1 Functionalized Hyperbranched Polyethyleneimines as

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Functionalized Hyperbranched Polyethyleneimines as Thermosensitive Drug Delivery Nanocarriers with Controlled Transition Temperatures Zili Sideratou, Maria Agathokleous, Theodossis Athanassios Theodossiou, and Dimitris Tsiourvas Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01325 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 9, 2018

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Functionalized Hyperbranched Polyethyleneimines as Thermosensitive Drug Delivery Nanocarriers with Controlled Transition Temperatures

Zili Sideratou†, Maria Agathokleous†, Theodossis A. Theodossiou†,‡, Dimitris Tsiourvas*†

†

Institute of Nanoscience and Nanotechnology, National Centre for Scientific Research

“Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece. ‡ Current address: Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, NO-0379 Oslo, Norway.

* To whom correspondence should be addressed Institute of Nanoscience and Nanotechnology, National Centre for Scientific Research ‘‘Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece. Tel.: +30-210-6503616; Fax: +30-210-6511766. E-mail:[email protected]

1 ACS Paragon Plus Environment

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Abstract The low critical solution temperature phase transition (Tc) that is exhibited by thermosensitive polymers is strongly dependent on polymer concentration, pH, ionic strength, as well as on the presence of specific molecules or ions in solution. Therefore, polymers with Tc values above 37 oC that are useful for hyperthermia therapy are not readily available. In the present study, temperature-sensitive hyperbranched polyethyleneimine derivatives were developed through stepwise functionalization with isobutylamide groups. Although factors such as the concentration of polymer, sodium chloride, phosphate ions, or pH, considerably affect the transition temperature, it was possible to obtain a hyperbranched derivative having the required Tc (38-39 oC) for the given aqueous medium required in cell experiments, by the careful selection of the degree of substitution. This thermosensitive derivative can encapsulate doxorubicin (DOX), a well-known anticancer agent, and was further studied as a temperaturetriggered drug delivery system. While the polymeric carrier showed no notable toxicity at temperatures either below or above the transition temperature, the thermoresponsive drugloaded formulation exhibited increased DOX cellular uptake and improved in vitro cytotoxicity at 40oC.

Keywords: drug delivery systems, hyperbranched polyethyleneimine, thermosensitive polymers, hyperthermia.


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Introduction In local mild hyperthermia therapy a malignant tissue is heated at slightly elevated temperatures (40–41 ◦C) complementing and improving the efficacy of anticancer drugs by increasing tumor blood flow and vascular permeability, and by upregulating drug influx into the tumor.1,2 Several methods such as microwave irradiation, high-intensity focused ultrasounds, or radiofrequency thermal ablation, are employed for increasing the temperature topically. This treatment is expected to afford increased therapeutic outcomes and reduce side effects of chemotherapy when combined with drug delivery systems that could preferably release their cargo at the above temperature range. As a result, the drug load would be released almost exclusively in the heated area achieving high local concentrations and avoiding its release in non-malignant tissues, which would further minimize pharmacological side effects. For this reason a large number of thermoresponsive or thermally-triggered drug delivery systems have been tested, mainly liposomes3,4and polymers. The latter group consists of either polymer-drug conjugate systems or polymeric (nano)particles, i.e. polymeric micelles, polymersomes and hydrogels.5,6,7 Thermosensitive or thermoresponsive polymers are characterized, among others, by a reversible phase transition, i.e., at low temperatures are water soluble while above a critical transition

temperature

(Tc),

they

disolvate,

change

their

conformation,

their

hydrophilic/hydrophobic balance and, finally, phase separate forming aggregates.8,9,10 Specifically, above Tc partial miscibility occurs and two separate phases, a polymer-rich and a water-rich phase, coexist in equilibrium. These so-called thermosensitive or temperatureresponsive polymers belong to the broader class of responsive polymers, which exhibit rapid and reversible changes in their conformation in response to an external stimulus and have attracted the attention of the scientific community during the last decade.11,12,13 The most prominent example is that of linear poly(N-isopropylacrylamide), PNIPAM,



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that exhibits a Tc in the temperature range of 30-34 οC and has been extensively studied in relation to its ability to act as a drug carrier.14 Although a large number of different polymers have been also studied, it is still not easily attainable to develop polymers with transition temperatures within the required tight temperature range of 38-40 οC, particularly if one takes into account that the transition temperature strongly depends on the concentration of the polymer in water medium as clearly evident in every polymer binary solution phase diagram,15 as well as on the pH, and the presence of the encapsulated drug and of specific ion(s) dissolved in the aqueous phase.16 The last, is the result of the effect of specific interactions, first studied by Franz Hofmeister, describing the effectiveness of various ions to either solubilize or precipitate proteins, between ions and macromolecules and between ions and the water molecules that are directly in contact with the macromolecules.17,18,19,20,21 Their effect on the transition temperature is of particular importance for bio-applications since a variety of ions is present both in pharmaceutical formulations as well as in the biological fluids. To overcome this drawback, various copolymer systems have been proposed in order to provide macromolecular systems with tailored transition temperatures.11,12,13,14,22,23,24,25 A separate, intensively explored, category of macromolecules26,27,28 with a tree-like topology in the molecular level is that of dendritic polymers (dendron = tree) that encompasses dendrons, dendrimers, dendrigrafts and hyperbranched polymers. Among them, hyperbranched polymers are characterized by their ease of preparation, generally by a low cost one-step polymerization reaction, and by the existence of a large number of end groups. These macromolecules due to their structural features are able to physically encapsulate in their interior small molecules, such as bioactive compounds, and are therefore for many years studied as prospected drug delivery systems. Additionally, the possibility to modify their end groups with appropriate functional groups that can impart a desired specific characteristic in the macromolecule, enables the design and development of drug delivery systems with


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desirable and well defined properties, paving the way for future clinical applications.29,30,31 Taking advantage of the ease of functionalization, the introduction of appropriate hydrophobic groups on the end groups of water-soluble hyperbranched polymers can result in a modification of hydrophobic/hydrophilic nature of the polymer. Upon careful selection of the hydrophobic group and of the degree of functionalization, well-known dendrimers, such as poly(amidoamine) or poly(propylene imine),32 or hyperbranched polymers, such as poly(glycidol)33 or polyethyleneimine,34,35,36,37 exhibiting Tc in aqueous solutions were obtained. Due to this property, this class is of interest in drug delivery applications since they can, at least in principle, exhibit thermally-triggered drug release.9,10 The objective of this work was to synthesize a series of polyethyleneimine (PEI) derivatives bearing isobutylamide groups and explore the possibility to accurately tailor the Tc of these derivatives by precisely controlling the degree of functionalization. For this reason a series of isobutylamide functionalized PEI with different degrees of substitution was synthesized, and their transition temperature was studied in aqueous solutions as a function of a variety of parameters and specifically of polymer concentration and pH, as well as the concentration of phosphates and sodium chloride, i.e. the typical inorganic salts present in biological fluids and commonly employed in cell culture experiments for providing the necessary buffering capacity and osmolarity. The detailed study of their effect on the polymer response is essential for optimizing the design of a specific drug delivery system encapsulating doxorubicin (DOX), a widely used anticancer drug.38 Among the synthesized derivatives, a DOX loaded thermoresponsive derivative was selected that had a transition temperature of 38 oC, under the conditions required for the in vitro experiments. This system was administered to MCF-7 cells and cell uptake as well as cell viability was studied both above and below Tc.



