Dual Controlled Delivery of Gemcitabine and Cisplatin using Polymer

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Dual Controlled Delivery of Gemcitabine and Cisplatin using Polymer Modified Thermosensitive Liposomes for Pancreatic Cancer Mandana Emamzadeh, Mina Emamzadeh, and George Pasparakis ACS Appl. Bio Mater., Just Accepted Manuscript • Publication Date (Web): 12 Feb 2019 Downloaded from http://pubs.acs.org on February 12, 2019

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ACS Applied Bio Materials

Dual Controlled Delivery of Gemcitabine and Cisplatin using Polymer Modified Thermosensitive Liposomes for Pancreatic Cancer Mandana Emamzadeh, Mina Emamzadeh, and George Pasparakis University College London, School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX

Correspondence: George Pasparakis University College London, School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX Tel 0044 (0) 20 7753 5816 Email [email protected]

Abstract: Although combinational anticancer chemotherapies have been proven to improve the life expectancy of patients in the clinic, their full potential is severely limited by the additive toxicities of the drug molecules. Targeted drug delivery systems could alleviate this major limitation by the design of nanocarriers that can cocarry multiple drug molecules in order to augment drug synergism at the site of interest while reducing the systemic side-affects. In this study, we report on a thermoresponsive polymer coated liposome nanocarrier that is capable to co-carry two potent anticancer drugs and release them via a thermally triggered mechanism. A synthetic polymer [poly(diethylene glycol) methacrylate-co-poly(oligoethylene glycol) methacrylate]-b-poly(2-ethylhexyl) methacrylate) was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization and was used as a thermoresponsive polymer coating shell on thermosensitive liposome carriers. The formulations were tested in vitro against two pancreatic cancer cell lines, MiaPaCa-2, and BxPC-3, and their cytotoxic potency was studied in respect to their targeted release properties as well as their biological interactions with cellular organelles. The polymer modified liposomes (PMTL) could co-carry and release Gemcitabine (Gem) and cisplatin (Cis) in a thermally controlled rate and were also found to exhibit specific hydrophobic interactions with the cell membranes above the temperature 1 ACS Paragon Plus Environment

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transition of the formulations. In addition, the nanocarriers were found to induce more than 10-fold improvement of the IC50 of both drugs, either as monotherapies or in combination, in both cell lines tested, in a temperature dependent manner. The proposed formulations constitute a potent nanomedicinal approach for the co-delivery of multiple drug molecules and could find potential uses as thermally triggered drug delivery systems for precision medicine and oncology and also as modulators of drug efficacy at the cellular level owing to their unique interactions with the cell membranes.

Keyword: Thermosensitive liposomes, Polymer, targeted delivery, combinational therapy, gemcitabine, cisplatin, synergistic effect, pancreatic cancer.

Introduction Pancreatic cancer is one of the most lethal malignancies with very low survival rate, not exceeding 5 years for the majority of patients by the time of diagnosis and is now ranked as the fourth most common cause of cancer mortality.1 Gemcitabine (Gem) is the front line drug for pancreatic cancer but with limited therapeutic effect due to the rapid deamination of the molecule in blood plasma which renders it inactive. Hence, to compensate for the moderate activity, Gem is administered in high doses as monotherapy (with typical doses of 1000 mg/m2)2 or in combination with other drugs. Recently, such combinational therapies have been shown to significantly improve the overall survival rates not only in pancreatic cancer patients but also in many other forms of cancer malignancies.3, 4 The rationale of combinational therapeutics lies on the potential synergism of multiple drug molecules via different biomolecular pathways which can exert an augmented therapeutic effect. Considering that there are ca. 250 approved anticancer drug molecules, it is easy to conceive that their respective doublet or triplet potential combinations gives rise to an exponentially growing number of possibilities likely to be translated to clinically useful therapeutic modalities which are yet to be explored.5-7 A notable example is the FOLRIFINOX therapy comprising multiple drug molecules resulting in superior survival rates compared to Gem monotherapy.8 Despite the clinical outcome, FOLFIRINOX therapy (and even dual-drug combinational or sequential modalities) poses serious clinical complications owing to the additive drug toxicities which often limit the

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group of patients that can benefit from such therapies; of particular interest is the combinational therapy of Gem with cisplatin (Cis) which has been shown to significantly increase the overall survival rates and also to improves the quality of life of patients.9, 10

Despite its broad range of use, Cis monotherapy is often associated with severe nephrotoxicity which poses a limitation to the maximum tolerated dose.11, 12 Therefore, the encapsulation of Cis in suitable nanocarriers could potentially alleviate toxicity issues and maximize the therapeutic potency.13 In fact, the concept of drug encapsulation is even more urgent in the case of combinational therapies such as Gem-Cis, where additive toxicities from multiple drug molecules could limit the potential synergism and hamper the overall efficacy. In principle, drug nanoencapsulation strategies aim to alleviate exposure of the drug molecules to healthy tissues while accumulating at higher rates at the primary tumor sites via the enhanced permeation and retention effect or by active targeting of cancer cells with the use of targeting ligands.14-16 In this regard, many studies have been reported to encapsulate chemotherapeutic drugs in suitable nanocarriers of various forms17 such as polymer nanoparticles18, 19, or liposomes.20, 21 Recently, the Huang group reported on nanomedicinal formulations of Gem derivatives and Cis for combinational therapy of bladder cancer with significant synergism effects with the use of ratiometrically precise nanoparticulate vehicles.22, 23 In our study we utilized liposomes as the main component of our proposed nanofomulation as they are considered widely translational with several nanomedicinal products in the market (i.e. Doxil®, ThermoDox®, Lipoplatin™)24 owing to their simple preparation and good biocompatibility.

