Self-Healing pH- and Enzyme Stimuli-Responsive Hydrogels for

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Self-Healing pH- and Enzyme Stimuli-Responsive Hydrogels for Targeted Delivery of Gemcitabine to Treat Pancreatic Cancer Panayiotis Bilalis, Dimitrios Skoulas, Anastasios Karatzas, John Marakis, Athanasios Stamogiannos, Chrisida Tsimblouli, Evangelia Sereti, Efstratios Stratikos, Konstantinos Dimas, Dimitris Vlassopoulos, and Hermis Iatrou Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00959 • Publication Date (Web): 10 Aug 2018 Downloaded from http://pubs.acs.org on August 11, 2018

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Self-Healing pH- and Enzyme Stimuli-Responsive Hydrogels for Targeted Delivery of Gemcitabine to Treat Pancreatic Cancer Panayiotis Bilalis,1 Dimitrios Skoulas,1 Anastasios Karatzas,1 John Marakis,2 Athanasios Stamogiannos,3 Chrisida Tsimblouli,4 Evangelia Sereti,4 Efstratios Stratikos,3 Konstantinos Dimas,4 Dimitris Vlassopoulos2 and Hermis Iatrou*,1

1. University of Athens, Department of Chemistry, Panepistimiopolis, Zografou, 15771, Athens, Greece 2. FORTH, Institute for Electronic Structure and Laser, Heraklion 70013, Greece and University of Crete, Department of Materials Science & Technology, Heraklion 70013, Greece 3. National Centre for Scientific Research Demokritos, Patriarhou Gregoriou and Neapoleos 27, Agia Paraskevi 15341, Athens, Greece. 4. Department of Pharmacology, Faculty of Medicine, University of Thessaly, Larissa, Greece

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Abstract. A novel, multifunctional hydrogel that exhibits a unique set of properties for the effective treatment of pancreatic cancer (PC) is presented. The material is comprised of a pentablock terpolypeptide of the type PLys-b-(PHIS-co-PBLG)-PLys-b-(PHIS-co-PBLG)-bPLys which is a non-cytotoxic polypeptide. It can be implanted via the least invasive route and selectively delivers gemcitabine to efficiently treat PC. Simply mixing the novel terpolypeptide with an aqueous solution of gemcitabine within a syringe results in the facile formation of a hydrogel that has the ability to become liquid under the shear rate of the plunger. Upon injection in the vicinity of cancer tissue, it immediately reforms into a hydrogel due to the unique combination of its macromolecular architecture and secondary structure. Due to its pH responsiveness, the hydrogel only melts close to PC, thus the drug can be delivered directionally towards the cancerous rather than healthy tissues in a targeted, controlled and sustained manner. The efficacy of the hydrogel was tested in-vivo on human to mouse xenografts using the drug gemcitabine. It was found that the efficacy of the hydrogel loaded with only 40% of the drug delivered in one dose, was equally or slightly better to the peritumoral injection of 100% of the free drug delivered in two doses, the typical chemotherapy used in clinics so far. This results suggest that the hydrogel can direct the delivery of the encapsulated drug effectively in the tumor tissue. Enzymes lead to its biodegradation, avoiding removal by resection of the polypeptidic carrier after cargo delivery. The unique properties of the hydrogel formed can be predetermined through its molecular characteristics, rendering it a promising modular material for many biological applications.