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2. Experimental 2.1. Materials and Methods Isobutyric acid (≥99.5%) and isobutyryl chloride (98%) were purchased from Fluka and Sigma-Aldrich Ltd. (Poole, UK), respectively. Triethylamine (TEA) and ammonium hydroxide solution, 28% NH3 in H2O, were purchased from Μerck Schuchardt OHG. Ν,Ν΄dicyclohexylcarbodiimide

(99%,

DCC),

Ν-hydroxysuccinimide

(98%,

NHS),

N,N-

diisopropylethylamine (DIPEA), dialysis tubes (molecular weight cut-off: 1200), RPMI 1640, Opti-MEM without phenol red, fetal bovine serum (FBS), penicillin/streptomycin, Lglutamine, phosphate buffer saline (PBS), thiazolyl blue tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Ltd. (Poole, UK). Nhydroxybenzotriazole (HOBt) and 2-(1H-benzotriazole-1-yl)- 1,1,3,3-tetramethyluronium (HBTU) were purchased from Anaspec (San Jose, USA). Doxorubicin hydrochloride (DOX) and branched polyethyleneimine (PEI) with a molecular weight of 5000 Da (Lupasol® G 100) were kindly donated by Regulon SA (Athens, Greece) and BASF (Ludwigshafen, Germany), respectively. All solvents used were distilled before use, while all aqueous solutions were prepared using Millipore water. 1

H and

13

C NMR spectra were recorded in D2O or CDCl3 by a Bruker Avance DRX

spectrometer operating at 500 and 125.1 MHz, respectively. Inverse-Gated 13C NMR (IG 13C NMR) was employed to determine the ratio of primary:secondary:tertiary amino groups of PEI, as well as of the number of isobutyl groups attached on the functionalized PEI derivatives. FTIR studies were performed using a Nicolet 6700 spectrometer (Thermo Scientific, Waltham, MA) equipped with an attenuated total reflectance accessory with a diamond crystal (Smart Orbit, Thermo Electron Corporation, Madison, WI). Samples were firmly pressed against the diamond and spectra were recorded at 4 cm-1 resolution. A minimum of 64 scans were collected and signal averaged.


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Dynamic light scattering measurements were performed on a ALV/CGS-3 Compact Goniometer System (ALV GmbH, Germany), using a JDS Uniphase 22 mW He-Ne laser, operating at 632.8 nm, and an Avalanche photodiode detector at an angle of 90°, interfaced with a ALV-5000/EPP multi-tau digital correlator with 288 channels and a ALV/LSE-5003 light scattering electronics unit for stepper motor drive and limit switch control. For each dispersion at least five light scattering measurements were acquired, and the autocorrelation functions were analyzed using the CONTIN algorithm to obtain the apparent hydrodynamic radii distribution. Typically, correlation functions were collected for 20 s. Fits to the correlation functions were made using the software provided by the manufacturer. ζ-potential measurements were conducted at temperatures above the phase transition (typically 38-40 oC) using the ZetaPlus of Brookhaven Instruments Corp. (Long Island, NY, USA) equipped with a 35 mW solid state laser emitting at 660 nm and with an in-built temperature controller able to stabilize the temperature within ± 0.5 oC. In a typical experiment, 125 µL dispersions of polymers kept at temperatures above the phase transition were added into 1.5 mL of water kept also at the same temperature to a final concentration of 0.15 mg/mL. From the determined electrophoretic mobility, the ζ-potential of the dispersions was calculated using the Smoluchowski equation. Ten measurements were collected for each experiment, and the results were averaged. The turbidity of solutions of PEI derivatives in various aqueous media was measured at 500 nm using a Cary 100 Conc UV-Visible spectrophotometer (Varian Inc., Mulgrave, Victoria, Australia). Quartz cuvettes of 1 mm path length were inserted in water thermostated cell holder connected to a Julabo ME-4 heating circulator (JULABO GmbH) with a temperature stability of ± 0.01 °C. The heating rate of the sample cell was maintained at 1 οC min-1 and the onset of the transition, taken as the initial break point of the obtained turbidity vs. temperature curve, was determined using Origin® (MicroCal, Northampton,



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MA). In all cases three independent measurements were performed and the obtained standard deviation was better than ± 0.4 oC. In the few exceptional cases that the transition temperature was higher than 70 oC, solutions were kept in sealed glass cuvettes to avoid water evaporation during the measurements. Scanning electron microscopy studies were conducted employing a Jeol JSM 7401F Field Emission Scanning Electron Microscope equipped with Gentle Beam mode. Gentle Beam technology reduces charging, improving resolution, signal-to-noise ratio, and beam brightness especially at low beam voltages (down to 0.1 kV). For the preparation of SEM samples, 5 µL of a polymer aqueous dispersion (0.2 mg/mL) that was kept above its phase transition temperature (at 40 oC), were casted on a conductive tape pre-mounted on the metallic substrate that was cooled with liquid nitrogen. The rapidly frozen drop was immediately placed in a freeze-drier and lyophilized. Images were obtained with an acceleration voltage of 0.5 kV. Transmission electron micrographs were obtained utilizing a Philips FEI CM20 TEM operating at 120 kWatt. For the preparation of TEM samples, 5 µL of a polymer aqueous dispersion (0.2 mg/mL) that was kept at 40 oC were casted on a PELCO® Formvar grid that was also kept at 40 oC and allowed to dry at 40 oC for 2 h. 2.2. Synthesis of isobutyl functionalized poly(ethyleneimine) derivatives The introduction of isobutyl groups (iBu) to PEI was realized in two steps as shown in Scheme I. In the first step, isobutyric acid was interacted with all primary amines of PEI affording PEI-iBu 100-0, while in the second step the secondary amines of this PEI derivative was reacted with isobutyryl chloride affording derivatives with various degree of substitution (DS) affording a series of PEI-iBu 100-x compounds (Scheme I). Specifically, in the first step, isobutyric acid (0.02 mol) was dissolved in 10 mL of dry DMSO in the presence of triethylamine (0.02 mol). To this solution, an equimolar quantity of DCC, dissolved in 2 mL


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of dry DMSO, was added dropwise and the reaction mixture was allowed under inert atmosphere for 1 hour. Subsequently, 0.02 mol of NHS dissolved in 2 mL of dry DMSO was also added. The solution was allowed to react overnight, under inert atmosphere, at room temperature. The mixture was filtrated to remove the by-product dicyclohexyl urea and directly added to a CHCl3 solution containing (0.4 mmol) PEI, which was previously degassed three times. The final mixture was also degassed for another time and allowed to react overnight, under vacuum, at room temperature. The solvent was partially removed under vacuum and the crude product was twice precipitated with diethyl ether. The solid was dissolved in aqueous solution containing TEA (pH=11) and was subjected to dialysis (mol. weight cut-off: 1200) against water. The final product (PEI-iBu 100-0) was received after lyophilisation and its structure was established by 1H and

13

C NMR. The degree of

substitution was determined by inverse-gated 13C NMR. 1

H NMR (500 MHz, CDCl3): δ =3.00-3.55 (CH2 relative to amide groups), 2.15-2.90 (m, CH2

of PEI scaffold and CH of iBu), 1.05 (s, CH3). 13

C NMR (125.1 MHz, D2O): δ =180.6 (C=O), 53.8-50.1 (CH2 relative to tertiary groups),