Liposomes are spherical vesicles that consist of a phospholipid bilayer surrounded by polar head groups in the interior core and exterior shell, respectively. They are capable of encapsulating hydrophilic drugs within their hydrophilic compartment or hydrophobic drugs in the lipophilic bilayer.18,

25

In 1978 Yatvin et al.

introduced the first class of thermosensitive liposomes which are now referred to as traditional thermosensitive liposomes (TTL). TTL consist of a lipid membrane that undergoes a phase transition from solid (ordered) to liquid (disordered) phase when heated above their transition temperature (Tm).26 However, TTL formulations require high heating in order to reach their transition temperature to become permeable. As a result, the high temperature can result in hemorrhage and trigger necrosis to neighboring healthy tissue.



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Hence, new approaches have been developed to reduce the transition temperature of thermosensitive liposomes to the range of mild hyperthermia (39-42° C).20,

21

A promising modification to overcome these

problems is the coating of the surface of TTLs with polymers. Polymer modified thermosensitive liposomes (PMTLs) exert significantly better colloidal stability and protein repellence in blood plasma which is mostly achieved by PEGylation strategies.27, 28 Of particular interest is the coating of TTLs with thermoresponsive polymers (Figure 1); the latter consist of block copolymers with a hydrophobic block acting as an inserting anchor in the lipid bilayer used to install and stabilize the polymer on the surface of the liposomes, and a second thermoresponsive block which is soluble and protein repellent below a critical temperature (so called, lower critical solution temperature, LCST) and becomes insoluble above the LCST. The thermally induced transition induces disruption of the lipid bilayer which in combination with the Tm of the lipids augments the drug release at the site of the heat stimulated area. This concept is particularly useful in combinational therapies where tumors are ablated by thermal means (such as radiofrequencies or ultrasound) with the drugs acting as a complementary means to kill primary or bystander cells.29,

30

Despite the powerful

conceptual merit of this approach in terms of therapeutic potency, the number of studies on PMTLs that cocarry multiple drug molecules and also exert thermally triggered drug release mechanism are very limited.31

Recently we reported on a thermoresponsive polymeric micellar nanocarrier that could co-carry two potent hydrophobic drug molecules, namely squalene-gemcitabine and paclitaxel, and we demonstrated that the carrier could not only facilitate triggered drug release profile but also exerted specific interactions with the cell membrane.32 In this work, we formulate the block copolymer with liposome carriers and we demonstrate that the polymer retains its cell membrane interacting capability. The proposed formulation can co-carry Gem and Cis, both water soluble anticancer drugs, and it is demonstrated that the thermally triggered drug release is accompanied by active cell membrane interaction which in turn augments the drug synergism in vitro. To the best of our knowledge, this is the first type of study on thermoresponsive PMTLs with multiple drug molecules, but more importantly, the reported results suggest that the proposed polymer induces a “generic” type of cell membrane interaction regardless of its formulation type (that is, embedded as micellar (i.e. as shown in our previous study32) or liposomal component (this study).


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Materials and methods Materials 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[amino(polyethyleneglycol)-2000] (ammonium salt) (DSPE-PEG2000), 2,2′-azobis(2-methylpropionitrile) (AIBN), 2-butanone, 2-cyano-2-propyl benzodithioate (CTA/RAFT agent), 2-ethylhexyl methacrylate (EHMA), acetic acid, cisplatin, cholesterol (Chol), chloroform, deuterated chloroform (CDCl3), diethyl ether, di(ethylene glycol) methyl ether methacrylate (DiEGMA), dulbecco’s modified eagle’s medium- high glucose (DMEM), Dulbecco’s phosphate buffered saline (DPBS), fetal bovine serum (FBS), fluorescein O-methacrylate, hexane, l-Glutamine, l-α-phosphatidylcholine, hydrogenated (Soy) (HSPC), methanol, NaCl, oligo(ethylene glycol) methyl ether methacrylate (OEGMA300), phosphate buffered saline (PBS) tablets, penicillinstreptomycin, tetrahydrofuran (THF), thiazolyl blue tetrazolium bromide (MTT) were purchased from SigmaAldrich. Gemcitabine.HCl was purchased from Sequoia Research Products Ltd. MiaPaCa-2 and BxPC3 cell line were purchased from ATTC company. The experimental procedures for the polymer synthesis (including the fluorescent polymer batch used for the cellular uptake assessment), nuclear magnetic resonance (NMR) spectroscopy, size exclusion chromatography (SEC), the determination of the polymers’ LCST, transmission electron microscopy (TEM), and characterization by dynamic light scattering (DLS) (experiments conducted at 37 oC and 40 oC to probe the effect of temperature on PMTLs) have all been described in detail in our previously published work.32 Preparation of polymer modified thermo-sensitive liposomes (PMTL) Liposome formulations consisted of DPPC/HSPC/Chol/DSPE-PEG2000 (100/50/30/6 molar ratio). The colloidal suspensions were prepared by dissolving the lipid mixture (10 mg) in a Chloroform:Methanol (3:1 v/v) solvent mixture, using a 25 mL round bottom flask. The solvents were evaporated with a rotatory evaporator to form a thin layer of lipid film on the inner wall of the flask. Gem or Cis (3 mg) were dissolved in 0.9% NaCl and this solution was used to rehydrate the lipid film (for co-encapsulation of GemCis 1.5 mg of each drug was used). The temperature of the solution was kept above the transition temperature (Tm) of the lipid mixture (at 50 °C) to ensure high permeability through the liposome membrane and to enhance the drug entrapment efficiency. After stirring the lipid film in 1 mL of 0.9% NaCl solution at 50 °C for 1 hr, the obtained