Keywords: synthesis of polypeptides, self-healing hydrogels, targeted drug delivery, pH- and enzymatically stimuli-responsive, cancer 2 ACS Paragon Plus Environment

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INTRODUCTION While the overall incidence of cancer mortality has steadily decreased over the last several decades for most types of cancer, the mortality of pancreatic cancer (PC) is still increasing. PC is the fourth leading cause of all cancer-related deaths in the United States, fifth in Europe, and the seventh worldwide, while the 5-year survival rate is only 8%.1 Compared to other cancers, PC has the unique property of high stromal-to-epithelial ratio which inhibits the formation and the function of blood vasculature, thereby diminishing drug delivery via perfusing blood, and thus through nanoparticles as nanocarriers.2-4 Due to these barriers, the fraction of nanoparticles that penetrate the tumor cells may be less than 0.7%.5 It is clear that with this tumor in particular, it is necessary to apply a local administration technology through drug-loaded implants rather than nanoparticulate drug carriers. Drug-loaded implants are administered directly at the site of disease, offering the advantages of controlled and prolonged drug release, direct delivery to the site of disease, hence minimizing drug waste and reduced side effects due to the avoidance of systemic circulation of chemotherapeutic drugs. The best approach to achieve local drug release is through polymeric hydrogels because they can achieve effective, selective and sustained release in order to reduce patient morbidity.6 Nearly all of the self-healing, pH- and temperature-responsive hydrogels considered so far are characterized by an ability to turn from viscous liquids in healthy tissue environment to hydrogels when injected within a cancer tissue. The sol-gel transition is due to shear thinning properties of the materials,7-8 the higher temperature9-15 and/or the lower pH16-17 of the cancer tissue.18-20 However, this mode of hydrogel formation can wound the cancer tissue, often leading to metastasis. Clinical oncologists face two different situations: in the first, the PC tissue is developed in a localized place on the pancreas, which can be eliminated by resection. In this case a localized drug delivery system that will be placed on the resected tissue and selectively eliminate 3 ACS Paragon Plus Environment


remaining cancer cells that could cause relapse of the primary cancer or even initiate further metastasis is preferred. In the second situation, cancer is spread over the pancreas. Here, it is not possible to resect a part of the organ, and oncologists prefer a localized drug delivery system that would deliver drugs only to the pathological site but would remain inactive towards healthy tissue. In addition, the implant carrying the drug should be administered close to the cancer tissue with the most facile, controlled and less invasive way in order to reduce patient morbidity. To meet these challenges, we envisaged a hydrogel that would operate in a completely different way when compared to most systems utilized so far. The non-cytotoxic hydrogel will exhibit shear-thinning properties, in order to be injected in-situ and transferred with the thinnest possible needle in order to be implanted in the least invasive way. The hydrogel should have the shortest possible self-healing time so as to be reconstructed quickly within the body and achieve a controlled deposition mechanism close to the cancer tissue. It should respond to the lower extracellular pH values of the cancer tissue, so as to melt in the vicinity of the pathological tissue, thus selectively delivering the drugs to PC tissue. The hydrogel would remain intact when in contact with the healthy tissue, minimizing the loss of the drug from this site. It should have buffering capacity in order to exhibit slow degradation rates and achieve slow and sustained drug release. In addition, it should be enzymatically biodegradable, so as not to require removal through resection after drug delivery (Scheme 1). Recently, we have demonstrated the controlled polymerization of histidine. Poly(Lhistidine) (PHIS) is the only pH-responsive amongst all homopolypeptides that undergoes a solubility transition within the physiological and tumor tissue pH and possesses many of the desired properties envisaged for the hydrogel.21-27 Herein, by using precise synthesis and living polymerization techniques, we present the synthesis of a series of novel amphiphilic pentablock terpolypeptides. After investigating their 4 ACS Paragon Plus Environment

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properties, the nature of the amino acids as well as the molecular characteristics were carefully modified to develop a polypeptide that forms a multifunctional hydrogel exhibiting the optimum properties combined in one material in order to achieve effective drug delivery. As a proof of concept, we show that it is suitable to be used for the targeted local delivery of gemcitabine to PC without effecting healthy tissues, as confirmed by in-vivo studies in mice exhibiting human PC xenografts.

Scheme 1. Illustration of the chemical treatment of PC: the hydrogel with the encapsulated drug is implanted in the least invasive way and melts only in the vicinity of the cancerous tissue due to the lower pH, leading to a targeted and directional delivery.