48.2-44.5 (CH2 relative to secondary groups), 39.0-36.6 (CH2 relative to amide groups), 35.5 (NHCOCH), 18.7 (CH3). In another approach, the coupling of isobutyric acid with PEI was achieved as following. Isobutyric acid (0.018 mol) was dissolved in 15 ml dry DMF. Solid HOBt (0.02 mol) was added under cooling (0 oC) and stirred for 15 min. HBTU (0.02 mol) and freshly distilled DIPEA (0.04 mol) were added sequentially. The reaction mixture was degassed three times and kept at room temperature for 30 min under stirring. PEI (0.4 mmol) was dissolved in 10 ml dry DMF and this solution was also degassed three times. This PEI solution was added to the reaction mixture and the final mixture was degassed for another time. The mixture was allowed to react overnight, under vacuum, at room temperature. The solvent was partially



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removed under vacuum and the crude product was twice precipitated with diethyl ether. The solid was dissolved in ammonium hydroxide aqueous solution (pH=11) and was subjected to dialysis (mol. weight cut-off: 1200) against water. The final product (PEI-iBu 100-15) was received after lyophilisation and its structure was established by 1H and 13C NMR. The degree of substitution was determined by inverse-gated 13C NMR. 1


of PEI scaffold and CH of iBu), 1.05 (s,). 13

C NMR (125.1 MHz, D2O): δ =180.7 (C=O), 53.5-50.0 (CH2 relative to tertiary groups),

48.2-44.5 (CH2 relative to secondary groups), 39.0-36.6 (CH2 relative to amide groups), 35.5 (NHCOCH), 29.8 (NCOCH), 18.8 (CH3). In the second step, PEI-iBu 100-0 (0.1 mmol) was dissolved in 15 ml dry CHCl3 in the presence of triethylamine (TEA) and the mixture was degassed three times. To this solution various appropriate quantities of isobutyryl chloride were added under cooling and degassed for another time. The reaction mixture was allowed to warm up and kept under stirring at room temperature for 4 hours. The solvent was partially removed under vacuum and the crude product was precipitated with diethyl ether. The solid was dissolved in aqueous solution of triethylamine and was subjected to dialysis (mol. weight cut-off: 1200) against water. The final products, PEI-iBu 100-x, x = 85, 93 and 97, were received after lyophilisation, while the products with x = 25, 40, 43, 48, 55, 60, 65 and 73 were received following centrifugation of their water solutions at elevated temperatures (30-40 oC depending on the derivative) in order to remove traces of derivatives with lower or higher degrees of substitution that could also be formed during the reaction. The structures of PEI derivatives were established by 1H and 13C NMR. The degree of substitution was determined by inverse-gated

13

C NMR with an

estimated accuracy of ±2%. 1



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of PEI scaffold and CH of iBu), 1.05 (s,). 13

C NMR (125.1 MHz, CDCl3): δ =178.1 (C=O), 56.7-49.3 (CH2 relative to tertiary groups),

49.2-42.4 (CH2 relative to secondary groups), 39.6-36.0 (CH2 relative to amide groups), 35.2 (NHCOCH), 30.1 (NCOCH), 19.7 (CH3).

Scheme I

2.3. Doxorubicin encapsulation and release For in vitro studies, DOX-loaded PEI-iBu systems were prepared as follows: to 1 mM polymer solution in phosphate buffer saline, 1 mg·mL-1 doxorubicin hydrochloride was dissolved and the solution was allowed under stirring for two hours. In order to remove nonencapsulated DOX, the obtained polymeric solution was centrifuged (20.000 rcf, 20 min) at 40 oC, i.e. above the transition temperature of the system. The supernatant containing the nonencapsulated DOX was discarded and the resulting pellet was washed and centrifuged as above, for a further three times to ensure complete removal of non-encapsulated DOX. The pellets were finally resuspended in 1 mL of Opti-MEM, and syringe-filtered through sterile



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nylon membrane filters of 0.20 µm pore size for in vitro experiments. In separate experiments, the resulting pellets were dissolved in Opti-MEM and their transition temperature in OptiMEM and at various polymer/DOX concentrations was determined by turbidity measurements. In addition, for the determination of encapsulated DOX in PEI-iBu by UV-Vis spectrophotometry, a calibration curve for various DOX concentrations (10–100 µM) in PBS was constructed at 25 oC. For every DOX-loaded PEI-iBu system aliquots (50 µL) were diluted in 700 µL of PBS at 25 oC and their absorbance at 561 nm was registered.39 The release of DOX encapsulated in PEI-iBu 100-97 (TPEI) was studied at different temperatures by monitoring the intrinsic fluorescence intensity of DOX both above and below the Tc, which in this case (TPEI concentration 0.5 mg/mL, DOX concentration 0.5 µΜ, pH: 7.4) was 34.9 oC (onset temperature). The fluorescence of free (non-encapsulated) DOX molecules that are released in the aqueous phase is effectively quenched (>95%) on the addition of trimethylamine, a known efficient quencher, at low concentrations (2 % v/v). DOX release is monitored by registering the fluorescence intensity decrease over time at different temperatures both above and below the phase transition. 2.4. In vitro cell experiments The cells used in this study were the human breast cancer cell line MCF-7. The cells were grown in RPMI 1640 without phenol red, with 10% FBS, penicillin/streptomycin at 37 °C in a 5% CO2 humidified atmosphere. For the cytotoxicity assessment, MCF-7 human breast cancer cells were inoculated (20×103) into 96-well plates and left to incubate in complete media containing 10% FBS for 24h. Cells were then treated with TPEI-DOX (10 µM PEI-iBu 100-97 corresponding to 0.1 mg/mL, encapsulating 110 nM DOX), DOX (110 nM) or TPEI (PEI-iBu 100-97, 10 µM) for 3 h or 6 h in Opti-MEM (without phenol red). During these periods, half the cell groups were incubated at 37 °C and the other half at 40 °C. Subsequently Opti-MEM was in all cases


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replaced by complete media and cells were returned to the incubators (one set to 37 and the other to 40 °C). The mitochondrial redox function of all cell groups was assessed by the MTT assay after 48 h or 72 h. This was carried out by replacing cell media with complete media containing 1 mg·mL-1 MTT and incubating at 37°C in a 5% CO2 humidified atmosphere for 2 h. MTT media were then removed from all cells and the produced formazan was solubilized with 100 µL DMSO per well. The plates were subsequently shaken for 10 min at 100 rpm in a Stuart SI500 orbital shaker, and the endpoint absorbance measurements at 562 nm were performed in an Infinite M200 plate reader (Tecan group Ltd., Männedorf, Switzerland). Blank values measured in wells with DMSO and no cells, were in all cases subtracted. For epifluorescence microscopy studies, MCF7 cells were inoculated on 22 mm cover slips housed in 35 mm petri dishes (10×105) and left to grow overnight in complete RPMI 1640 (2 mL). Subsequently, the cells were incubated at 37 or 40 oC with various concentrations of free DOX or TPEI encapsulated DOX for 4 h in OPTIMEM. After washing the cover slips with PBS, they were inverted onto microscope slides and placed under the Olympus UPLFLN40× objective (NA 0.75) of an Olympus BX-50 microscope coupled with a Mercury USH 102D lamp (Ushio Inc.) and an Olympus DP71 digital color camera. Both brightfield and fluorescence microscopy images were acquired. Fluorescence emission of DOX was imaged using the rhodamine isothiocyanate (RITC) filter (Chroma Technology Corp). For confocal microscopy studies, MCF-7 cells were inoculated on 22mm cover slips housed in 35 mm petri dishes (10×105) and left to grow overnight in 2 ml of complete RPMI 1640 in the same conditions as elaborated earlier in the cell culture section. Subsequently, cells were incubated with either TPEI-DOX (10 µM PEI-iBu 100-97 encapsulating 110 nM DOX) or DOX (110 nM) for 6h in Opti-MEM. During these 6h, half the cell groups were incubated at 37 °C and the other half at 40 °C. After washing the cover slips with PBS, they 13 ACS Paragon Plus Environment


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were inverted onto microscope slides; the slides were placed under the ×60 oil immersion quartz objective (NA 1.3) of a Biorad MRC 1024ES laser scanning confocal microscope. Intracellular DOX was excited using the 488 nm argon–krypton ion laser line. DOX fluorescence was collected after a long-pass filter at ≥ 585 nm. During image acquisition a level 3 (three iterations per image) Kalman smoothing routine was applied in all occasions to eliminate spurious signals.

