPs

22

as the normal visualization of heavy metals under TEM.


17


Page 18 of 31

1

2 3

Figure 2. AFM histograms (2.5x2.5) of (A) AuNP-PpIX, (B) PpIX-FA and (C) AuNP-PpIX-FA

4

vesicles.

5

Cell Culture Studies

6

Cell viability. Cytotoxicity of theranostic vesicles on FR(+) HeLa and FR(-) A549 cell lines was

7

evaluated via conventional MTT assay. For the comparison, the cytotoxicity of AuNP-PpIX and

8

PpIX-FA was also evaluated. Cells were exposed to a wide concentration range of test samples

9

for 24 h. The obtained MTT data in Figure 3 clearly indicates that cellular viabilities for both

10

HeLa and A549 cells decreased after 5.0 µM PpIX bearing niosome vesicles to 60% viability

11

levels. Within the comparison of control groups and AuNP-PpIX-FA vesicles, encapsulated


18

Page 19 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

PpIX created an effect upon both cell lines while it has similar effect on some cancer types. In a

2

reported study, free PpIX treatment upon HeLa cells without light irradiation showed its efficacy

3

after 9.0 µM whereas synthesized AuNPs (7.0 nm) also revealed a slight decrease in viability

4

until 80% compared to control.24 In the case of our findings from MTT assay without any

5

treatment, quasi results were obtained after encapsulation of them, while PpIX-FA have not been

6

effective at its last concentration. The reason which handicaps this might not be only from

7

AuNPs and it might be occurred due to the niosomes' main material, Tween80. According to

8

another study with Tween80 niosomes, it was indicated that Tween80 could create toxicity after

9

19 µM whereas maximum Tween80 amount for the highest concentration in our results is 58

10

µM.25 Hence, the initial reduction point (10 µM PpIX) of cellular viability which contain also ̴15

11

µM Tween80 is in accordance with the previous study. Likewise, the effect of carrier material

12

presents similar reductions for the final theranostic vesicle formulation. Another important issue

13

is that cell viability of A549 cells seems more decreased according to HeLa cells in Figure 3C

14

after the treatment of AuNP-PpIX-FA. Concomitantly, statistical analysis also showed that there

15

is a significance for the last two concentrations (25 and 50 µM PpIX containing niosomes). On

16

the other hand, the difference between AuNP-PpIX and AuNP-PpIX-FA analyzed via one-way

17

ANOVA method and there were no significant difference between those groups. As

18

aforementioned, it might be due to the Tween 80 and also the possible accumulation of high

19

concentrated samples over cells for 2D cell culture platforms which can generate stress

20

conditions for cells. Notably, in the general aim of the study was to investigate the combined

21

therapeutically effect of those formulations by using their non-toxic levels without any light

22

treatment or irradiation. Therefore, 5.0 µM PpIX containing AuNP-PpIX-FA vesicle

23

concentration were introduced to the cells with its control samples for the rest of the study.


19


Page 20 of 31

1 2

Figure 3. Dose-dependent toxicity of (A) AuNP-PpIX, (B) PpIX-FA and (C) AuNP-PpIX-FA

3

vesicles for HeLa and A549 cells. X axis means the concentration of PpIX inside vesicles. Error

4

bars mean ± standard deviation (n=4).

5

Combined therapy of developed niosomes. The challenges during treatment of patients may

6

occur with only chemotherapy or combined modalities with different drugs in variable doses.

7

The advancing understanding of cancer biology has led us to the development of molecularly

8

targeted anticancer drugs. In the final step of our work, theranostically designed AuNP-PpIX-

9

FA vesicles was introduced to the FR(+) HeLa and FR(-) A549 cell lines to generate a novel

10

candidate in the war of cancer specifically. Since higher amounts of drugs/formulations may

11

reveal extra side-effects, it is preferable to combine different modalities with the strategy of


20

Page 21 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

"all-in-one" concept. Figure 4 illustrates this "all-in-one" approach by using PDT and RT

2

together thanks to the unique properties of encapsulant materials in niosome vesicles. After

3

every sample with 5.0 µM PpIX checked without any treatment for both cell lines to keep cell

4

viability as 100%, therapy modalities were conducted to cell in the presence of theranostically

5

engineered AuNP-PpIX-FA and control vesicles, accordingly. For that, we used the 2.5 Gy

6

irradiation with 5 min light photo-irradiation as the RT and PDT treatments as optimized in our

7

previous paper.8 As expected from RT results, the presence of both AuNPs and PpIX illustrates

8

encapsulant materials' RT activities by decreasing the viability under 50%, while control group

9

without any samples remains their high viability around 75%. Concomitantly, the another

10

important issue is AuNP-PpIX and AuNP-PpIX-FA do not contain same AuNP amount,

11

however, it seems FA tagging might enable an important difference by active targeting for

12

FR(+) HeLa cells in the comparison of cellular viabilities (p

Theranostic Niosomes as a Promising Tool for Combined Therapy and

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