EXPERIMENTAL SECTION Materials and Methods Materials. Boc-HIS(Trt)-OH (>99%) was purchased from SENNCHEM. Triphosgene (99%) was purchased from Acros Organics. Thionyl chloride (>99%), L-Lysine (>99 %), γ-BenzylL-glutamate and H-Leu-OH (>99 %) were purchased from Aldrich. Triethylamine (>99%, 5 ACS Paragon Plus Environment


Acros Organics) was dried over calcium hydride for one day and then distilled and stored in the vacuum line over sodium. The appropriate quantity needed was freshly distilled in a vacuum line. Purification of THF (dried, max 0.005% water, Merck) was performed using standard high vacuum techniques reported elsewhere.1 Ethyl acetate (>99.5%, Merck) was fractionally distilled over phosphorous pentoxide. Hexane (>99%, Merck) was fractionally distilled over sodium. Dichloromethane was purchased by Merck. DMF (99.9+ %, special grade for peptide synthesis with less than 50 ppm of active impurities), the polymerization solvent, was purified by short-path fractional distillation under vacuum in a custom-made apparatus. The middle fraction was always used. Thiophene free benzene was treated with calcium hydride and it allowed to be stirred overnight to react with moisture. Then, it was distilled under high vacuum in a nearby flask containing n-BuLi, which reacted and stored.1,6-diaminohexane (98%, Alfa Aesar) was distilled fractionally under high vacuum and it was treated with sodium at room temperature for one day. Then, it was subsequently distilled into pre-calibrated ampules with break-seals. It was then diluted with DMF to the appropriate concentration in a sealed apparatus equipped with pre-calibrated ampules and kept away from light. Diethyl ether was purchased from Aldrich. Trifluoroacetic acid (TFA) (>99 %) was purchased by Merck. Fluorescamine and Trypsin were purchased from Aldrich.

Instrumentation. Size-exclusion chromatography. Size-exclusion chromatography (SEC) was used to determine the Mn and Mw/Mn values. The analysis was performed using a SEC equipment composed of a Waters 600 high pressure liquid chromatographic pump, Waters Ultrastyragel columns (HR-2, HR-4, HR-5E and HR-6E), a Waters 410 differential refractometer detector and a Precision PD 2020 light scattering detector at 60oC featuring two detectors at 150 and 900 (TALLS). A DMF solution containing 0.1N LiBr was used as an eluent at a rate of 1mL/min, operating at 60 oC. 6 ACS Paragon Plus Environment

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NMR spectroscopy. 1H NMR spectroscopy (300 MHz) were performed using a Varian Unity Plus 300/54 spectrometer. The spectra of the NCAs were taken in CDCl3 at room temperature. FTIR. FTIR measurements were performed with a Perkin Elmer Spectrum One instrument, in KBr pellets at room temperature, in the range 450-4000 cm-1. Circular Dichroism. Circular Dichroism was performed with a Jasco J-815 model, featuring a Peltier model PTC-423S/15 thermo stabilizing system. The cell used was 1 mm Quartz Suprasil. The aqueous solution of the PBLG/PHIS polymer with ratio of the monomeric units 50/50 had a concentration of 3.3x10-4 g/mL, while the aqueous solution of the PBLG/PHIS polymer with ratio of the monomeric units 70/30 at every pH had a concentration of 2.5x10-4 g/mL. The adjustment of pH was performed through addition of diluted HCl or NaOH. Scanning electron microscopy. Scanning electron microscopy/SEM–EDS of the hydrogels was performed with a Jeol JSM-5600 equipped with Oxford EDS. Carbon grids were used. The preparation of the samples was performed first by quick deep freezing of a small amount of the hydrogels with liquid nitrogen, followed by freeze-drying to remove the water. Sputter coating of the samples with gold occurred prior the measurements. Enzymatic Degradation-Fluorescence measurements. All fluorescence measurements were performed using a TECAN SPARK 10M multimode microplate reader using 96-well black microplates (Greiner). To measure the enzymatic degradation of the hydrogel by either Trypsin or Leucine Aminopeptidase (LAP), 2mg of hydrogel was mixed with 0,1nM Trypsin or 100nM LAP in PBS and incubated at 37 oC for 1 hour or 24 hours respectively. At the end of the incubation, a sample of 10 µL from the reaction was mixed with 90 µL of a solution of fluorescamine containing 1 mg/ml fluorescamine in acetonitrile, 15 µL of 0.1M borate buffer at pH 8.0 and 145 µL of MilliQ water. After 5 min, the fluorescence was measured using excitation at 405 nm and emission at 485 nm.