3. Results and Discussion 3.1. Synthesis and characterization of hyperbranched polyethyleneimine derivatives Due to the presence in the parent hyperbranched polyethyleneimine chemical structure of both primary, secondary, and tertiary amino groups, IG

13

C NMR was initially employed to

determine the ratio of primary to secondary to tertiary amino groups (ΝΗ2:ΝΗ:Ν) according to the literature (Figure S1 in Supporting Information)40. Integration of the carbon peaks located at the a position relative to the different types of amino groups allowed the determination of the ratio of ΝΗ2:ΝΗ:Ν = 1.06:1.26:1.00. The degree branching was found to be 0.68 and its average number of primary and secondary amino groups were determined as 37 and 44, respectively. In order to gradually functionalize with the isobutyl group initially the primary amino groups and, at a second step, the secondary ones, the reaction of PEI with isobutyric acid in the presence of TEA, DCC and NHS was selected as it was found that under these conditions no secondary amines were functionalized. It should be noted that if the same reaction takes place in the presence of HOBt/HBTU a number of secondary amino groups are also functionalized. It was also found that it is necessary to completely (100%) functionalize the primary amino groups in order to obtain the thermoresponsive derivative PEI-iBu 100-0, which, however, exhibits a transition temperature at high temperatures (> 100 oC, see below).


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Therefore, the attachment of isobutyl groups at the secondary amino groups was followed, which was achieved by the reaction of PEI-iBu 100-0 with isobutyl chloride in the presence of TEA. Careful monitoring of isobutyl chloride: PEI-iBu 100-0 molar ratio and of the reaction conditions, allowed the synthesis of a series of derivatives with different substitutions of the secondary groups (PEI-iBu 100-x, x = 15 – 97 %). Furthermore, centrifugation of their water solutions at suitably chosen elevated temperatures enables the removal of small amounts of either lower or higher degree of substitution that could be also be formed during the reaction. The successful introduction of iBu to PEI was established by proton and carbon NMR spectroscopy (Figures 1 and Figures S2-S4 in Supporting Information) as well as by FTIR spectroscopy (Figure 2). Specifically, the substitution of PEI was confirmed by the appearance in the 1H NMR spectrum of a new singlet peak at 1.05 ppm, attributed to methyl protons of iBu. Additionally, a new broad peak between 3.00 and 3.70 ppm was ascribed to the α-methylene protons relative to the primary and secondary amino groups.34,35 More information about the purity of the PEI derivatives and the exact position of the attachment of iBu was obtained by

13

C NMR spectra. In this context, it should be noted that isobutyl

chloride can easily be hydrolyzed to isoburyric acid which then will protonate the amino groups of PEI. This can lead to erroneous results regarding the determination of degree of substitution using NMR. Indeed, the 1H NMR signal of methyl protons of isoburyric acid appears in the same position as that of covalently attached iBu group. Therefore, it is mandatory to verify the absence of the peak at 185 ppm (a in Figure 1) assigned to the carboxyl group of isoburyric acid in the spectra of all derivatives and the presence of the peak at ~180 ppm, attributed to the carbon of the newly formed amide group. These findings provide strong evidence that all derivatives are pure and also the covalent attachment of iBu groups is successful. Moreover, the attachment of iBu groups to primary and secondary amino



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groups of PEI was established by the signals of methine carbons of iBu moieties at ~35 and ~30 ppm (f, g in Figure 1), respectively. In the case of PEI-iBu 100-0, only the signal at ~35 ppm was observed, proving that the introduction of iBu moieties took place exclusively to the primary amino groups of PEI. Finally, precise determination of the degree of substitution (DS) was attained by IG 13C NMR (Figure 1). Comparing the integrations of the signals of the methine carbons (35 and 30 ppm) and the signals of all methylene carbons of PEI scaffold (57-49, 49-42 and 40-36 ppm), it was able to determine the degree of substitution to the primary and secondary amino groups of PEI. It was found that the substitution of primary amino groups of all derivatives is 100%, while different substitutions of the secondary groups ranging from 15% to 97 % were calculated.


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O

O NH

O

HN

NH

d

d

O

N

d

N HN

N

N

N

N N

HN

HN

a

O

h

N NH

O N

NH

N

b

c

HN

e a

O

NH

O

O

HN

g

NH O

h

f

h a f 195

190

185

180

175

170

165

δ (ppm)

b c

70

60

de

50

40

30

20

10

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Figure 1. IG 13C NMR spectrum of ΡΕΙ-iBu 100-0 (upper part), ΡΕΙ-iBu 100-48 (middle part) and PEI-iBu100-97 (lower part). 17 ACS Paragon Plus Environment


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FTIR spectroscopy was also used to follow the gradual increase of PEI functionalization with the iBu groups. Overall the spectra of the various PEI-iBu derivatives (Figure 2) are comprised of both the characteristic bands of PEI and isobutyl groups and, in addition, provide proof of the new amide bonds formed at the expense of the primary and secondary amino groups of PEI. The asymmetric (νas) and symmetric (νs) stretching bands of the CH2 groups of PEI observed at 2932 and 2884 cm-1, respectively,41,42,43,44 are still present, as expected, in the spectra of the derivatives. Of special interest is the band at 2807 cm-1, which normally should be detected at about 2850 cm-1, attributed to the vs of CH group that interacts with the lone electron pair of the nitrogen atom of amines that are in a trans-position to it.41,45,46 Given that this interaction does not take place when the lone pair is delocalized or donated into a vacant orbital,43,45,46 which is indeed taking place upon the formation of amide groups, this band progressively lowers in intensity (Figure 2, upper part) upon increase of the degree of functionalization. Therefore, the functionalization of amino groups does not only affect their bands but also this particular C-H stretching mode. Moreover, a new band at 2968 cm−1 attributed to the CH3 stretching vibration of the isobutyl groups becomes the prominent band of this region while the peaks at 2932 and 2884 cm-1 also increase in intensity due to the superposition of the PEI peaks with the νas and νs CH2 vibrations of the isobutyl moiety since these bands appear in the spectra of isobutyric acid at 2938 and 2880 cm-1 respectively (Figure 2, upper part)41. In an analogous fashion, the band attributed to the bending mode of CH2 groups of PEI observed at 1455 cm−1, is progressively overlapped by the two strong bands at 1470 and 1429 cm-1 attributed to the asymmetric and symmetric CH3 deformation bands of the isobutyl group (Figure 2, middle part).41-44 Furthermore, a new band at 1238 cm-1 attributed to the strong skeletal ν2 vibration of the methyl groups of the (CH3)2CH- moiety41 is evident in the spectra of the PEI-iBu 100-x derivatives although it is overlaid with the Amide III band (see below).


