Paclitaxel-loaded self-assembled lipid nanoparticles as targeted drug

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Biological and Medical Applications of Materials and Interfaces

Paclitaxel-loaded self-assembled lipid nanoparticles as targeted drug delivery systems for the treatment of aggressive ovarian cancer Jiali Zhai, Rodney Luwor, Nuzhat Ahmed, Ruth Escalona, Fiona H Tan, Celesta Fong, Julian Ratcliffe, Judith A. Scoble, Calum J. Drummond, and Nhiem Tran ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08125 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 2, 2018

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ACS Applied Materials & Interfaces

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Paclitaxel-loaded self-assembled lipid nanoparticles as targeted drug delivery systems for the treatment of aggressive ovarian cancer Jiali Zhai1, Rodney B. Luwor2, Nuzhat Ahmed3, 4, 5, 6, Ruth Escalona3, 5, 6, Fiona H. Tan1, 2, Celesta Fong1,7, Julian Ratcliffe7, Judith A. Scoble8, Calum J. Drummond1, Nhiem Tran1, *

1

School of Science, College of Science, Engineering and Health, RMIT University,

Melbourne, VIC 3000, Australia 2

Department of Surgery, Royal Melbourne Hospital, University of Melbourne, Melbourne,

VIC 3052, Australia 3

Fiona Elsey Cancer Research Institute, Ballarat, VIC 3353, Australia

4

Federation University Australia, Ballarat, VIC 3010, Australia

5

The Hudson Institute of Medical Research, Clayton, VIC 3168, Australia

6

Department of Obstetrics and Gynaecology, University of Melbourne, Parkville, VIC 3052,

Australia 7

CSIRO Manufacturing, Clayton, VIC 3168, Australia

8

CSIRO Manufacturing, 343 Royal Parade, Parkville, Victoria 3052, Australia

*Corresponding author: Dr Nhiem Tran School of Science, RMIT University GPO Box 2476, Melbourne, Vic 3000, Australia Ph: +61 3 9925 2131 Email: [email protected] Keywords: lipid nanoparticles; ovarian cancer; paclitaxel; drug delivery; EGFR

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Abstract Chemotherapy using cytotoxic agents such as paclitaxel (PTX) is one of the most effective treatments for advanced ovarian cancer. However due to non-specific targeting of the drug and the presence of toxic solvents required for dissolving PTX prior to injection, there are several serious side effects associated with this treatment. In this study, we explored selfassembled lipid based nanoparticles as PTX carriers, which were able to improve its antitumour efficacy against ovarian cancer. The nanoparticles were also functionalised with antiEGFR fragments to explore the benefit of tumour active targeting. The formulated bicontinuous cubic and sponge phase nanoparticles, which were stabilised by Pluronic F127 and a lipid-PEG stabiliser, showed a high capacity of PTX loading. These PTX loaded nanoparticles also showed significantly higher cytotoxicity than a free drug formulation against HEY ovarian cancer cell lines in vitro. More importantly, the nanoparticle-based PTX treatments, with or without EGFR targeting, reduced the tumour burden by 50% when compared to PTX or non-drug control in an ovarian cancer mouse xenograft model. In addition, the PTX loaded nanoparticles were able to extend the survival of the treatment groups by up to 10 days compared to groups receiving free PTX or non-drug control. This proof of concept study has demonstrated the potential of these self-assembled lipid nanomaterials as effective drug delivery nano-carriers for poorly soluble chemotherapeutics such as PTX.


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1. Introduction Nearly 200,000 women die annually world-wide from ovarian cancer 1. The high mortality rate in ovarian cancer patients results from the diagnosis of disease at an advanced stage when the cancer has already spread into the peritoneal cavity. The gold standard treatment of ovarian cancer patients after debulking surgery is the systemic administration of maximum tolerated doses (MTD) of taxane based (paclitaxel (PTX)) or platinum based (cisplatin or carboplatin) drugs 2. Although, 80% of ovarian cancer patients respond well to the standard treatment, almost all suffer from severe side effects of the MTD regimen of PTX. Patients surviving chemotherapy treatment enjoy a short-lived period of remission with asymptomatic minimal disease. However, the microscopic residual disease persisting after the first line chemotherapy usually gives rise to consecutive episodes of recurrent tumour and eventual death. Disappointingly, this results in a five year survival period of as low as ~40%, which has remained unchanged for the last thirty years 3. In addition, many side effects caused by the drugs and solvents that are required to dissolve these poorly-soluble drugs prior to treatment, cause further burden to the patients. For example, commercially available formulations of PTX contain solubilising agents such as Cremophore EL (polyethoxylated castor oil) and dehydrated ethanol in Taxol and polysorbate 80 (Tween 80) in Taxotere

4-5

.