multilamellar vesicles were submitted to ten cycles of freezing (with dry ice) and thawing (with oil bath at 50 °C). The resulting colloidal suspension of the multilamellar vesicles was extruded (Avanti polar lipids, Inc) through 800 μm and 100 μm pore size polycarbonate membrane filters to transform them to unilamellar vesicles. The untrappted 0.9% NaCl solution was removed by centrifugation at 13300 rpm (Thermo Scientific Heraeus Fresco 17) for 1 hr at 4 °C. Subsequently the loaded liposomes were incubated with the TR polymer at ratio polymer : lipid (1:1) for 1 hr at 30°C. The unattached TR polymer was removed by centrifugation at 13300 rpm for 1 hr at 4 °C. Amount of polymer bound on the surface of PMTL The fluorescent poly (DiEGMA-co-OEGMA300)-b-(EHMA-co-FOMA), polymer, was used to determine the degree of polymer modification on the surface of the PMTLs. The amount of the attached polymer on the surface was determined by fluorescence spectrophotometry (SpectraMax multi-mode microplate reader, Molecular device); the excitation and emission wavelengths were set to 490 and 525, respectively. Briefly, the liposomes were incubated with the fluorescent polymer at a ratio of polymer:lipid of 1:1, for 1 hr at 30 °C. The unattached fluorescent thermo-responsive polymer was removed by centrifugation at 13300 rpm for 1 hr at 4 °C. The intensity of the fluorescence signal from the surface of the liposomes was determined from a calibration curve constructed from previously prepared polymer solutions of known concentrations. HPLC analysis for drug loading and release determination The HPLC assay for the quantification of both Gem and Cis was developed using an Agilent Technologies 1200 Series HPLC system. The data was recorded and analyzed using ChemStation for LC software, also by Agilent Technologies, UK. The chromatographic separation was achieved using a Discovery® HS F55

15cmx 4.6 mm, 5 μm (SUPELCO Analytical). Buffer and DMF (95:5) were introduced to the column as

mobile phase under gradient conditions (Table 1). The aqueous buffer was prepared by dissolving 3.86 g of ammonium acetate (0.05 M) and 1 g of sodium-1-octane-sulphate in 1 liter of HPLC grade water and adjusted to pH 4 with glacial acetic acid. The mobile phase was pumped through the column at a flow rate of 1 mL/min. The UV detector was set at 300 nm and the injection volume was 20 μL. The GEM and CIS standard solutions used for quantification were prepared by suitably diluting a 100 μg/mL working standard of each drug in the buffer solution. Drug loading efficiency


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The drug loading efficacy of the PMTLs was analysed by HPLC in order to measure the amount of drug entrapped within the liposome’s core. The encapsulation efficiency (EE) was measured using formula:

EE

Amount of entrapped drug

(%) = Amount of drug added initialy x 100

(1)

Determination of in vitro drug release The drug release profile from the PMTLs was evaluated using a Slide-A-Lyzer dialysis cassette (MWCO=7 kDa, Spectrum laboratories). Drug loaded liposomes dispersed in 1 mL of 0.9% NaCl solution were transferred in the dialysis cassette by using a syringe which was immersed in 200 mL of 0.9% NaCl solution. The drug release experiments were performed under mild stirring below and above the LCST, at 37°C and 40°C respectively, in order to compare the drug release profiles at different temperatures. At predetermined time intervals, aliquots (0.1 mL) were withdrawn from the cassette which was replenished with fresh 0.9% NaCl solution. The collected samples were centrifuged (Thermo Scientific Heraeus Fresco 17) at 13300 rpm for 10 min and analyzed by HPLC to measure the released drug. In vitro cytotoxicity assay The cytotoxicity of the PMTL was evaluated by the MTT assay. Liposomes with LCST at 40°C were prepared and investigated for cytotoxicity, stability in serum-containing media and cellular uptake using MiaPaca-2 and BxPC-3 cell lines. The cells were seeded in a 96-well plate at a density of 1×104 cells per well. MiaPaca-2 cells consumed Dulbecco’s modified eagle’s medium- high glucose supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1% L-glutamine. BxPC-3 cells consumed RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Both cells were incubated either at 37°C or 40°C in humidified atmosphere with 5% CO2 for 24 h before the assay. The cells were incubated with medium of PMTL with different concentrations of drug ranging from 0.0001 to 100 μM. After incubation for 72 h, the medium was replaced by 100 μL of fresh medium and 25 μL of MTT stock solution (5 mg/mL in PBS) and incubated for an additional 4 h. Subsequently, the medium was removed and the formazan crystals were dissolved in 200 μL of DMSO. The plates were shaken for 2 min at room temperature before measuring the optical density at 570 nm on a SpectraMax® M2/M2e multimode microplate reader, with SoftMax® Pro Software. 7 ACS Paragon Plus Environment


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The combination index The combination index (CI)33 was investigated in order to measure the combinatorial therapeutic effect resulting from the co-delivery of GemCis. CI > 1 denotes antagonistic behavior, CI = 1 corresponds to additive behavior and CI < 1 represents synergistic drug activity. The CI was calculated based on the IC50 values obtained from the MTT assay by using the following equation.

CI =

IC50 (A + B) IC50 (A)

+

IC50 (A + B) IC50 (B)

(2)

Where IC50 (A) and IC50 (B) are the IC50 values obtained from each drug separately. IC50 (A+B) is the IC50 value of both drugs in combination. Thermo-dependent cellular uptake of fluorescent liposome The effect of temperature on cellular uptake of thermo-responsive liposomes below and above their thermal transition temperatures was studied using fluorescence microscopy. MiaPaCa-2 and BxPC-3 cells were seeded in 6-well plates at a concentration of 1x105 cells/well and incubated at 37 °C and 5% CO2 to allow the cells to attach and reach confluency. The media in each well was replaced with a stock culture medium dispersion containing 1 mg of fluorescent liposomes (as prepared with the use of the fluorescent polymer) in 2 mL of culture medium. After 30 min, the media were removed and the cells were rinsed with DPBS once at room temperature. The washed cells were fixed using 4% paraformaldehyde phosphate buffer solution for 20 min and rinsed twice with DPBS. The cells were observed in 6-well plates using the EVOS® FL Imaging System. Flow Cytometry analysis MiaPaCa-2 and BxPC-3 cells were plated in 6 well plates at a seeded density of 1x105. After the cells reached confluency, they were treated with 2 mL of culture medium containing fluorescent liposomes (1 mg). After 30 min, the cells were washed with cold PBS (4°C) several times to stop the intake. 1 mL of cold PBS (4°C) was added, and the cells were scraped and transferred into Nalgene® centrifuge tubes to be analyzed by a MACSQuant Analyzer 10 Flow Cytometer. Clonogenic cell survival assay 2x105 MiaPaCa-2 or BxPC-3 cells were seeded in a 25 cm2 T-flask (using 5 mL of culture medium). Once the cells reached confluency, each flask was treated with different concentration of drugs in combination. 8 ACS Paragon Plus Environment