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Furthermore, upon functionalization of the amino groups of PEI, the NH asymmetric and symmetric bands of PEI at 3350 and 3278 cm-1 as well as the shoulder at 3188 cm-1 (δ ΝΗ overtone) are surpassed by a new band attributed to the NH stretching vibration (Amide A) of hydrogen-bonded trans secondary amides of a trans configuration[41-44]; the observed band is centered at 3282 cm-1 for the PEI-iBu 100-0 derivative (it is actually the overlay of both the amide band and of the NH stretching band of yet unreacted amino groups) and gradually shifts to 3302 cm-1 for the PEI-iBu 100-97 derivative. In addition, a rather low intensity broad band centered at approx. 3490 cm-1 suggests the presence of free (non-hydrogen bonded) secondary amides (Figure 2, upper part).41 In the carbonyl absorption region (Figure 2, middle part), the band at 1642 cm-1 is assigned to the Amide I band of secondary amides formed upon functionalization of the primary amines, while the evolving band at 1625 cm-1 is due to the formation of tertiary amides resulting from the functionalization with iBu units of the secondary amino groups.41-44 The Amide II band originating only from the secondary amides is, as expected, of the same intensity for all PEI-iBu 100-x derivatives and is located at 1539 cm-1. Likewise, the Amide III band is registered at about 1240 cm-1 although it is overlapped with the 1238 cm-1 band of the iBu group as described above. The fact that the Amide II band is constant for all the PEI-iBu 100-x derivatives synthesized, suggests that this band can be used as an internal reference to estimate the degree of functionalization (x) of the secondary amino groups. Indeed, it is found that the I1622/I1539 ratio has an almost linear dependence on x, while the I2814/I1539 ratio can also follow quite satisfactory the degree of functionalization (Figure 2, lower part). Both ratios can therefore be used as an initial rapid estimation of the degree of functionalization of such compounds, before resorting to the more time consuming IG 13C NMR experiments.


Biomacromolecules

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Figure 2. FTIR spectra of isobutyric acid, PEI and PEI-iBu 100-x (where x is the degree of functionalization; x = 0, 25, 48, 73, 97) in the 3600-2500 cm-1 (upper part) and the 1800-1000 cm-1 (middle part) region. Spectra are shifted vertically for clarity. Lower part: plot of the intensity ratios I1622/I1539 and I2814/I1539 vs. the degree of functionalization x.


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3.2. Phase transitions of isobutyl functionalized HEI derivatives All isobutyl functionalized HPEI derivatives exhibited phase separation upon heating their aqueous solutions (c = 5 mg·mL-1, 50 mM phosphate buffer, pH=7.4) with the exception of PEI-iBu 100-0 suggesting that functionalization of only the primary amino groups does not lead to thermoresponsive properties. Derivatives with partially substituted secondary amino groups at percentages above ~30% exhibit phase separation temperatures, taken as the transition onset throughout this manuscript, in the range from ca. 20 to 70 oC which are wellcorrelated with the degree of substitution (Figure 3). Even lower substitution percentages (1525%) afford derivatives with transition temperatures close to the b.p. of water, Figure 3 (these particular experiments were performed in hermetically closed tubes to avoid water loss due to evaporation during the experiment). The phenomenon is fully reversible as upon cooling complete re-dissolution of the polymer-rich phase is observed (Figure 3, insert). It is interesting to note that in the presence of 100 mM NaCl (i.e. at isotonic solutions) the transition temperature is slightly decreased in all cases by ca. 3-5 oC due to the so-called salting out effect (vide infra). It should be also emphasized that a 50 mM phosphate buffer was deemed necessary to ensure the buffering capacity required to maintain the pH value (7.4). Lower concentrations were not adequate to ensure neutral pH especially for the derivatives with low degrees of substitution. As shown in Figure 3, at the low degree of substitution region (15-48%) the Tc significantly varies with degree of substitution (DS) by some 60 oC, while at medium to high DS (~50-97%) the reduction of Tc is not so extensive (approx. ~15 oC). This is in line with the observed variation of Tc upon butyrylation of branched PEI (Mw=25,000) where increase in n-butyl groups substitution from 55-91% resulted in a reported decrease of ~ 12 oC.35 Substitution with iBu groups changes the hydrophilic/hydrophobic balance, rendering the system more hydrophobic and inducing thermo-responsiveness. Their effect in Tc, in absolute


Biomacromolecules

numbers, is depended on the introduction of hydrophobic end-groups. Their presence even in small amounts introduces hydrophobicity in an otherwise hydrophilic polymer and therefore Tc is crucially affected at low DS. On the other hand at high densities of iBu groups, apparently located both in the outer surface of the hyperbranched structure as well as in its interior, the relevant change of the hydrophilic/hydrophobic balance is not so strong and Tc is less affected.

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Figure 3. Transition temperatures vs. degree of functionalization of secondary amino groups with isobutyl groups of PEI-iBu 100-x solutions (c=5 mg·mL-1, 50 mM phosphate buffer, pH=7.4) registered either in the absence (closed symbols) or presence (open symbols) of NaCl. Insert: Photograph showing a PEI-iBu 100-48 polymer/aqueous system above and below the phase separation temperature.

The transition is well-known to depend on polymer concentration since the phase separation temperature (or the cloud point) varies significantly in the temperaturecomposition phase diagram.15 In addition, this transition is also depended on a rather large number of aqueous phase variables that are usually encountered in cell culture media or drug formulations as such as the ionic strength (NaCl concentration), or the concentration of phosphates that are usually used in in vitro experiments for providing buffering capacity and 22 ACS Paragon Plus Environment

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isotonicity, and are also present in biological fluids. Finally, the pH value(s) differ from the normal physiological value in some tissues or subcellular compartments and this result in the alteration of the degree of ionization of the amino groups and therefore the conformation and hydrophilicity of polyamine-type polymers.47,48 Therefore, the effect of the above parameters on the transition temperature was evaluated in order to identify which of the above derivatives could be the most appropriate as a potential drug delivery system. The effect of polymer concentration on the transition is clearly evident in Figure 4. Low concentrations lead to higher transition temperatures as well as broader transition widths. The increase in phase separation temperature upon decrease of polymer concentration clearly suggests that the concentrations employed are lower that the Tc which is the shared minimum of the concave up spinodal and binodal (or coexistence) curves. This is also reflected in the observed broadening of the transition since in the phase diagram the coexistence region is increased for concentrations far from Tc (Figure 4, lower part).