These solubilising agents are clinically associated with serious side effects such as hypersensitivity, nephrotoxicity and neurotoxicity

6-8

. Thus, there is an urgent need to

develop methods that will allow delivery of specifically targeted chemotherapeutic agents to ovarian cancer cells to achieve long-term disease-free survival without inducing cytotoxic effects on normal tissues. Nanomedicine has emerged as the next-generation treatment for cancer, offering unique drug delivery properties to eliminate or reduce the generic toxicity of chemotherapeutics via selective targeting and controlled release 9. A vast number of nanoparticle-based platforms


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using materials including lipids, polymers, metals, and proteins have been investigated with more than a dozen of these nanomedicine-based anti-cancer therapeutics currently in clinical use 10-11. Among many classes of nanoparticle-based drug carriers, lyotropic liquid crystalline lipid nanoparticles (LCNPs) stand out due to their ability to encapsulate a wide range of hydrophilic and hydrophobic drugs12. They also require a simple formulation process and they are biocompatible because of the main components being food grade lipids

13-14

. These

lipid nanoparticles are formed by a self-assembly process, creating internally ordered, nanostructured mesophases such as the inverse bicontinuous cubic phase and the inverse hexagonal phase in an excess water environment 15-18. With the aid of steric stabilisers, these bulk mesophases can be dispersed into stable nanoparticles called cubosomes (CB) or hexosomes that contain the internal cubic phase or the hexagonal phase, respectively

19-22

.

Furthermore, their internal nanostructure can be fine-tuned by additives that enable control of the drug diffusion rate, size selectivity and stimuli responsiveness of the nanoparticles 23-25. In the context of active tumour targeting, it has been demonstrated that epidermal growth factor receptor (EGFR) antibody fragment can be functionalised on CB and hexosomes through thiol-maleimide bioconjugation 26. Since a number of ovarian cancer cell types express a high level of EGFR 27, this surface modification technique may help in enhancing the interaction of nanoparticles with ovarian cancer cells in close proximity, leading to higher cellular uptake of drugs and cell death. Despite a vast number of in vitro studies demonstrating the functional advantage of non-lamellar lipid nanoparticles as drug delivery vehicles

28-33

number of studies examining their efficacy and safety in vivo

, there has been a limited

28, 34-38

. These in vivo studies,

however, varied widely regarding the nanoparticle composition, the type of cancer, the administration route, and the location of the tumour. For example, Cervin et al. were able to load docetaxel (DOX) at 5 wt% (to lipids) in a phosphatidyl choline and diolein-based


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formulation

5

34

. They showed that in a prostate cancer mouse model, the nanoparticle-based

treatment delivered intravenously resulted in a much lower tumour volume compared to the control group, which was treated with Taxotere. In another study, PTX was formulated into monoolein hexosomes at 1 wt% (to lipids) in the presence of ethanol and the nanoparticles were examined in a mouse model bearing Ehrlich Ascites tumours.35 A smaller of the tumour size of 500 mm3 was found in mice treated with nanoparticles, compared to a tumour size of 1500 mm3 in the free PTX control group and of 2400 mm3 in the untreated control group. In the current study, we examined the efficacy of CB as targeted delivery vehicles for PTX in an aggressive ovarian cancer mouse model. These unique CB were made from food grade lipid monoolein (MO) and were stabilised by both Pluronic F127 triblock copolymers and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEGMw=3400-maleimide (DSPE-PEGmal) (Figure 1). MO is a non-toxic, biocompatible and biodegradable lipid commercially used as a food emulsifier

39-40

. The addition of DSPE-PEG-mal polymers also allowed for

further surface functionalisation with EGFR antibody fragment. A previously established HEY cells-derived ovarian cancer xenograft model was used to study the efficacy of the nanoparticle based PTX treatment and to determine whether EGFR targeting provided additional advantages

41-42

. This study contributes significantly to the development of

nanoparticle-based drug delivery systems, particularly ones that deliver poorly soluble chemotherapeutic drugs such as PTX for treating aggressive ovarian cancer.



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Figure 1: Chemical structures of MO, DSPE-PEG-Mal, PTX, and Pluronic F127 (A) and the schematic illustration of a cubic phase lipid nanoparticles and the molecular arrangement within its lipid bilayer (B).

2. Materials and Methods 2.1 Materials MO was purchased from Nu-check-Prep, Inc (Elysian, MN, USA). DSPE-PEG-mal was purchased from Nanocs, Inc (New York, NY, USA). Pluronic F127, phosphate buffer saline (PBS, pH 7.4), ethanol, chloroform, and Tween 80 was purchased from Sigma-Aldrich (St Louis, MO, USA). PTX was purchased from Melone Pharmaceutical Co., Ltd (Dalian, Shandong, China). EGFR 528 monoclonal antibody was a gift from the CSIRO Protein Production Facility (Parkville, Vic, Australia). Milli-Q H2O (18.2 MΩ) was used for all aqueous preparations. All compounds were used without further purification. 2.2 Preparation of nanoparticle formulations


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The name and composition of the MO-based nanoparticles used in this study are shown in Table 1. MO and PTX stock solutions were prepared in ethanol. DSPE-PEG-mal stock solution was prepared in chloroform. Desired amounts of MO, PTX, and DSPE-PEG-mal were then mixed in a vial and organic solvents were removed under vacuum at 40°C overnight. Pluronic F127 stock solution was prepared in PBS pH 7.4 and desired amounts of F127 was added to the dried lipid and drug mixture. Nanoparticle was made by sonication of the mixture using a probe sonicator (Qsonica, Newtown, CT, USA.), at a frequency of 20 kHz, with a 5 s-on, 5 s-off mode for a total sonication time of 3 min. Table 1: Name and composition of MO-based nanoparticles.

