Fluorescein nanocarriers based on cationic star copolymers with acetal linked sugar cores. Synthesis and biochemical characterization

Abstract

Well-defined star-shaped copolymers consisting of acetal derivatives of methyl α-D-glucopyranoside cores and polymethacrylate arms containing oxirane pendant groups have been modified with ethylenediamine (EDA) to form amphiphilic and water soluble polycations (positive zeta potential). Normal human dermal fibroblasts (NHDF) and human colon cancer cells (HCT-116) were employed to investigate the cellular uptake and cytotoxicity of diamine-functionalized star copolymers. Star-shaped polymers exhibited low cytotoxicity in NHDF cells, whereas in the case of HCT-116 cell line MTS assay resulted in the decrease of cell proliferation, which was also confirmed by Annexin-V assay indicating the increased rate of HCT-116 cell apoptosis. In the next step, the incorporated amine groups were applied for covalent conjugation of fluorescein isothiocyanate (FITC) with an efficiency of 60–85% estimated by ¹H NMR. The additional conjugation experiments were performed by isothermal titration calorimetry (ITC) yielding 45–100% of polymer labeling by fluorescent FITC. The confocal laser scanning microscopy proved cell internalization of the fluorescein-conjugated star copolymers was successful. The model studies showed that this type of star copolymers can be promising carriers for the delivery of drugs.

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Introduction

Functionalization reactions are an important tool in modern polymer chemistry to design well-defined polymers with properties required for applications. Specific functional groups can be incorporated into a polymer chain, which is especially important in cases when their presence is not convenient during the polymerization process itself, for example in the highly restrictive anionic mechanism. However, the improved functional group tolerance of pseudoliving/controlled radical polymerization methods allows the synthesis of polymers bearing a wide range of functional groups that can be quantitatively and selectively modified using relatively mild conditions without any side reactions. Post-polymerization transformation reactions are related to standard organic reactions (hydrolysis, esterification, amination, azidation, glycosylation and etc.) and coupling reactions via “click” mechanism.¹

In biomedical applications the functionalized polymers are used for conjugation reactions with biologically active compounds and fluorescent dyes. It is worth to notice that a large number of polymer-based conjugates of proteins, polypeptides, DNA or RNA as well as small molecules have since been reported to be more efficient and stable than their free forms.^2,3 There are several commercially available polymer-based conjugates, in which poly(ethylene glycol) and poly(D,L-lactide-co-glycolide) are the carriers in contemporary drug delivery systems (DDS).^4,5 In addition the use of polymer in DDS is limited by their cytotoxicity and cellular uptake as well as a carrier size, composition, surface charge and surface hydrophobicity.³

In recent years, there has been growing research interest in developing fluorescent dye-labeled macromolecules as probes for various bioimaging applications such as diagnostic imaging of cancers,^6,7 tagging of stem cells,^8,9 and imaging of pathogenic cells.¹⁰ Fluorescein derivatives as nontoxic fluorescent dyes are commonly used as fluorescent indicators and tags, pH probes of intercellular fluids, fluorescent probes and sensors for biogenic matter.^11,12 Fluorescein based sensors were studied to visualize ions (Ca²⁺, Zn²⁺) at the molecular level in cell biology and neurology,¹³ as protein sensors,¹⁴ or in dopamine detection.¹⁵ In targeting drug delivery purpose the fluorescein-labelled polymers were used to quantify functional groups for bioconjugation and verify localization in cell cultures, as it was reported for poly(N-vinyl pyrrolidone)-graft-(aminopropyl)methacrylamide microgels/nanogels,¹⁶ galactosylated polycaprolactone-graft-dextran micelles as a liver drug carrier,¹⁷ mannosylated micelles of poly(ethylene glycol)-b-poly(ε-caprolactone) to recognize lectin glycoreceptors,¹⁸ water-dispersible chitosan nanoparticles as a specific for human leukemia cells,¹⁹ or chitosan nanocarrier surface for cancer cells.²⁰ The more composed polymeric systems are based on dual functionalities as it was demonstrated for fluorescein-polyethyleneimine coated gadolinium oxide nanoparticles as magnetic resonance imaging-cell labeling dual agents,²¹ or poly(ethylene glycol)-b-poly(N-isopropylacrylamide) functionalized with biotin group for attaching the polymers to streptavidin and fluorescent dyes for polymer monitoring in various environments.²²