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Figure 4. The influence of polymer concentration on thermosensitivity. Left part: onset transition temperatures of isobutyl functionalized ΡΕΙ-iBu 100-x derivatives as a function of concentration (50 mM phosphate buffer, pH=7.4). Right part: indicative temperature dependence transmittance curves for ΡΕΙ-iBu 100-48 solutions having concentrations of 2, 5, and 10 mg·mL-1. 23 ACS Paragon Plus Environment

Biomacromolecules

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40

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pH Figure 5. The influence of phosphates, NaCl, and pH on thermosensitivity. Onset transition temperatures of isobutyl functionalized ΡΕΙ-iBu 100-x derivatives as a function of phosphates concentration (upper part: polymer concentration: 5 mg·mL-1; pH=7.4), NaCl concentration (middle part; polymer concentration: 5 mg·mL-1; 50 mM phosphate buffer, pH=7.4), and pH (lower part: 5 mg·mL-1; 50 mM PB, 100 mM NaCl). 24 ACS Paragon Plus Environment

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This series of derivatives bears both hydrophilic (amine) and hydrophobic (isopropyl) groups and the effects of salts on the Tc have been related to the Hofmeister series, specifically due to the direct interactions of ions (more significantly anions compared to cations) with the macromolecule’s functional groups (e.g. amide groups) and with the water molecules that form the immediately adjacent hydration shell of the polymer.18,19 In this respect it is observed that both chlorine and phosphate anions are causing Tc decrease as expected from the Hofmeister effect of ions, and as also observed for both the linear poly(Nisopropylacrylamide), PNIPAM, or the dendritic analogues of iBu functionalized dendrimeric or hyperbranched polyethyleimines.19,34-37 The obtained Tc exhibits a linear dependence on phosphates concentration at pH 7.4, Figure 5 and Figure S5 in Supporting information, as is also the case for linear PNIPAM. Dihydrogen phosphate is considered a kosmotropic anion, and is known shown to result, as all strong kosmotropes, in significant solubility decrease and a linear dependence of Tc on its concentration.18 In line with the Hofmeister series, the effect of Cl- is less strong as, in contrast to dihydrogen phosphate, it is usually considered the inbetween anion between kosmotropic and chaotropic anions. The variation of Tc with NaCl concentration (Figure 5 and Figure S6 in Supporting information) is clearly non-linear for all the derivatives tested. This non-linear behaviour is not observed in the case of the linear PNIPAM macromolecules, but is reported for the respective hyperbranched analogues.37 It is apparent that Tc changes significantly at low NaCl concentrations, while above 150 mM the change is less prominent. Also, the effect is more pronounced for derivatives with low degrees of functionalization. This behavior is clearly due to the macromolecular dendritic architecture of these polyelectrolytes and to their property to assume either an open (or hollow core) structure at low salt concentrations due to strong electrostatic interactions between the positively charged groups, or a dense core structure at high salt concentrations due to



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screening of these interactions.49 Evidently, an open structure filled with water molecules is more hydrophilic leading to high Tc values, while a dense core structure results in removal of water molecules and enhances aggregation of macromolecules at lower temperatures.37 This is also corroborated by the fact that the effect is more pronounced in less substituted derivatives that due to the large number of tertiary and secondary amines are more charged and assume a more open structure. Indeed this is more intense at DS less that ~45% and is depicted clearly in the abrupt increase of Tc for these derivatives as shown in Figure 3. The effect of pH on transition temperatures follows also a, definitely, non-linear trend (Figure 5 and Figure S7 in Supporting information). As previously the Tc change is not significant at pH values above 7.4 but becomes more effective in low pH values. The same general trend is also observed iBu functionalized poly(amidoamine) and poly(propylene imine) dendrimers.32 Decrease of pH results in increase of the degree of protonation of secondary (unreacted) amines as well as in the tertiary amines of PEI derivatives. It is known for polypropylene imine dendrimers, their functionalized derivatives as well as for associated oligo-amines that protonation proceeds initially at the external secondary amino groups and at the core tertiary nitrogens, while protonation of the intermediate tertiary amino groups takes place gradually at pH values between 7 and 5.50,51 This increases the hydrophilicity of the system and induces an open structure having a hollow water-filled interior, both resulting in water solubility and Tc increase.47,48 The effect is more prominent for derivatives with a large number of non-reacted secondary amino groups (low degrees of substitution) but it is still clearly noted in derivatives with high DS such as the ΡΕΙ-iBu 100-x, x = 93 or 97. This supports the proposition that tertiary amine protonation in PEI derivatives as well, is starting taking place at these pH values which concomitantly leads to increase of Tc as pH values become lower than neutral. However, it should be noted, that although in the relevant literature only the H2PO4- anions are discussed extensively, both H2PO4- and HPO42- species


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are present in solution at varying percentages at different pH values. Indeed, the H2PO4/HPO42- ratio is approx. 1 at pH 7.4, while at the pH of 6 the ratio is 15.52 It is therefore clear that considerably more H2PO4- species are present at low pH values, while the situation is totally different at neutral to slightly basic values. It is reasonable to assume that these two species will not interact identically with either the bound water molecules or the amide groups of the polymer since their electrostatic potential in solution is different and this could also result in a far from linear variation of Tc vs. pH.

3.3. Dynamic light scattering studies Dynamic light scattering studies were performed on selected polymer/aqueous systems to examine the effect of the various parameters, i.e. degree of functionalization, polymer, NaCl and phosphate concentration, as well as of pH on the hydrodynamic radii of polymer entities vs. temperature. In each experiment the same general trend is observed (Figures 6 and 7). At temperatures below the transition temperature the registered size (mean hydrodynamic diameter) of polymer in solution is 4.4±0.3 nm for all polymer derivatives of this series and irrespective of NaCl and phosphate concentration. After the onset of the transition and within the temperature range of the transition, two distinct size distributions are observed, that of the dissolved polymer which gradually reduces in intensity and finally disappears within 3-4 oC after the onset of the transition, as well as a second one which gradually increases in intensity and shifts to higher hydrodynamic radii. Further temperature increase results in a single, rather sharp size distribution of aggregated polymer molecules. Their size continues to increase with temperature reaching plateau values that depend significantly on the aqueous media. However the size distributions are monomodal and narrow (Figure 6A and Figure S8 and S9 in Supporting Information).


Biomacromolecules

Intensity (a. u.)

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Figure 6. Hydrodynamic diameter size distributions of ΡΕΙ-iBu 100-48/aqueous system (5 mg·mL-1; 50 mM PB, pH=7.4), upper part. Plot of their mean hydrodynamic diameters as a function of temperature, lower part.

Comparing the sizes of derivatives ΡΕΙ-iBu 100-x having different degrees of functionalization (x=43, 48, 55) under identical conditions (5 mg·mL-1; 50 mM PB; pH=7.4) it is clear that the transition temperatures registered using turbidity measurements are in agreement with DLS results (Figure 7 upper part, and Figure S8 in Supporting Information). Their transition temperatures decrease upon increasing of the degree of substitution while the 28 ACS Paragon Plus Environment

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temperature range of the transition decreases. Below their transition temperatures the size of polymers in solution are approximately the same (~ 4 nm), within experimental error; upon increasing the temperature above the transition, aggregates are formed which increase rapidly with temperature reaching a final value of >800 nm within approx. the next 5 oC.

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Figure 7. Upper part: Hydrodynamic mean diameters of ΡΕΙ-iBu 100-x /aqueous systems (5 mg·mL-1; 50 mM PB, pH=7.4) as a function of temperature; x = 43 (red), 48 (green), 55 (blue). Lower part: Hydrodynamic mean diameters of ΡΕΙ-iBu 100-48 aqueous systems (pH=7.4) as a function of temperature under different conditions: 5 mg·mL-1/50 mM PB (green), 2 mg·mL-1/50 mM PB (red), 5 mg·mL-1/50 mM PB/100 mM NaCl (blue).