Synthesis of the Terpolypeptides.

A. Synthesis of the Monomers. The synthesis of the monomers, i.e. ε-tertbutyloxycarbonyl-L-lysine N-carboxy anhydride, Nim–trityl-L-histidine N-carboxy anhydride, γ-benzyl-L-glutamate N-carboxy anhydride as well as L-leucine N-carboxy anhydride has been presented in our previous publication,21 and are given briefly in the SI.

B. Synthetic Approach of the Polypeptides. The polypeptides were synthesized using high vacuum techniques.28-29 The polymerization reactors were designed to have volumes at least three times larger than that of the CO2 generated by each polymerization. Polymerizations were carried out with 1,6-diaminohexane as the initiator. Briefly, in a solution of ε-tert-butyloxycarbonyl-L-lysine N-carboxy anhydride in DMF (Lys-NCA), an α,ω-hexamethylenediamine solution in DMF was added. After completion of polymerization (middle block), appropriate amounts of Nim-trityl protected Lhistidine N-carboxy anhydride (HIS-NCA) and γ-benzyl-L-glutamate N-carboxy anhydride (BLG-NCA) (molar ratio 1:1) were dissolved in DMF and added to the reaction flask. The polymerization was allowed to continue for 6 days (two hydrophobic blocks). Lys-NCA was then added, followed by precipitation in diethylether after the completion of the polymerization. The selective cleavage of trityl and tert-butyloxycarbonyl groups from poly(L-histidine) and poly(L-lysine) segments, respectively, was achieved by treating with trifluoroacetic acid (TFA), followed by addition of triethyl silane. The solution was precipitated in diethylether, and the white solid was filtered and dried. The deprotected pentablock was then mixed with MilliQ water and dialyzed (membrane molecular weight cut off 3500 Daltons) against dilute HCl solution (pH = 3) for 2 days, dilute NaOH solution (pH = 8) for 2 days and DI water for 2 days (for each step the water changed every 12 h). Finally, the solution was lyophilized in order to obtain the final pentablock terpolypeptide as a white


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powder. A similar procedure was followed for the synthesis of the terpolypeptides exhibiting poly(L-leucine) (PLEU) instead of PBLG.

Formation of Empty Hydrogels and Hydrogels Loaded with Gemcitabine. Pentablock terpolypeptide hydrogels were prepared very simply by adding MilliQ water in a vial containing the solid polypeptide. After 2-3 hours, the hydrogel was ready without requiring any other treatment. The water swelled the polypeptide, keeping the general shape of the initial solid. Pentablock terpolypeptide hydrogels loaded with gemcitabine (Hydrogem) were prepared by first dissolving the appropriate amount of gemcitabine in pyrogen-free MilliQ water (gemcitabine:polypeptide ratio 1:1 (w/w)), at 35 oC, followed by addition of the warm gemcitabine solution to the polypeptide. The temperature was kept at 35 oC until complete swelling of the polypeptide and formation of homogeneous hydrogel, about 3-4 hours. In order to ensure the formation of a homogeneous mixture, the hydrogel was inserted into a syringe and passed between two syringes multiple times using a syringe connector, prior to the in-vivo tests. The hydrogel was stable at room temperature for several days, while the same gemcitabine solution in MilliQ water precipitated after 0.5 hours when the temperature was lowered to 20 oC.