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One flask remained untreated as a control. After 24 hours, the treated and untreated cells were trypsinized, counted and subsequently 250 cells from each flask were seeded in the 6 well plate. After 14 days, the colonies were washed with PBS and then fixed using methanol and acetic acid in ratio 3:1. The colonies were stained with 0.5% crystal violet solution (diluted with methanol) for 5 min. The stained plates were rinsed in the tray with distilled water and left in the fume hood over night to dry. Colonies appeared as clusters of violet stained cells visualized with the naked eye. The number of air-dried colonies for the average of three colony counts for each plate was recorded. A cluster of 50 or more cells were counted as one colony. The plating efficiency (PE) was calculated by dividing the number of colonies counted by the number of cells plated and then multiplying by 100:

% Plating efficiency =

No. of colonies counted No. of cells plated

(3)

x100

PE was determined to investigate the percentage of single cells seeded in the plates that could form a colony. By determining PE, the survival fraction of the single cells seeded in the plates was also calculated by dividing the PE of the treated cells by the PE of the control and then multiplying by 100:

% Survival fraction =

PE of the treated cells PE of the control

x100

(4)

Results and Discussion The polymer synthesis rationale was based on our previous findings that oligoethylene glycol methacrylate polymers have a tuneable cell membrane capability32 and hence we sought to test the concept in a different formulation format, that is, in liposomes. Also, OEGMA type of polymers are well known to resemble the protein repellent properties (and hence prevent rapid opsonization) of PEG below the LCST owing to their structural similarity, which has been shown to enhance their circulation times in the bloodstream.34 Above the LCST, the coil-to-globule transition of the polymer can facilitate the disruption of the liposome bilayer and induce thermally triggered type of drug release (Figure 1b). The thermoresponsive block was synthesized by RAFT polymerization (Figure 1a) at a monomer feed ratio of DiEGMA:OEGMA300 of 60:40 (Mn 31,600 Da, Đ 9 ACS Paragon Plus Environment


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1.3) which was optimum to adjust the LCST onset at 40oC. Subsequently, the hydrophobic EHMA block was grown to serve as the lipophilic anchor in the liposomal bilayer. The liposomes were prepared by wellestablished solvent evaporation methods with appropriate amounts of polymer incorporation in the lipid bilayer. A fluorescent block copolymer [poly(DiEGMA-co-OEGMA300)-b-(EHMA-co-FOMA)] was synthesized in order to track the amount of polymer that was possible to anchor on the lipid bilayer. Fluorescence microscopy showed that the amount of immobilized polymer was 0.43 ± 0.07 mg per mg of lipid contents which is adequate to probe its role in the thermal properties of the formulation.

Transmission electron microscopy

TEM was used to determine the morphology, and the size distribution of the PMTL (Figure 2a-c). The images showed almost complete absence of multilamellar liposomes with the majority of the particles appearing as unilamellar vesicles with a clear lipid bilayer shell. The size of 150 liposomes was measured to plot the histogram of the diameter range distribution for each drug-loaded PMTL. The frequency distribution curve of the liposomes showed Gaussian size distribution. The mode represented the liposome size that appeared most frequently in the dataset of the measured liposomes. The most frequent particle size obtained from TEM histogram of Gem, Cis and GemCis loaded PMTL were 145.0, 141.0 and 145.0 nm, respectively (Figure 2d-f). The average liposome diameter size, was measured to be 140.4 ± 9.7, 140.0 ± 5.5 and 143.4 ± 10.9 for Gem, Cis and GemCis loaded PMTL, respectively (Figure 2d-f).

Dynamic light scattering

DLS was used to determine the size and size distribution of the liposomes (Figure 2 g-I, Table 2) as a complementary method to TEM. As expected, the TR polymer attached on the surface of the blank PMTL increased the hydrodynamic diameter of the liposomes to 137.0 ± 2.11 nm (vs 131.7 ± 13.3 nm for the nonpolymer coated TTL samples35 presumably due to the additive effect of the hydrodynamic diameter of the polymer. Furthermore, the average particle size for Gem and Cis and GemCis co-loaded PMTL was measured to be 149.4 ± 3, 145 ± 4, 146.8 ± 3.1, respectively (Table 2). The average diameter of PMTL was


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increased after drug loading however all formulations retained narrow PDI. Overall, the DLS results corroborate with the values obtained by TEM. The hydrophobic interaction above the LCST (40oC) was also confirmed by DLS experiments of PMTLs (figure S1) suspended in aqueous media which showed significant increase of their average hydrodynamic diameter as a result of inter-particle aggregation36 due to the collapsing of the polymeric coronae at 40oC; this hydrophobic interaction is the enabling driving force to augment the interaction of PMTLs with the cell membranes which in turn leads to higher cellular uptake rates, as we demonstrate further in our study.