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For the same derivative, a decrease in its concentration results not only in an increase of the transition temperature as shown in the previous section, but also in a substantial decrease of the obtained aggregates above the transition. As shown in Figure 7 (lower part) and ion the corresponding hydrodynamic diameter size distributions that are presented in Supporting Information, Figure S9, the hydrodynamic sizes reach plateau values of ca. 450 nm when the concentration is 2 mg·mL-1 compared to sizes of ~900 nm when the concentration is 5 mg·mL-1. On the other hand, the presence of NaCl not only reduces the transition temperature as expected due to the “salting out effect” of the hydrating water molecules but also, rather unexpectedly, affects considerably the final size of the aggregates. It, therefore, appears that the observed continuous increase of the aggregates size even after the completion of the transition can be at least in part dominated by such interactions and that the presence of NaCl stabilizes their size to lower mean values. This can be rationalized on the basis of the dense core conformation, and the reported concomitant reduction of the mean radius of gyration of dendritic polyelectrolytes at high salt concentrations.49 Overall, both low polymer concentrations and high salt values contribute to lower sizes of the aggregates formed above transition which can be beneficial in drug delivery applications through passive targeting taking advantage of the so-called Enhanced Permeability and Retention (EPR) effect. In this case, nanocarriers with sizes ranging from 200-800 nm are considered appropriate as they can extravasate in the interstitial space of tumors by passing through the endothelial pores (10 to 1000 nm in diameter) of tumor vasculature and, further, accumulate there due to the reduced lymphatic drainage that is also observed in most tumors.53,54,55

3.4. DOX encapsulation and release studies The hyperbranched polymeric structures are known to behave as unimolecular micelles since they can solubilize, or encapsulate, in their interior water insoluble or poorly-soluble


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organic compounds, including drugs.56,57 This procedure has been suggested to encompass non-covalent interactions such as hydrophobic interactions between the interior of the hyperbranched polymer (PEI) and the hydrophobic bioactive molecule (DOX). It should be noted that doxorubicin is a weak base (pKa = 8.2-8.3)58,59 and in this form is water insoluble. Therefore, although the DOX hydrochloride employed in this study is water soluble, at this pH (7.4) and taking as pKa a mean value of 8.25, approximately 14 % of these molecules will be present in free base form in solution, as calculated from the equilibrium equation. The free base DOX molecules, due to their hydrophobicity, are prone to reside inside the hyperbranched interior, albeit in a small amount. As described in the experimental section, a number of DOX-loaded PEI-iBu 100-x systems were prepared and initially tested with respect to their DOX encapsulation ability and their transition temperatures in Opti-MEM, which was the reduced serum media that would be used in the following in vitro experiments. Given that the DOX concentration was decided to be kept in the nanomolar range, the polymer concentration was concomitantly low and this led to the selection, after making a series of preliminary experiments and taking into consideration the concentration of the polymer, the specific medium appropriate for in vitro cell experiments, and the requirement to perform in these studies at 40 oC, of the PEI-iBu 100-97 system (denoted as TPEI in the following) at a concentration of 10 µM corresponding to 0.1 mg·mL-1. At this concentration, TPEI is able to encapsulating DOX at a concentration of 110 nM (TPEI:DOX molar ratio = 90:1). This system, denoted as TPEI-DOX in the following and used in all following in vitro experiments, due to its low polymer concentration and to the presence of various ionic species in Opti-MEM, was found to have a transition temperature onset of 38 oC. It should also be noted, however, that the encapsulation efficiency can be increased if higher concentrations of TPEI were to be used, e.g. at a concentration of



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10 mg/mL (1 mM) of TPEI, the encapsulated DOX concentration is considerably higher (40 µΜ) (TPEI:DOX molar ratio = 25:1). The obtained TPEI-DOX was further characterized through a series of physicochemical studies before progressing to in vitro experiments. ζ-potential values of TPEI nanoparticles at 40 oC, i.e., above the phase transition, were found to be 5.8±1.5 mV denoting a slightly positive charge of the phase separated nanoparticles due to the presence only of tertiary nitrogen groups of TPEI, given that all the primary and secondary groups of the parent polymer are substituted in this derivative by amide groups. DOX loaded TPEI nanoparticles at the same temperature range exhibit somehow higher ζ-potential values (6.3±1.4 mV) attributed to the encapsulation of doxorubicin hydrochloride which, being a week base, contributes to the observed increase of positive charge. Dynamic light scattering studies of TPEI or DOX loaded TPEI nanoparticles (0.2 mg/mL, pH 7.4) at temperatures above the phase transition reveal, for both cases, a monomodal particle size distribution (Figure S10, Supporting information) with mean hydrodynamic values at 40 oC of 100±10 nm and polydispersity indexes of 0.12 and 0.10, respectively. The considerably lower values than those reported above for the PEI-iBu derivatives (see section 3.2.) are due to the significantly lower polymer concentration that is employed in this case (0.2 mg/mL vs. 2-5 mg/mL). SEM images of samples kept at 40 oC and rapidly frozen and lyophilized on the sample holder are in line with the DLS experiments, revealing almost spherical particles with diameters ranging from 55 to 130 nm (Figure S11, Supporting information). TEM images of samples allowed drying at 40 oC temperatures point to a rather uniform morphology of globular particles (Figure S11, Supporting information). However, their diameters, ranging from ca. 20 to 50 nm, are considerably lower than those registered in DLS and SEM experiments. This can be attributed to the fact the TEM samples are prepared by placing a


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drop of aqueous suspension on the grid and allowing the solvent to evaporate. During this procedure, the polymer particles shrink due to the removal of water. Similar differences in sizes measured by DLS and TEM are also frequently reported in the literature both for nonthermoresponsive and thermoresponsive micellar or star-shaped polymeric systems.60,61,62,63 The release of DOX encapsulated in TPEI was studied at temperatures both above (37 and 40 oC) and below (26 and 34 oC) the Tc, which in this case (TPEI concentration 0.5 mg/mL, DOX concentration 0.5 µΜ, pH: 7.4) was 34.9 oC (onset temperature). The employed approach is based on the fact that the fluorescence of free DOX molecules is effectively quenched by the presence of small amounts of an efficient quencher, in this case trimethylamine, while DOX encapsulated in TPEI fluoresces since the selected quencher, a tertiary amine that is protonated in this pH, is not able to approach and interact with the positively charged TPEI entities and the DOX molecules that are encapsulated within its structure. This is due to the fact that, either static or dynamic, quenching requires contact between the fluorophore and the quencher.64 Therefore, DOX release in the aqueous media is manifested as a continuous decrease of the registered fluorescence intensity. Given that under the experimental conditions employed (DOX concentration 0.5 µΜ), the fluorescence intensity is linearly dependent on DOX concentration and that trimethylamine can quench the fluorescence of free DOX quantitatively (>95%), the registered fluorescence intensity can be considered as depicting with acceptable accuracy encapsulated DOX. From the profiles obtained (Figure 8) it is evident that within the first ca. 10 min a first burst release is taking place which accounts for about 10% of total DOX at temperatures below Tc and for ca. 20% at temperatures above Tc. This initial rapid release can be attributed to the most loosely bound DOX molecules as it is shortly followed by a rather stable phase where release almost levels off, as is apparent especially in the low temperature release profile (27 oC). Following this initial phase, the release progressively follows, in all cases, a simple


Biomacromolecules

exponential decay profile. The exponential time constants determined at temperatures below Tc (285±15 and 255±10 min-1 at 26 and 34 oC, respectively) are significantly different to those registered at temperatures above Tc (110±5 and 90±5 min-1 at 37 and 40 oC, respectively). While it is obvious that temperature increase increases DOX release, it is interesting to note that below and above the phase transition the difference in the profiles is very large and cannot be attributed simply to temperature increase (cf. the difference of profiles obtained at 34 and 37 oC to that of profiles at 37 and 40 oC). Evidently, above Tc the release of DOX is enhanced and more that ~80% is released at 40 oC within the 3 h period that is employed in in vitro experiments (see below) in contrast to ~ 50% that is released at temperatures below the transition.