Culture of Pancreatic Cancer Cells AsPC-1 human PC cell line (adenocarcinoma derived from ascites) purchased by ATCC (ATCC-LGC, Germany), was cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 100U/ml penicillin + 100µg/ml streptomycin (Gibco, Grand Island, NY, USA), 2mM L-glutamine (Gibco, Grand Island, NY, USA), 5% fetal bovine serum (Lonza, Verviers, Belgium), at 37°C in a humidified atmosphere containing 5% CO2.



In Vivo Studies. For the in-vivo studies, NOD.CB17-Prkdcscid/J mice purchased from Jackson Laboratory (The Jackson Laboratory 600Main Street Bar Harbor, Maine 04609 USA) were used. All animals for in vivo experiments were kept under specific pathogen free (SPF) conditions in type IIL cages, at the experimental unit of our department (Department of Pharmacology, EL42-BIO_Exp03, protocol license 5542/22806-30/11/2015) in a climate-regulated environment (21 ± 1°C; 50-55% relative humidity) under a 12 h/12 h (lights on at 7:00 AM) light/dark cycle and allowed ad libitum food and water. Female and male mice, 6–8 weeks old, were used in the studies described here. Handling and experimentation of animals complied with Greek laws (PD 56/2013 and Circular 2215/117550/2013) and the guidelines of the European Union (2013/63/EU).

Acute Toxicity Testing of the Empty Hydrogel. Acute Toxicity Testing was performed following the guidelines of the NCI (NCI, NIH, USA) protocol30 for acute toxicity dosing. A single female mouse was given a single injection of 400 mg/kg, a second mouse received a dose of 200 mg/kg and a third mouse received a single dose of 100 mg/kg. The mice were observed for a period of 2 weeks. They were sacrificed if they lost more than 20% of their body weight or if there were other signs of significant toxicity. In a preliminary experiment to select the least toxic form of hydrogel between PLEU and PBLG, three female mice received a single dose of hydrogel subcutaneous (sc) at the dorsal area of gemcitabine at 400, 200 and 100mg/kg (320, 160 and 80µl, respectively) of a 1/30 (w/w) hydrogel formulation and one received hydrogel intra-peritoneally (ip, 200mg/kg). Mice were observed daily for a total period of 2 weeks. For the needs of the experiments, the parameters probed and recorded were survival, weight loss and behavioral changes.


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Gemcitabine Toxicity Testing. In the first experiment, gemcitabine (Gemsar VL7501, Farmaserve-Lilly) was administered to three male mice at three different doses (one dose/mouse). Gemcitabine (400, 200 and 100mg/kg) was administered in a single dose either ip or sc, and the mice were observed daily to detect and record any signs of toxicity. Sub-chronic Toxicity of Gemcitabine. Gemcitabine was further evaluated for toxicity according to the administration schedule adopted for the efficacy studies. For this purpose, three mice received two injections of gemcitabine, ip or sc, at a week interval. Again, three gemcitabine doses were tested, 200mg/kg, 100mg/kg and 50mg/kg (one mouse per dose).

In-Vivo Efficacy Testing. To generate xenografts, exponentially growing cultures of AsPC-1 human PC cells were subcutaneously injected at the axillary region of the rear flank of 8 week-old male NOD.CB17Prkdcscid/J