Sample name

MO Pluronic F127 DSPE-PEG-mal PTX EGFR (mg) (mg) (mg) (mg) (µg)

Final Volume in PBS (mL)

CB

10

1

1.6

-

-

0.5

PTX-CB

10

1

1.6

1

-

0.5

EGFR-PTX-CB

10

1

1.6

1

55

0.5

After the PTX-loaded nanoparticle dispersions were made, EGFR 528 antibody binding fragment (Fab’) was conjugated to the particle surface according to a method by Zhai et al. 26 Briefly, freshly reduced EGFR Fab’ exposing free thiol groups was incubated with the maleimide-functionalised CB at room temperature overnight. The conjugation was enabled by a thiol-maleimide reaction and confirmed by SDS-PAGE gel electrophoresis as previously described.[1] The three nanoparticle formulations prepared were named CB, PTX-CB, and EGFRPTX-CB. Their compositions are given in Table 1. 2.3 Cryogenic transmission electron microscopy


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Cryogenic transmission electron microscope (cryo-TEM) was used to visualise the nanoparticles. Copper grids (200-mesh) coated with perforated carbon film (Lacey carbon film, ProSci Tech, Australia) were glow discharged in nitrogen to render them hydrophilic and placed in a laboratory-built humidity-controlled vitrification system. Aliquots of the samples were applied onto the grids and after a 30 s adsorption time, grids were then blotted manually by filter paper for approximately 3 s. The grids were then plunged into liquid ethane cooled by liquid nitrogen. Images were recorded using a Tecnai 12 TEM operating at 120 kV, equipped with an FEI Eagle 4k×4k CCD, (FEI, Eindhoeven, The Netherlands). At all times low dose procedures were followed, using an electron dose of no more than 10 electrons/Å2. Cryo-TEM images were analysed using ImageJ software (NIH). 2.4 Small angle X-ray scattering (SAXS) Synchrotron SAXS measurements were performed at the SAXS/wide-angle X-ray scattering (WAXS) beamline at the Australian Synchrotron. The X-ray had a beam with a wavelength of λ = 1.032 Å (12.0 keV) with a typical flux of approximately 1013 photons/s. Samples were loaded in a 96-well, half-area UV-clear plate (Greiner Bio-One, Germany) and positioned in a custom-designed plate holder with the temperature controlled at either 25°C or 37°C via a recirculating water bath as previously described.17,

22, 43-44

The sample to

detector distance was chosen as 1.6 m, which provided a q-range of 0.01-0.5 Å-1. The exposure time for each sample was 1 s. 2D X-ray diffraction patterns were recorded on a Decris-Pilatus 1-M detector of 10 modules. The scattering images were integrated into onedimensional plots of intensity versus q for phase identification. SAXS data were analysed using the IDL-based AXcess software package to examine the identity and the lattice parameter (LP) of the internal lyotropic liquid crystalline phase.45 A silver behenate standard (d = 58.38 Å) was used for calibration. 2.5 In vitro cytotoxicity of PTX carrying nanoparticles


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2.5.1 Cell line The HEY cell line was originated from a human ovarian cancer xenograft (HX-62) formerly grown in the peritoneal deposit of a patient with moderately differentiated papillary cystadenocarcinoma of the ovary,46 and has been described previously 41, 47. The HEY cell line was maintained and propagated in complete RPMI 1640 (Sigma-Aldrich, Sydney, Australia) supplemented with 10% (v/v) heat inactivated foetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 2mM L-glutamine (Murdoch Childrens Research Institute (MCRI), Victoria, Australia) and 1% (v/v) penicillin and streptomycin (MCRI). Cells were grown in a sterile, non-pyrogenic BD Falcon® polystyrene tissue culture flasks (BD Biosciences, North Ryde, NSW, Australia) in a humidified atmosphere at 37˚C in the presence of 5% CO2. 2.5.2 Cell viability detected by Cell-Titre Glo assay To assess the cytotoxicity of CB, PTX-CB, and EGFR-PTX-CB in ovarian cancer HEY cells, the cell viability was measured by the CellTiter-Glo Luminescent Cell Viability assay (Promega, Wisconsin, USA). Briefly, the cells at a density of 1x104 cells/well were seeded onto 96-well plates for overnight incubation at 37°C and 5% CO2. After 24 h, the medium was replaced with 100 µL of varying concentrations of CB, PTX-CB and EGFR-PTX-CB. Free PTX, which was dissolved in ethanol and Tween 80 (1:1), and PBS were used as controls. After 72 h incubation, the media was replaced with 50µL of the Cell-Titre Glo lysis buffer and was incubated for 20 minutes on a gentle rotational shaker at 4°C. An aliquot (40µL) was transferred to a fluroblok (Microlon®) 96 well plate and was read on the Glomax 96-well luminometer (Promega) to assess the levels of ATP used as an indicator for cell viability. Cell viability was plotted against MO concentration or PTX concentration depending on whether the tested nanoparticles contain PTX or not. 2.6 Animal studies