3.5. Drug loading and triggered release studies

The EE of the drug loaded TTL and PMTL samples was measured by HPLC as calculated using Eq. 1 (Table 3). The EE achieved was 16.37 ± 3.66, 18.52 ± 2.09 for Gem loaded TTL, Cis loaded TTL respectively and overall 22.05 mg/mL for GemCis loaded TTL. The EE of PMTL was further measured to be 11.06 ± 2.46, 17.90 ± 1.66 for Gem loaded PMTL, Cis loaded PMTL respectively and overall 17.19 mg/mL for GemCis loaded PMTL. From this data, it is concluded that the polymer modification of the TTL samples, has a slightly negative impact on the EE for both drugs and their combination, however, the EE was still satisfactory and on par with similar studies on PMTL.35 We then proceeded to perform drug release experiments with the TTL (Figure 3a) and PMTL (Figure 3b) samples at temperatures above (40°C) and below (37°C) the polymer’s LCST. It was found that Cis was released almost linearly in the first hours with minimum variation from the temperature change; after 3 hours the release plateaued in both temperatures. The effect of temperature was more pronounced in the release profile of Gem where it was observed that it was released linearly in the first 2 hours above the LCST before starting to plateau at a maximum release of ca. 74% (figure 3a); below the LCST, the release of Gem proceeded in a considerably lower rate with a linear release profile for 3 hours before plateauing at a maximum release of 65% after 4 hours (figure 3a). These results are quite expected in that it is already established that TTL are thermosensitive at the tested temperature window. Interestingly, the impact of temperature on the release profile was significantly higher in the case of PMTL samples (figure 3b). Cis was



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again released in a rather linear manner below the LCST for up to 3 hours until it turned constant and release profile of ca. 74% at 5 hours. Above the LCST, Cis was released at a higher rate for the first 2 hours, but intriguingly, the maximum release at 5 hours was virtually the same with the sample below the LCST. In the case of Gem, below the LCST It showed a parabolic release curve, however above the LCST a sharp linear release profile was observed in the first hour followed by a slower release profile. At 5 hours the total release was only slightly higher compared to the sample below the LCST. However, above the LCST, the total Gem release at five hours was nearly quantitative reaching 93%. From these results, it is concluded that the polymer coil-to-globule transition has a significant impact on the release profile of the drugs which is only limited to the first 1-2 hours as it does not seem to affect significantly the release rate in the longer term (i.e. after 4 hours). Also, the polymer seems to render the liposomes leakier as the release profiles at 5 hours are relatively higher compared to the TTL samples. These results are in accord with previous studies by Ta et al. that demonstrated that the macroscopic increase of the leakiness of the liposomes is attributed to the synergism between the coil-to-globule transition of the polymer above the LCST and the gel-to-liquid transition of the lipid bilayer at similar temperatures; conversely, polymer coated liposomes with temperature-insensitive bilayer do not exhibit measurable increase of the release rate.35 Overall, from these experiments it is apparent that the temperature has indeed an important role in the release profile of both drugs but the relative margin differences below and above the LCST are not so sharp to justify a potential effect in in vitro studies, as we observed (see below).

In vitro cytotoxicity studies

MiaPaCa-2 and BxPC-3 cells were treated with the drug loaded formulations and the IC50 values were calculated based on the MTT assay (Table 4). Initially, free Gem was found to have similar IC50 at the temperatures tested, namely, at 37oC (3.0 ± 0.8 µM) and 40oC (2.8 ± 1.1 μM) on MiaPaCa-2 cells (Table 4). After encapsulation of Gem in TTL and PMTL the IC50 increased only marginally to 7.3 ± 1.5 µM and 5.4 ± 0.5 µM, respectively. However, by application of


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hyperthermia at 40°C the IC50 was further reduced to 1.7 ± 0.4 µM in TTL which is significantly lower than the parent drug. PMTL liposomes reduced the IC50 comparably down to 1.6 ± 0.3 µM. As Cis is not a first line drug for the treatment of pancreatic cancer, expectedly, it showed relatively high IC50 when used as free drug either at 37oC (13.0 ± 3.8 µM) or 40oC (12.7 ± 2.6 μM) (Table 4). At 37°C, IC50 was not achieved in the Cis loaded TTL because the cell death was only obtained at very high concentrations (Table 4). The IC50 of the Cis loaded PMTL was also not achieved because the formulation did not inhibit the cell growth up to 50% of the cell population. Remarkably, at 40°C, the IC50 was reduced to 11.0 ± 3.5 µM in TTL and drastically reduced to 2.0 ± 0.4 µM in PMTL (Table 4) which is almost 6 fold improvement compared to the parent drug. It is interesting to notice in this dataset the marked role of the formulation which completely alleviates the toxic potency of Cis but enhances it significantly at higher temperatures.

Finally, treatment of the cells with the two parent drugs (GemCis) resulted in a comparative reduction of the IC50 to 1.5 ± 0.1 µM and 1.3 ± 0.1 µM, at 37oC and 40oC, respectively (Table 4). Encapsulation of GemCis followed by incubation at 37°C increased the IC50 to 3.0 ± 0.8 µM and 7.5 ± 1.6 µM in TTL and PMTL, respectively. Whereas, The IC50 was significantly enhanced at 40°C and the concentration required to inhibit 50% of cell growth reached down to 1.0 ± 0.3 µM and 0.5 ± 0.1 µM in TTL and PMTL, respectively. To sum up, in all formulations once the drug(s) was encapsulated in the liposomes and incubated BL (at 37°C), the formulation reduced or even alleviated the toxicity compared to free drugs resulting from the ability of the liposomes to retain the drug molecules within their aqueous compartment so that it is not directly uptaken by cells BL. On the other hand, encapsulation of the drugs in the liposomes followed by incubation AL (at 40°C), improved the chemotherapeutic effect and supressed the cancer cells growth. These marked improvements on the IC50 were significantly more pronounces in the PMTL samples.