mice

from

our

animal

facility

(1x106 cells/injection,

one

injection/mouse). When the tumors reached a size of about 200mm3 (advanced stage model) the mice were arbitrarily divided into 5 groups, each group consisting of 7 mice (7 tumors/group). Mice received A) empty hydrogel sc in the vicinity of the tumor (peritumoral administrations, pt) B) 100mg/kg gemcitabine ip C) 100mg/kg gemcitabine pt and D) 100mg/kg of gemcitabine loaded in hydrogel pt. All mice received two injections, once a week except for the group D mice that received only one injection at the beginning of the administration schedule. Another group of 7 mice received no treatment and served as the control group. In order to study the in-vivo effect of the compounds, mean tumor volume and standard deviation (SD) for all groups were calculated and growth curves were plotted as a function of time. Tumor volume was calculated according to the formula [(axb2)/2], where a=length and b=width of the tumor as measured with a vernier’s caliper (measurements were performed twice a week). Activity of the compounds was further evaluated by calculating the 11 ACS Paragon Plus Environment


tumor growth inhibition (% DΤ/DC ratio).31 The DT/DC ratio represents the mean tumor volume for the treated groups versus control. DT and DC were calculated as the (mean tumor volume at day X) - (mean tumor volume at the first day of the treatment), in treated and control groups respectively. Mice remained in the study until the tumor volume reached a size of approximately 1600-1800mm3 and were then euthanized. Mice were also weighed to monitor toxicity twice a week.

Rheology. The polypeptides were characterized by means of dynamic oscillatory measurements using a sensitive strain-controlled rheometer (ARES 100FRTN1 from TA, USA). They included a) Dynamic frequency sweeps with a strain ߛ = ߛ଴ sin ߱‫ ݐ‬in order to probe the viscoelastic relaxation spectrum, i.e., the frequency dependent storage (‫ܩ‬′) and loss (‫ܩ‬′′) moduli in the range 0.01 – 100 rad/s. The stress response was ߪ = ߪ଴ sinሺ߱‫ ݐ‬+ ߜ ሻ = ߛ଴ ሺ‫ܩ‬′ sin ߱‫ ݐ‬+

‫ܩ‬′′ cos ߱‫ݐ‬ሻ with ߪ଴ being the stress amplitude and ߜ the phase angle (ߜ = ‫ ܩ‬ᇱᇱ /‫ܩ‬′); b) Dynamic strain sweeps at constant frequency ω (typically 1 rad/s) and continuously increasing strain amplitude ߛ଴ from 10-3 to 3 strain units, in order to determine the limits of linear viscoelasticity and at the same time to explore the nonlinear response; c) Dynamic time sweeps at low frequency (typically 1rad/s) and a linear strain amplitude, with duration long enough to ensure that steady state was reached. In order to ensure that the measurements were reproducible, they were proceeded by a time sweep at large strain amplitude in the nonlinear regime (typically at strain amplitude of 2 strain units) or steady shear measurements at different rates (from 1‫ି ݏ‬ଵ to 100‫ି ݏ‬ଵ ) in order to shear-melt the structure (rejuvenation process), and a subsequent linear time sweep in order to reach steady state (the kinetic process toward steady state is the physical aging of the sample and, depending of its nature, it may relate to self-healing).

This protocol allowed to erase the sample’s mechanical history 12 ACS Paragon Plus Environment

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(possible residual stresses during sample preparation and loading) and ensuring reproducible initial conditions for the measurements. The measurements were performed with a stainless steel cone-and-plate geometry (diameter 25 mm, cone angle 0.1 rad) and the temperature was controlled by means of a Peltier element. To minimize the risk of solvent evaporation, two approaches were implemented depending on sample availability. One consisted of effectively sealing the measred specimen with a non-volatile immiscible low-viscosity fluid (silicon oil of viscosity 5 mPas) by means of a home-made cyclindrical collar that was placed around the cone. The other made use of a home-made solvent trap that saturated the atmosphere around the specimen with solvent. Statistical Analysis. Statistical analysis was performed using ANOVA or student’s t-test and the GraphPad Prism ver. 6 software package (GraphPad Software, Inc. La Jolla, USA). A p

Self-Healing pH- and Enzyme Stimuli-Responsive Hydrogels for

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