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This study was carried out in strict accordance with the Care and Use of the Laboratory Animals manual of the National Health and Medical Research Council of Australia. The experimental protocol was approved by the University of Melbourne’s Animal Ethics Committee (Project-1413207.2). 2.6.1 Tumour growth inhibition study Twenty female Balb/c nu/nu mice (age, 6–8 weeks) were obtained from the Animal Resources Centre, Western Australia and housed in a standard pathogen-free environment with access to food and water. The mice were injected intraperitoneally with 5 x 106 viable ovarian cancer HEY cells similar to that previously described.42 After 14 days, the mice were divided into four random groups of five mice: Group 1- PBS (saline control), Group 2 – PTX solution (dissolved in 3% ethanol and 3% Tween 80 aqueous solution), Group 3 - PTX-CB, and Group 4 - EGFR-PTX-CB. The detailed composition of all nanoparticle formulations used in the subsequent animal studies is given in Table 1. Each mouse received a treatment of 120 µL via intraperitoneal injection once a week for two weeks. Each treatment contained an equivalent PTX dose of 5 mg/kg body weight for Groups 2-4 whereas Group 1 received PBS. All samples were freshly prepared and filtered prior to injection. The mice were inspected daily and the overall body condition and body weight were measured. At the endpoint, mice were euthanized and organs (such as liver, large bowel, pancreas and spleen) and solid tumours were collected and placed in 10% formalin for Hemotaxylin & Eosin (H&E) and immunohistochemistry analysis. The average weight of the solid tumours was used to compare the effectiveness of each treatment in inhibiting tumour growth. 2.6.2 Survival rate study In order to study whether the treatments improved the overall survival rate of mice bearing intraperitoneal HEY ovarian cancer cells, we performed a similar experiment to tumour growth inhibition (see Section 2.8.1) with endpoints determined by the health of the


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mice. For this, twenty five female Balb/c nu/nu mice (age, 6–8 weeks) were injected intraperitoneally with 5 x 106 HEY cells. Tumour-bearing mice were randomly divided into five groups: Group 1 – PBS; Group 2 – PTX solution (3% ethanol and 3% Tween 80 aqueous solution); Group 3 – CB (only cubosomes, no PTX); Group 4 – PTX-CB; Group 5 – EGFRPTX-CB. Each group contained five mice. Each mouse received a treatment of 120 µL via intraperitoneal injection once a week. Each treatment contained a PTX dose of 5 mg/kg body weight for Groups 2, 4 and 5. Mice in Group 3 received CB solution containing the same amount of MO as the ones in Group 4 and 5, which were 50 mg/kg body weight per injection per mouse. All samples were freshly prepared and filtered prior to injection in mice. Mice were monitored daily until their condition deteriorated to the point that the experiments were deemed unethical to continue. At this point the mice were euthanized by over inhalation of CO2 and the number of surviving days was recorded. 2.6.3 Histology of mouse tumours and tumour infiltrated organs Hemotaxylin & Eosin (H & E) staining of mouse tumours and organs was performed by the staff at the Anatomical Pathology Laboratory Services located at The Royal Children’s Hospital, Melbourne, Australia according to the standard H&E protocol.48 Briefly, paraffin embedded tissues were sectioned at 4µm thickness, deparaffinised by submerging the sections in xylene for 3 min, rehydrated in graded ethanol and stained for 3 mins with Haematoxylin (Australian Biostain Pty Ltd, Traralgon, VIC, Australia). Slides were then rinsed with water, followed by a rinse with 0.25% Acid Alcohol (70% alcohol, 25% Hydrochloric Acid) and Scott’s Tapwater Substitute (Sodium hydrogen carbonate, Magnesium Sulphate, Tap Water and Thymol). Sections were stained with Eosin (Amber Scientific, Midvale, WA, Australia) for 2 min before final rinsing with absolute alcohol and xylene. A qualified pathologist assessed the possibility of metastatic development of intraperitoneal injected cells in the organs.



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For immunohistochemistry, formalin-fixed, paraffin embedded 4µm-sectioned mouse tumour xenografts were stained using a Ventana Benchmark Immunostainer (Ventana Medical Systems, Inc. Tucson, Az, USA) as described previously 41. Briefly, tumour sections were de-waxed with Ventana EZ Prep and endogenous peroxidise activity blocked using the Ventana Universal DAB inhibitor. Primary antibodies against Ki-67, CA125 and CD31 were diluted as per manufacturer’s instructions. The sections were counter-stained with Ventana Haematoxylin and Blueing solution, and primary antibody staining was detected using the ultraView Universal DAB detection Kit (Roche, Basel, Switzerland). Negative controls were prepared by incubating each tumour section without primary antibodies. Sections of human breast tissue, high-grade ovarian tumours and human tonsils were used as positive controls to determine the staining efficacy of primary antibodies. Immunohistochemistry images were taken using an Aperio ImageScope (Leica Microsystems, Mt Waverly, Australia) with the associated digital pathology viewing software. DAB staining was measured using the open source image processing package Fiji (https://fiji.sc/) with a plug-in developed to recognize DAB staining for 4 randomly captured image per section. DAB staining intensity of the negative control subtracted from the DAB staining of the antibody of interest. 2.7 Statistical analysis The results were reported as mean ± standard deviation. Student’s t-test in Excel was used to analyse the statistical significance of the tumour growth inhibition data. 3. Results and Discussion 3.1 Characterisation of PTX loaded CB For the MO based lipid nanoparticles, Pluronic F127 triblock polymer at 10 wt% to MO and DSPE-PEG-mal at 16 wt% to MO were used as the stabilising agents to impart colloidal stability. In this study, PTX was first loaded into the nanoparticles at a concentration range of 1-10 wt% to MO and the resulting samples were examined visually. It is evident that the MO