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3.5. In vitro studies Initial experiments involved epifluorescence microscopy studies on MCF7 cells utilizing the inherent fluorescence of DOX to verify that TPEI (PEI-iBu 100-97) encapsulated DOX is 34 ACS Paragon Plus Environment

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indeed stable both below (37 oC) and above (40 oC) the transition temperature, that can penetrate the cells more efficiently than free DOX, and to perceive its subcellular localization. Images of MCF7 cells incubated at 37 oC with 110 nM DOX show minimal cell uptake, hardly observable under the experimental conditions employed, in sharp contrast to cells treated with DOX encapsulated in TPEI (TPEI concentration 0.1 mg/mL corresponding to 10 µΜ, DOX concentration 110 nM) which clearly show increased DOX fluorescence signals in the cell nuclei (Figure S12, Supporting information). More importantly, the same experiments conducted at 40 oC unequivocally indicate increased cell localization within cell nuclei both for free DOX, giving further proof that hyperthermia induces increased drug uptake, as well as for the DOX encapsulated in TPEI (Figure S13, Supporting information). In this particular case, TPEI-DOX internalization is significantly increased compared to free DOX providing a first evident that DOX encapsulation is strong and could improve its cytostatic efficiency towards cancer cells despite the low concentration attained. The increased internalization can be attributed both to the elevated temperature during incubation, as well as to the membrane transporting property of positively charged dendritic polymers,65 which was, among others, greatly utilized in their exploitation as gene delivery systems.66 Further increase in the concentration of TPEI, up to 0.2 mg/mL corresponding to 20 µΜ, of free DOX (2210 nM) and of TPEI-DOX did not improve the DOX fluorescence signal in the respective cell images (Figure S14, Supporting information) and, therefore, further experiments employing these concentrations were not pursued. Representative confocal microscopy images of MCF7 cells incubated with either free DOX (110 nM) or DOX encapsulated in TPEI (TPEI-DOX: 10 µΜ PEI-iBu 100-97, 110 nM DOX) for 6h at 37 °C or 40 °C are shown in Figure 9. From these microphotographs it is evident that both free DOX or TPEI-DOX fluorescence is localized in cell nuclei, while substantially higher intensities are evident when incubation is taking place at 40 °C (Figure 9



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C, D) than at 37 °C (Figure 9 A, B). Specifically, free DOX is localized in cell nuclei as is already well established,67,68 and incubation at 40

o

C further enhances intracellular

localization which, to our knowledge, was not previously shown for free DOX. This increase in DOX uptake upon incubation at 40 °C (Figure 9, C vs. A) could be due to the increase of permeation of the membrane bilayer as a function of temperature, or, to an associated phase transition of the cell membrane which further enhances the permeability of the bilayer as is well-established for liposomal membranes.3,4,69 The increase of DOX localization for the TPEI-DOX system at 37 °C (Figure 9, B) can be easily rationalized if one takes into consideration that positively charged functionalized PEI derivatives (e.g. quaternary or guanidinium functionalized PEI or poly(propyleneimine) dendrimers) have been shown to effectively transport through cell membranes and preferably localize in cell nuclei.70,71 The first conclusion from the representative images in Figure 9 is that even at 37 oC, TPEI encapsulation results in enhanced accumulation of DOX in cell nuclei compared to incubation with free DOX. Incubation of cells with TPEI-DOX at 40 °C, leads to further increase of intranuclear fluorescence possibly due to enhanced permeability of the membrane, as previously discussed, while, in addition, intense perinuclear fluorescence sites were also observed (Figure 9, D). This is consistent with vesicle inclusion, which could be the result of enhanced endocytocis due to the formation of TPEI-DOX nanosized particles in response to temperature increase above Tc.


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Biomacromolecules

Figure 9. Representative confocal microscopy images of MCF-7 cells incubated for 6 h at 37 o

C (A, B) or 40 oC (C, D) with free DOX (left column, 110 nM) and TPEI-DOX (right

column, 10 µΜ DOX-loaded PEI-iBu 100-97, 110 nM DOX): λex = 488 nm; λem ≥ 585 nm.

Cell viability experiments performed following 6h incubation with TPEI, DOX and TPEIDOX at 37 or 40 °C and assessed 72 h post incubation are shown in Figure 10. At 37 °C incubation, the toxicity for DOX was low (cell survival ~80%) due to its low concentration employed (110 nM) which, however, is increased at 40 oC incubation (cell survival ~60%). This is in line with the corresponding cell internalization profiles obtained from fluorescence microscopy (Figure 9 A and C, respectively) that indicate increased uptake at high temperature and further corroborates with the notion that hyperthermia increases drug efficiency. The TPEI-DOX toxicity data at 37 °C are alike the toxicity observed for DOX (cell survival ~80%) although confocal microscopy images denote increased internalization of DOX in this case. This implies that DOX molecules could be still encapsulated within the carrier and not available to interact with DNA which would further reduce cell viability. On the other hand, incubation at 40 °C significantly increases the toxicities for both TPEI-DOX 37 ACS Paragon Plus Environment

Biomacromolecules

(cell survival ~55%) and DOX (cell survival ~65%), and although these values are close, the increased toxicity for TPEI-DOX is statistically significant (p=0.002). This is also consistent with the higher loading profiles, registered at 40 °C, of TPEI-DOX compared to DOX as shown in the representative confocal images (Figure 9 D and B, respectively). It should be noted, however, that a large fraction of vesicular DOX observed in Figure 9D, is located outside of the cell nucleus, at least within the time frame of the experiment, and is therefore not available to interact with DNA and result in chemotherapeutic enhancement. Additional experiments in which cell viability was assessed 48 h post incubation show that incubation at 40 °C results again in higher toxicities. However, incubation either at 37 °C or 40 °C leaded to statistically equivalent cell toxicity for DOX and TPEI-DOX (Figure S15 in Supporting Information). This points to a sustained release of DOX encapsulated in the thermoresponsive polymer since prolonged incubation periods (72 vs 48 h) are required for encapsulated DOX to be released and reach increased toxicity levels. 37-37 oC 40-37 oC

100

Survical (% control)

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** 80

60

40

20

0

TPEI

DOX

TPEI-DOX

Figure 10. MCF7 cell survival assessed by standard MTT assays 72 h after the end of a 6h incubation period with TPEI (PEI-iBu 100-97, 10 µM), DOX (110 nM) or TPEI-DOX (10 µΜ of DOX-loaded PEI-iBu 100-97, 110 nM DOX). Cells were incubated for 6 h either at 37 °C or at 40 °C and subsequently remained at 37 °C until cytotoxicity assessment (72 h). The statistical significance between the designated cell groups follows the assignment: ** p

1 Functionalized Hyperbranched Polyethyleneimines as

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