Similar trends were observed in BxPC-3 cells. Interestingly, Gem was virtually impotent against BxPC-3 cells in the non-encapsulated form, at both temperatures tested. However, the IC50 of Gem loaded TTL and PMTL at 37°C was found to be 6.4 ± 0.8 µM and 1.9 ± 0.5 µM, respectively (Table 4). At 40°C the IC50 was decreased to 3.8 ± 0.2 µM and 0.4 ± 0.1 µM, for TTL and PMTL, respectively which shows the marked cytotoxic response of the formulation by the thermal stimulus, especially in the polymer coated samples.



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Free Cis had an IC50 of 8.1 µM at both temperatures whereas Cis loaded TTL could not inhibit 50% of cell growth. However, Cis loaded PMTL increased the IC50 up to 12.4 ± 3.3 µM. Cis loaded TTL at 40°C, showed an IC50 of 14.6 ± 3.3 µM, similar to Cis loaded PMTL at 37°C but higher than the parent drug. Nevertheless, Cis loaded PMTL at 40°C reduced the IC50 (2.8 ± 0.3 µM) significantly by almost 3 fold compared to the free drug. As expected, the combinational co-delivery of the two drugs, GemCis loaded in TTL and PMTL, required less concentration of drugs to supress the cell viability when compared to each of the free drugs separately.

By application of free GemCis, again no sigmoidal curve was obtained and an IC50 was not calculated, in any of the temperature tested. The combinational application of GemCis reduced the IC50 compared to each drug separately (Table 4). The IC50 of GemCis co-loaded liposomes at 37°C was 2.1 ± 0.6 µM and 0.5 ± 0.2 µM for TTL and PMTL, respectively. At 40°C, the IC50 was further reduced to 0.6 ± 0.1 µM and 0.2 ± 0.1 µM for TTL and PMTL, respectively. The IC50 achieved from the PMTL was lower than that of TTL, showing the augmentation of the drugs’ cytotoxicity by the PMTL formulation. To conclude, the cytotoxicity of the drugs was unaffected by temperature when administered in a nonencapsulated form but was enhanced once loaded in liposomes followed by hyperthermia at 40°C. In addition, TR polymers on the surface of PMTL improved the cytotoxic effect of the chemotherapeutic drugs considerably. The IC50 of the native drugs was considerably increased when encapsulated in liposomes and incubated at 37°C, demonstrating reduced cytotoxicity of the liposomes below Tm in TTL and below the LCST in PMTL. However, the IC50 was either restored or even further reduced compared to the native drugs when the TTL and PMTL heated at 40°C.

The combination index Having established the cytotoxic potency of the formulations against the two cell lines, we sought to elucidate the potential synergism of the two drugs in the co-loaded form by calculating the drug CI based on the Chou-Talaley model.33 A CI1 represents drug antagonism (Eq. 2).


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Table 5 shows the CI calculated from the IC50 of liposomes on MiaPaCa-2 cells. It was found that the CI of the non-loaded GemCis was 0.62 and 0.57 at 37°C and 40°C respectively, which verifies the strong synergism of the two drug molecules. A CI value could not be obtained for the TTL and PMTL at 37°C (below the LCST) as no IC50 was obtained for the encapsulated drugs at 37°C. The CI of the TTL at 40°C was found to be 0.68, representing synergistic effect too. Interestingly, the CI value achieved by the PMTL above LCST at 40°C was considerably lower compared to the CI value of the free drugs or the loaded TTL sample; a CI of 0.56 indicated significant enhancement of the synergistic activity of GemCis in the formulated form.

As a result, the in vitro studies showed that combinational therapy of the two drugs, Gem and Cis, has synergistic effect on pancreatic cancer cells. Moreover, this synergism was further significantly enhanced by encapsulating the drugs in PMTL at 40°C. The CI values on BxPC-3 cells was also calculated (Table 6). Application of the free drugs did not exert any measurable effect due to the wide range of toxicity and the CI could not be calculated at any temperature. The CI of the loaded TTL at 37°C was also impossible to calculate due to the absence of IC50 value in one of the formulations. On the other hand, encapsulation of the drugs in TTL at 40°C had very low CI of 0.20 representing very strong drug synergism. In the PMTL samples the CI was as low as 0.30 and 0.57 at 37°C and at 40°C, respectively. Noticeably, the CI values obtained from the PMTL at 40°C were similar on both MiaPaCa-2 and BxPC-3 cells. These results show that the free drugs did not exert any additive or synergistic effect when applied directly to BxPC-3 cells but once encapsulated their synergistic cytotoxicity was significantly improved. In addition, the data show superior cytotoxic efficacy of the PMTL formulation especially above the LCST which however cannot be justified solely on the drug rather moderate increased drug release rates. Therefore, we hypothesized that the polymer itself might be actively interacting with the cell membrane in a temperature dependent manner which could further augment the drug uptake rates above the LCST and therefore fluorescence imaging was conducted to observe the early mechanism of the liposomal uptake.

Thermo-dependent cellular uptake of fluorescent liposome



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The fluorescently labelled PMTL samples were tracked with a fluorescence microscope (figure 4a-b and 4de) and it was shown that they could rapidly associate with the cell membranes as a characteristic green fluorescent pattern coinciding with the cellular silhouette periphery was observed in both MiaPaCa-2 and BxPC-3 cancer cells, followed by distinct fluorescence signal in the cytosol which is indicative of cellular internalization. The fluorescence signal was considerably more intense in the cells that were incubated with the nanocarriers at 40°C, that is, above the polymers’ LCST which presumably augmented the interaction of the cell membranes with the hydrophobic/collapsed polymer coronae of the liposomes. To fully verify the temperature dependent cell membrane association and ultimately the higher cellular uptake by the PMTL we performed flow cytometry analysis on both cell lines (figure 4c and 4f).