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CB formulation was able to incorporate PTX at the highest investigated concentration of 10 wt%. A milky white, homogenous colloidal dispersion of nanoparticles was achieved after the sonication step and no precipitation of PTX crystals was observed at room temperature. Three nanoparticle formulations (CB, PTX-CB, and EGFR-PTX-CB) with their compositions given in Table 1 were selected for further structural and efficacy studies. Cryo-TEM images of unloaded CB show (Figure 2A) well-ordered internal nanostructures of either cubic symmetry or hexagonal symmetry. An example of fast Fourier transform (FFT) analysis of the internal structures of the cryo-TEM image is given in Figure 2A-inset, displaying the characteristic hexagonal symmetry of a primitive cubic phase viewed along its [111] direction, consistent with previous studies.19, 49 Furthermore, the FFT analysis of the cryo-TEM images revealed a lattice parameter (LP) of around 138 Å, which was later confirmed to be in good agreement with the LP of a primitive cubic phase (space group Im3m) measured by synchrotron SAXS (Figure 2D). Both the presence of the primitive cubic phase and the corresponding LP values of the unloaded CB are consistent with previous studies 50. The loading of PTX to MO lipid nanoparticles greatly affected their nanostructures as clearly seen in cryo-TEM (Figure 2B), where a significant population of particles with the sponge (L3) phase was observed. The sponge phase has been reported previously in some LCNPs and represents a “melted cubic phase” that lacks long-range ordered nanostructures 5152

, hence the FFT analysis yields a “fuzzy” ring. Furthermore, a highly swollen cubic phase

with a LP of 180 Å was observed, significantly larger (38% increase) than the LP of the unloaded CB. PTX entrapment at high concentrations has therefore resulted in the disruption of the original cubic phase leading to co-existence of cubic nanoparticles with a sponge phase or a highly swollen cubic phase. This is consistent with the insertion of the bulky PTX ring structure into the lipid membranes of the nanoparticles. Following successful loading of PTX in the nanoparticles, a simple and efficient surface functionalistion was performed, whereby



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bioconjugation of EGFR Fab’ to the PTX-CB was achieved through a maleimide-thiol reaction similar to a previously reported study 26. Cryo-TEM images of EGFR-PTX-CB show the presence of both cubic phase and sponge phase nanoparticles similar to those of PTX-CB (Figure 2C), suggesting that the bioconjugation process did not significantly perturbed the structural integrity of PTX-CB. Additionally, these nanoparticles had hydrodynamic diameters in the range of 170-250 nm and polydispersity indices (PdI) of 0.15 as measured by dynamic light scattering (DLS) (Supporting information Figure S1), which are consistent with the cryo-TEM analysis. The stability of CB, PTX-CB, and EGFR-PTX-CB nanoparticles in PBS and cell culture media was also monitored over 72 h using DLS. No significant changes in the average particle size of these nanoparticles were seen in the examined timeframe (Supporting information Figure S1).

Figure 2: Representative cryo-TEM images of (A) CB, (B) PTX-CB and (C) EGFR-PTX-


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CB. Scale bars = 200 nm. Refer to Table 1 for detailed compositions. Arrows and insets show fast Fourier transform analysis of selected cubic and sponge phase particles. (D) Synchrotron SAXS profiles of CB and PTX-CB measured at 37 °C. (D-inset) The scattering peaks of CB with spacing ratios of √2:√4:√6:√10:√12:√14 can be indexed as the (110), (200), (211), (310), (222), and (321) reflections of an Im3m cubic phase. (E) SDS-PAGE confirming the conjugation of anti-EGFR Fab’ to PTX-CB (sample name: EGFR-PTX-CB). In a nonreduced condition, Fab’ conjugation resulted in an upward migration of the bands in the EGFR-PTX-CB due to increase in the effective molecular weight (Lane 1). Unconjugated PTX-CB showed no band as expected (Lane 2). In reduced condition, DTT broke the disulphide bonds and reduced the Fab’ into a heavy chain and a light chain (Lane 7). Reduced EGFR-PTX-CB shows upward migration of the band corresponding to the heavy chain rather than the light chain because the conjugation occurred at the hinge region of the Fab’ (Lane 5). Synchrotron SAXS was performed to study the internal nanostructures of the nanoparticles (Figure 2D). The scattering profile of CB without drug at 37oC contains peaks with spacing ratios of √2:√4:√6:√10:√12:√14 that can be indexed as the (110), (200), (211), (310), (222), and (321) reflections of a primitive cubic phase (space group Im3m). Although the established MO phase diagram shows a double diamond cubic phase with the space group Pn3m in excess water,40 it has been demonstrated that when Pluronic F127 is used as a stabiliser for creating nanoparticle systems, the polymer molecules insert into the internal MO bilayer, forcing a phase transformation to a primitive cubic phase.19,

44, 53

The SAXS

result in this study is therefore consistent with previous findings.19, 44, 53 The effect of PTX loading on the internal nanostructure of the CB was also evident in SAXS. The scattering profile of PTX-CB showed a diffused scattering peak that can be originated from the sponge phase. Combining the SAXS data and the cryo-TEM images, it can be concluded that the majority of the PTX-CB nanoparticles are sponge phase with a small additional number of primitive cubic phase. The successful conjugation of anti-EGFR Fab’ to PTX-CB was confirmed by SDSPAGE using a method previously published by Zhai et al.26 In a non-reduced condition, Fab’



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exhibited a band at 55 kDa, corresponding to its molecular weight (Figure 2E, lane 3). Bioconjugation of Fab’ to DSPE-PEG-mal through a thiol-maleimide reaction resulted in an upward migration of the band as seen in the EGFR-PTX-CB sample due to an increase in the molecular weight (lane 1). PTX-CB showed no antibody band as expected (lane 2). In the reduced condition, all samples were treated with a strong reducing agent (DTT) that broke the disulphide bonds in the Fab’ before SDS-PAGE was performed. As a result, the heavy chain and the light chain of the Fab’ were separated into two bands at lower molecular weights than the original 55 kDa of the non-reduced Fab’ (Figure 2E, lane 7). The reduced EGFR-PTX-CB sample showed upward migration of the band corresponding to the heavy chain rather than the light chain (lane 5), confirming that the bioconjugation occurred at the hinge region of the Fab’. As expected, no bands were observed in non-conjugated PTX-CB samples (lane 6). This result confirmed the successful bioconjugation of anti-EGFR Fab’ to PTX-CB nanoparticles. It is consistent with previous findings by Zhai et al., which demonstrated similar bioconjugation of anti-EGFR Fab’ to a phytantriol based cubic and hexagonal phase lipid nanoparticles through thiol-maleimide reactions 26. The greater incorporation of PTX at up to 10 wt% of MO in the current work over previous studies is a significant point of difference. It should be noted that relatively low loading capacities of PTX into LCNPs have been reported previously 28, 35. Jain et al. loaded a range of PTX varying from 0.01%-0.06% w/w to a CB formulation containing 5% w/w MO with respect to dispersion

35

. It was shown that PTX concentration beyond 0.05% w/w

resulted in a rapid precipitation of drug crystals in the aqueous dispersion, indicating a maximum entrapment at 1 wt% of PTX to MO. Another recent study loaded 50 µM PTX to 10 g of dispersions containing 1 wt% of host lipids, that is equivalent to 0.4 mg PTX to 100 mg of lipid (0.4 wt%) 28. The current study used two stabilising agents, i.e. Pluronic F127 and DSPE-PEG-mal, both of which are able to insert into the internal MO lipid membranes


54

,

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whereas previous studies have either focused on a single stabiliser or used various concentrations of the DSPE-PEG polymers for their manufacture. In this study, it was found that the usage of both DSPE-PEG-mal and Pluronic F127 for stabilising the MO nanoparticles significantly enhanced the PTX incorporation, as MO nanoparticles stabilised by either Pluronic F127 or DSPE-PEG-mal alone could not incorporate PTX at 10 wt% and showed rapid PTX crystallisation (data not shown). More studies are currently being undertaken to examine the effect of using multiple amphiphilic stabilisers in LCNP formulations on high loading of hydrophobic drugs such as PTX. 3.2 Effect of PTX loaded CB on HEY cell viability in vitro In order to study the in vitro toxicity profile of the PTX loaded nanoparticles, HEY cells, an aggressive serous ovarian cell line, were used. As demonstrated in Figure 3A, it is clear that, the CB containing no PTX did not significantly affect HEY cell viability in the tested concentration range, indicating that CB alone was not toxic to cells. The IC50 value for CB was over 5,000 µg/L (MO concentration) determined after 72 h incubation with HEY cells. On the other hand, the viability of HEY cells treated with PTX or PTX in nanoparticles decreased as drug concentration increased, highlighting PTX’s inhibitory effect on HEY cell viability. More importantly, the viability of HEY cells exposed to PTX-CB and EGFR-PTXCB decreased at significantly lower PTX concentrations compared to the free PTX, suggesting that nanoparticle based PTX formulations were more toxic to HEY cells than the free PTX. The IC50 values for free PTX, PTX-CB and EGFR-PTX-CB were approximately 25 µg/L, 0.2 µg/L, and 2.5 µg/L, respectively when measured by PTX concentrations. Curiously, the EGFR-PTX-CB nanoparticles appeared to be less toxic to HEY cells compared to PTX-CB nanoparticles. Nanoparticle-based treatments have been linked to higher cytotoxicity against cancer cells in vitro in previous studies. Yuan et al. showed that PTX-loaded PLGA nanoparticles



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and PTX-loaded PLGA-Tween 80 copolymer nanoparticles were both more toxic to A549 lung cancer cells than free PTX 55. Interestingly, this trend held for A549/T cells, which were resistant to PTX. This enhanced toxicity to cancer cells was attributed to higher uptake of nanoparticle- based PTX treatments, leading to more drug entering the cells and eventual cell death

56

. A similar observation was shared by Huang et al., who demonstrated that PTX-

loaded micellar nanoparticles reduced viability and reduced tumour growth in A549 cancer cells 57. Moreover, studies have also shown enhanced cytotoxicity effect of encapsulated PTX nanoparticles compared to free PTX in other cancer types including ovarian and Lewis lung carcinomas cell