Flow Cytometry analysis The fluorescent PMTL was incubated with cells for 30 min prior to analysis. In MiaPaCa-2 cells (figure 4c), the standard curve was divided into two curves, one with lower intensity (closer to zero) and another one shifting right towards higher intensity. The first bell shaped curve represents the cells which have PMTL adsorbed only on the cell membrane, and hence these cells have weaker fluorescent intensity. The second bell shaped curve represents the cells which have uptaken more PMTL in addition to their cell membrane adsorption. The two overlaid curves in figure 4c shows the cell count below LCST (green curves) and above LCST (red curves). The curve above the LCST showed more number of cells having PMTL adsorbed on the cell membrane when compared to the curve below the LCST. Moreover, the fluorescent intensity of the cells increased and the curve shifted towards the right showing higher uptake by the cells above the LCST.

The two overlaid curves in figure 4f shows the BxPC-3 cell count below LCST (green curves) and above LCST (red curves). Here, the number of cells that have uptaken the fluorescently tagged liposome increased significantly compared to MiaPaca-2 cells. This result is in accord with the cell viability studies, where BxPC3 cells were more sensitive to the liposomes and resulted in higher cell growth inhibition. Furthermore, the curve above the LCST showed substantial shifting to higher fluorescent intensity showing faster cellular association of the liposome at 40°C. The results showed that the PMTL was internalized in a temperature dependent manner and as the temperature increased from 37°C to 40°C, the liposome-cell interaction


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improved effectively which is reflected in the cytotoxicity data. It is apparent that the kinetics of the cellular uptake are significantly faster than the drug release rates as it is observed that (thermo- dependent) cellular uptake starts within 30 minutes of incubation where the drug release is not exceeding 10% (figure 3) implying that the dominant cytotoxicity mechanism is the heat-induced cellular uptake and to lesser extent the thermally triggered drug release which presumably takes place in the cytosol.

Clonogenic cell survival assay The clonogenic cell survival assay was conducted to investigate the proliferative ability of survived MiaPaCa2 and BxPC-3 pancreatic cancer cells post treatment (Figure 5, 6, and 7). The clonogenic cell survival assay measures the long-term cytotoxic effects of drugs by measuring their ability to propagate from a single cell to a clone and produce a viable colony37. According to the results obtained for each concentration of drugs, some single cells formed colonies (100-200 cells), some remained single and few stopped dividing after formation of small colonies (5-20 cells). The number of colonies produced after treatment is inversely proportional to the drugs concentration. Figure 7 shows the standard colonies formed by MiaPaCa-2 and BxPC-3 cells. MiaPaCa-2 colonies were larger in size compared to BxPC-3 colonies. In addition the BxPC-3 cells formed colonies smaller than 50 cells which were excluded from the calculations.

The survival fraction (SF) of MiaPaCa-2 cells was calculated (Eq.3 and Eq.4) after exposure to the different concentration of drugs (from 0.001 to 100 μM) (Figure 8a). The SF of cells after treatment was inversely proportional to the drugs concentration (μM) i.e. as the concentration of the drugs decreased the SF of cells increased and more colonies were formed. Almost all the formulations showed significant reduction in the number of colonies and cell survival as GemCis concentration increased from 10 μM and 100 μM. At 0.001 to 0.1 μM, a striking difference was observed between the SF (%) of cells treated with PMTL at 37°C (BL) and at 40°C (AL). The SF of MiaPaCa-2 cells exposed to PMTL (BL) were almost 100%, indicating that the cells fully retained their ability to reproduce. Conversely, PMTL (AL) showed statistically significant reduction in the SF of cells compared to free drugs from 0.01 to 1 μM. There was no significant difference between the cell survival of TTL (BL) and TTL (AL) at lower concentrations (0.001 to 0.1 μM). The SF of cells exposed to



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TTL (AL) was reduced as the drug concentration increased to 1 μM but the overall SF obtained from TTL formulations were higher than free GemCis and no improvement in cytotoxicity was observed. On the other hand, PMTL (AL) was the only formulation which could reduce the SF % to less than 50% in all the concentrations tested and showed statistically significant reduction in SF% compared to free GemCis.

Exposing BxPC-3 cells to different concentrations of drugs also showed a dose dependent SF (Figure 8b). The gradual and steady decrease in cell survival was observed with an increase in drug concentration, as expected. However, as demonstrated earlier, BxPC-3 cells showed more sensitivity to the formulations and at lower concentrations (0.001 to 0.1 μM) almost all the formulations reduced the SF % significantly compared to free GemCis. Moreover, PMTL (AL) significantly decreased the SF % compared to free GemCis. Free GemCis combinational treatment inhibited the colony formation from 1 μM to 100 μM. In contrast to SF obtained from MiaPaCa-2 cells, the SF of TTL (BL and AL) and PMTL (BL) was significantly lower than the SF of free GemCis in BxPC-3 cells. As the concentration increased to 10 μM, the SF reduced to 0% by free GemCis and PMTL (AL) only. In all the concentrations, the PMTL (AL) was the only formulation which could inhibit the colonies growth to less than 50%. Additionally, the SF remained the least when the PMTL was applied in combination with heat at 40°C (AL).

Conclusion Overviewing the performance of TTL and PMTL, the PMTL formulations in combination with heat at 40°C (AL) could reduce the cell viability (%), decrease the IC50 of the chemotherapeutic drugs, enhanced the cellular uptake of the drugs and inhibited the proliferation ability of cells after treatment compared to the parent drugs. The presence of thermosensitive polymers on the surface of liposomes rendered the formulations as effective nano-carriers for the co- delivery of chemotherapeutic drugs to cancer cells. Therefore our study extends the application of smart polymers in the targeted delivery of therapeutics not just in the form of monotherapies with the use of a single anticancer drug, but also as a means to precisely manipulate and control the synergism multiple drug molecules in a single formulation. This strategy could potentially alleviate systemic toxicity issues while maximizing the therapeutic potency in a highly targeted manner.


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Acknowledgments The UCL Excellence Fellowship program and the Engineering and Physical Sciences Research Council (EPSRC; EP/M014649/1) are acknowledged for funding this project (GP).