58-60

. Other anti-cancer drugs such as doxorubicin were also encapsulated in

lyotropic liquid crystal systems previously and demonstrated superior efficiency in killing cancer cells.30, 32 For example, Negrini et al. demonstrated that a pH-responsive MO-based lyotropic liquid crystal systems had a superior efficiency in killing human colon cancer cells.32 It is therefore reasonable to postulate that the PTX-CB in this study showed higher cytotoxicity compared to free PTX due to the higher uptake of drug carrying nanoparticles into HEY cells, leading to cell death. A few studies have investigated the drug release and cellular uptake mechanisms of cubosomes in vitro using a range of methods.29, 61 Although drug release from lipid lyotropic liquid crystal bulk systems follows a first-order kinetic profile through diffusion,62 experimental discrepancies exist in in vitro drug release studies of nanoparticle systems depending on the experimental method used in buffer conditions.61 For example, Boyd reported that equilibrium dialysis, one of the most common method to study drug release, incorrectly showed sustained release compared to other methods.61 In cultured cells, the uptake or endocytosis mechanisms of the nanoparticles are even more complex. Hinton et al. used confocal microscopy to investigate MO-CB uptake in CHO cells and found that these nanoparticles were internalised as discreet vesicles co-localised with endosomes or


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lysosomes.29 Because the MO-CB used in this study have PTX loaded and EGFR antibody fragments decorated on the surface, interactions with the cells will be significantly different. A follow-up in vitro study will be conducted to elucidate the release and uptake mechanisms of PTX-CB and EGFR-PTX-CB.

Figure 3: (A) In vitro cell viability of HEY ovarian cancer cells after 72 hr treatment with CB, PTX-CB, EGFR-PTX-CB nanoparticles, and free PTX. Data presented as % viability compared to untreated control cells. Data = Mean ± SD, N =3. (B, C) Smaller tumour size in mice with ovarian cancer after two treatments with PTX-CB and EGFR-PTX-CB compared to free PTX and control (PBS). The mice were treated with a PTX dose of 5 mg/kg body weight for two weeks. Data = mean ± SD. n = 5. *p < 0.05, **p < 0.01. Representative images of HEY cell induced ovarian cancer tumour removed after the experiment. (D) Improved survival rate of mice with ovarian cancer treated with PTX-CB (red line) and EGFR-PTX-CB (cyan line) compared to free PTX, no drug loaded CB, and PBS control. The PTX dosage for the treated mice was 5 mg/kg body weight once a week for two weeks. n = 5. Note: the CB and PTX lines are coincide.

3.3 PTX loaded lipid nanoparticles inhibit ovarian cancer tumour growth in vivo The chemotherapeutic efficacy of the developed nanoparticles was assessed in vivo using a HEY cell-derived ovarian cancer model in mice. The HEY cell line has been found to be one of the ovarian cancer cell lines that has cell expression of EGFR and therefore was



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chosen in this study to investigate the targeting effect of EGFR-PTX-CB 27. Figure 3B shows that by day 28 post inoculation of HEY cells, the untreated control mice developed tumours with an average mass of 2.2 g, whilst the treated groups, especially the ones received PTXCB and EGFR-PTX-CB, displayed much lower average tumour masses at the same time point. Mice treated with free PTX showed no significant reduction in tumour mass compared to the control untreated mice. However, both PTX-CB and EGFR-PTX-CB at an equivalent PTX dose were more effective in inhibiting tumour growth by approximately 50% (1 – 1.2 g on average) when compared to the PTX control group (Figure 3C). A statistical significance of p < 0.01 was observed between the PTX control group and both nanoparticle-based treatment groups (i.e. PTX-CB and EGFR-PTX-CB). In this context, it is important to mention that a recent study has demonstrated a 50% reduction in tumour burden in mice with ovarian cancer, which were treated with a weekly dose of PTX at 15 mg/kg body weight 63. A similar level of tumour size reduction was achieved here with the nanoparticle-based treatments at a PTX dose of 5 mg/kg body weight of mice. This indicates that PTX loaded CB may provide a significant advantage in reducing the cytotoxic side-effects of chemotherapy in patients without reducing anti-tumour efficacy. There was however, no significant difference between the tumour mass of the mice treated with PTX-CB and EGFR-PTX-CB. This result further reflected the in vitro cytotoxicity data, which suggested that the conjugation of EGFR antibody fragments to PTXCB did not provide additional benefit to the treatment with bare PTX-CB. Several studies have shown that anti-EGFR Fab’ conjugated nanoparticles were able to enhance the interaction of nanoparticles with cancer cells, leading to higher uptake and retention of nanoparticles in tumours 64-65. In our case, we suspect that the EGFR expression of HEY cells may have been too low for the targeting to be effective. Other ovarian cancer cell lines such as SKOV-6 or OVCAR-7 which have been shown to express much higher level of EGFR


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may be more suitable for testing active targeting by anti-EGFR Fab’ conjugated nanoparticles 27

. The adsorption of proteins on the nanoparticles surfaces may also be a reason for

ineffective specific targeting using anti-EGFR Fab’. Protein coronas are usually formed in vivo on nanoparticle surfaces, influencing their interaction with cells 66. The thickness of the protein corona may also impact the exposure of the targeting molecules, in this case the antiEGFR Fab’. However, the adsorption of proteins on self-assembled lipid nanoparticles, especially nanostructured particles such as CB or hexosomes, has not been studied. This should be a priority in future studies in order to understand the behaviour of these nanoparticles in vivo. 3.4 Nanoparticle based PTX treatments prolong survivability of ovarian cancer mice To determine the influence of the CB-based treatments on survivability, mice with HEY-derived tumours were divided into five groups, which received one of the following regimens; PBS, free PTX (dissolved in ethanol and tween 80), CB (no PTX), PTX-CB, or EGFR-PTX-CB weekly, until moribund. The weekly dose of PTX is the same as in the tumour growth inhibition study, which was 5 mg/kg of body weight per injection. The survival rate of ovarian tumour-bearing mice is presented in Figure 3D. Under the current regimen, PTX-CB group survived between 22 to 38 days, the longest amongst all groups, whilst non-treated or the PTX control groups had a shorter survival period of between 25 to 27 days. This represents a 10-day longer survival period, which is consistent with the tumour growth inhibition analysis. However, mice injected with EGFR-PTX-CB survived a maximum of 33 days indicating that addition of EGFR Fab’ to PTX-CB may not provide a survival advantage, as already suggested by the anti-tumour activity data (Figure 3B), for which the choice of another cell line with relatively higher expression of EGFR may provide further insight to EGFR-PTX-CB specific targeting capability. 3.5 Histological analysis



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After the tumour growth inhibition study (Section 2.3), the tumours and several tissues including liver, spleen, and large bowl were collected for histological evaluation, which provided insight into the effect of the treatments on tumour morphology and metastasis. Histological examination of tumour xenografts revealed morphological features resembling high grade serous carcinoma in women with ovarian cancer (Figure 4, first row). In general, the isolated tumours from all groups displayed extensive cellular budding, aneuploidy, very large and rounded nuclei. Tumour invasion was seen in the liver and spleen of all groups (Figure 4), but no infiltration into the pancreas was observed (data not shown). Infiltration of tumour cells into the liver of both PTX-CB and EGFR-PTX-CB groups showed clusters of tumour cells surrounded by fibrous tissue, which was more prominent in the PTX-CB group. Infiltration into liver in the PTX-CB and EGFR-PTX-CB groups was manifest as relatively defined smaller clusters of tumour cells, suggesting that CB-based drug targeting of tumour cells may be effective in keeping the tumour growth confined, to potentially alleviate intraperitoneal dissemination of ovarian cancer. Infiltration into the large bowel was observed only in control mice but not in any treatment group. In the treatment groups, tumour cells were visible around the large bowel without any obvious infiltration.


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Figure 4: Representative images of H&E stained tumour, liver, spleen, and large bowel infiltration by HEY ovarian tumour cells developed in mice (n=3 per group). Scale bars = 60µm. To further analyse the tumour phenotype following administration of PTX, PTX-CB and EGFR-PTX-CB in mice, the resected tumours were subjected to immunohistochemical analysis for the expression of cancer antigen 125 (CA125), proliferative marker Ki67 and cluster of differentiation 31 (CD31) (Figure 5). No significant change was observed for CA125 and Ki67 staining in the xenograft derived from PTX, PTX-CB, EGFR-PTX-CB treated mice compared to the xenograft of control untreated mice (Figure 5). Even though CA125 is routinely used to diagnose, screen and predict treatment outcomes in ovarian cancer patients, the levels of CA125 in the blood of some ovarian cancer patients can be absent even when the disease is present, or levels can be high when no malignant disease exists

67-68

.

Hence, changes in CA125 levels can be representative of ovarian cancer status, but it is not



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always a true indicator of disease status. However, a lack of variation in Ki67 staining, which indicates proliferation of cells in PTX-CB and EGFR-PTX-CB groups where 50% reduction of tumour burden was observed, cannot be explained. We also demonstrate that the encapsulation of PTX into nanoparticles increased CD31 staining when compared to the xenograft of control untreated mice and those treated with PTX alone. In the PTX-CB group, an increased trend in CD31 staining was observed but it was not significant compared to the control groups. The EGFR-PTX-CB group showed statistical significance in increasing the CD31 level (Figure 5). The enhanced CD31 staining in EGFR-PTX-CB and PTX-CB groups may indicate enhanced tumour vascularization because of the passive targeting of the nanoparticles that exploit the abnormal structure and architecture of the tumour’s blood vessels 69.


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Figure 5: Immunohistochemical analysis of CA125, Ki67, and CD31 expression in tumour xenografts derived from mice intraperitoneally injected with HEY cells and treated with PBS control, PTX, PTX-CB and EGFR-PTX-CB. Scale bars = 60µm. Quantification of CA125, Ki67, c-Kit, Oct3/4 and CD31 DAB staining was performed using Fiji software as described in the Experimental section. Results are expressed as the average DAB reading of positivelystained tumour cells subtracted by the average DAB staining of the negatively-stained cells for each xenograft ± SEM (n=3/group). Parametric One-way ANOVA with Tukey’s post-test was used for statistical analysis. Significance is indicated by *p

Paclitaxel-loaded self-assembled lipid nanoparticles as targeted drug

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