Disclosure The authors report no conflicts of interest in this work.

Table 1. HPLC gradient method Time (min)

Buffer (%)

DMF (%)

0

95

5

15

95

5

20

70

30

30

20

80

31

95

5

35

95

5

Table 2. Size and distribution of loaded PMTL liposomes obtained by DLS PMTL loaded liposome

Temp. (°C)

Diameter (nm)

PDI

Zeta potential (mV)

Gem

37

149.4 ± 3.0

0.147 ± 0.021

-1.27

Cis

37

145 ± 4.0

0.192 ± 0.019

-1.31

GemCis

37

146.8 ± 3.1

0.233 ± 0.033

-1.83

± : Standard deviation obtained from three measurements.



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Table 3. The average encapsulation efficiency of liposomes Average (%)

SD

Gem loaded TTL

16.37

± 3.66

Cis loaded TTL

18.52

± 2.09

Gem

10.99

± 1.80

Cis

11.06

± 1.05

Gem loaded PMTL

11.06

± 2.46

Cis loaded PMTL

17.90

± 1.66

Gem

8.11

± 1.21

Cis

9.08

± 0.94

GemCis loaded TTL

GemCis loaded PMTL


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Table 4. The IC50 values of native and encapsulated drugs in TTL and PMTL, on two different pancreatic cancer cell lines MiaPaCa-2 and BxPC-3

Anti-cancer Drug

Gemcitabine

Type of liposome

Temp. (°C)

IC50 (μM) MiaPaCa-2

IC50 (μM) BxPC-3

Not encapsulated

37

3.0 ± 0.8

Very wide

40

2.8 ± 1.1

Very wide

37

7.3 ± 1.5

6.4 ± 0.8

40

1.7 ± 0.4

3.8 ± 0.2

37

5.4 ± 0.5

1.9 ± 0.5

40

1.6 ± 0.3

0.4 ± 0.1

37

13.0 ± 3.8

8.1 ± 1.3

40

12.7 ± 2.6

8.1 ± 0.9

37

Very wide

*

40

11.0 ± 3.5

14.6 ± 3.3

37

*

12.4 ± 3.3

40

2.0 ± 0.4

2.8 ± 0. 3

TTL

PMTL

Not encapsulated

Cisplatin

TTL

PMTL



Not encapsulated

GemCis

TTL

PMTL

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37

1.5 ± 0.1

Very wide

40

1.3 ± 0.2

Very wide

37

3.0 ± 0.8

2.1 ± 0.6

40

1.0 ± 0.3

0.6 ± 0.1

37

7.5 ± 1.6

0.5 ± 0.2

40

0.5 ± 0.1

0.2 ± 0.1

*: Did not kill 50% of the cell population. ± : Standard deviation obtained from three measurements.

Table 5. CI values of GemCis loaded liposomes on MiaPaCa-2 cells

Free drugs

TTL

PMTL

Temp. (°C)

CI

37

0.62

40

0.57

37

-

40

0.68

37

-

40

0.56

- CI was not calculated due to the absence of IC50.

Table 6. CI values of GemCis loaded liposomes on BxPC-3 cells

Free drugs

TTL

Temp. (°C)

CI

37

-

40

-

37

-


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PMTL

40

0.20

37

0.30

40

0.57

- CI was not calculated due to the absence of IC50.



Figure 1. a) The chemical structure of the components used for the PMTL formulations and b) After application of heat, the TR block undergoes coil to globule transition and leads to disruption of lipid membrane and content release.

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Figure 2. TEM images of PMTL encapsulated a. Gemcitabine only b. Cisplatin only c. GemCis in combination. The scale bars show the size of 500 nm. The frequency histograms show the mode and average diameter of liposomes loaded with d. Gemcitabine only e. Cisplatin only f. GemCis in combination at 37° C using ImageJ. The DLS graphs show the size distribution of liposomes loaded with g. Gemcitabine only h. Cisplatin only i. GemCis in combination at 37° C in triplicate.



Figure 3. Combinational loaded a. TTL b. PMTL liposomes revealed controlled release profile below (BL) and above LCST (AL).

Figure 4. Fluorescence microscopy images of MiaPaCa-2 cells incubated with fluorescent liposome a. below, and b. above the LCST (Scale bars are 400 μm) with the respective flow cytometry graph in c. Fluorescence microscopy images of BxPC-3 cells incubated with fluorescent liposome d. below, and e. above the LCST (Scale bars are 1000 μm) with the respective flow cytometry graph in f.


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Figure 5. Digital photographs of the clonogenic assay of MiaPaCa-2 cells performed with the combination of free GemCis, loaded in TTL and PMTL below and above the LCST for 14 consecutive days in 6-well plates (the diameter of each well is approx. 34.8 mm).



Figure 6. Digital photographs of the clonogenic assay of BxPC-3 cells performed with the combination of free GemCis, loaded in TTL and PMTL below and above the LCST for 14 consecutive days in 6-well plates (the diameter of each well is approx. 34.8 mm).

Figure 7. Standard colonies formed in a. MiaPaCa-2 cells b. BxPC3 cells without application of formulations after 14 days. The scale bars show the size of 1000 μm.


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Figure 8. The percentage survival fraction of a. MiaPaCa-2 b. BxPC-3 cells post treatment with GemCis loaded TTL and PMTL after 14 days. P value 0.0001 to 0.001 indicated with *** means extremely significant; P value 0.001 to 0.01 indicated with ** means very significant; P value 0.01 to 0.05 indicated with * means significant. The P values were calculated from comparison of the percentage survival fraction of the nanoformulations with the free GemCis.

Supplementary Information Measurements of PMTLs by dynamic light scattering.



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We report on thermoresponsive polymer-coated liposomes for targeted combinational therapy of pancreatic cancer. 165x69mm (96 x 96 DPI)

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Dual Controlled Delivery of Gemcitabine and Cisplatin using Polymer

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