In literature the polymers with various architectures, i.e. linear block copolymers, branched copolymers (including hyperbranched, dendrimers, graft and star polymers), and cross-linked structures (polymer networks), are reported as carriers of small molecules in biomedical applications.²³ The investigation of a single macromolecule shape in relation to physicochemical properties revealed that the branched polymers in comparison to the linear ones represent unique solution behavior and enhanced cell uptake.^24,25

Our previous studies were focused to design the star-shaped polymers with narrow molecular weight distributions containing poly(methyl methacrylate-co-glycidyl methacrylate) (P(MMA-co-GMA)) arms and acetal derivatives of D-glucopyranoside in the cores.²⁶ The acetal groups in the core were introduced to regulate the number of pre-initiating groups (related to number of arms) and increase degradation rate of the polymer. The protecting role of acetal groups, next to orthoester, hydrazone, imine and cis-aconityl ones, is well known in organic synthesis and from the fact that they can be broken in acidic conditions. Such conditions can be found in tumor tissue, endosomes, and lysosomes what might induce the cleavage of acetal bonds in DDS. Hence, the polymers containing an acid-degradable linkage, mostly poly(amino acid)s, poly(ethylene glycol)s and polymethacrylamides, have been employed to create polymer–drug conjugates that can degrade under lysosomal and endosomal conditions, triggering the fast release of drugs or DNA.^27,28

In this paper, we report the studies on fluorescent nanocarriers based on amphiphilic and water soluble 3- and 4-armed star-shaped copolymers. The stars s-P(MMA-co-GMA) were modified by ring opening reaction of oxirane via aminolysis with ethylenediamine (EDA), and then the incorporated amine groups were used to form covalent bonds with fluorescein isothiocyanate (FITC) as a model molecule to generate fluorescent properties of star copolymers. The isothermal titration calorimetry (ITC) was used for alternative experiments of FITC conjugation. The cytotoxicity of polymers was tested on normal human dermal fibroblasts (NHDF) and human colon cancer cells (HCT-116). The proliferation MTS assay and apoptosis assay Annexin V (plasma membrane assay for flow cytometry) were performed to examine the pathway whereby EDTA-functionalized star copolymers inhibit the proliferation of cancer cells and induce apoptosis. The confocal microscopy of fluorescein-labeled stars facilitated to monitor the cellular uptake and localization of copolymer, that will be important in further studies on drug carriers in delivery systems.

Experimental

Materials

Dimethylsulphoxide (DMSO, Alfa-Aesar, 99%), ethylenediamine (EDA, Alfa-Aesar, 99%), triethylamine (TEA, Aldrich, >99%), borax anhydrous (Fluka, ≥98%), fluorescein isothiocyanate isomer I (FITC, Alfa-Aesar, 95%), NaCl (POCh), 0.01 M phosphate buffer saline (PBS, pH = 7.4, Sigma Aldrich), 1.0 M phosphate buffer solution (PB, pH = 7.4, Sigma Aldrich), 4′,6-diamidino-2-phenylindole (DAPI, Sigma Aldrich, ≥98%), Dulbecco's Modified Eagle's Medium (DMEM, PAN-Biotech) as cell culture media and supplements (Sigma Aldrich), and propidium iodide (PI, Invitrogen) were used as received. Dead Cell Apoptosis Kit with FITC Annexin V (Biomedica) and Cell Titer 96® Aqueous One Solution Cell Proliferation Assay (Promega) containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS) were applied in the cytotoxicity studies.

Synthesis and modification of star copolymers

Poly(glycidyl methacrylate-co-methyl methacrylate)s (S1–S5). The star-shaped copolymers have been synthesized according to previously reported procedure.²⁹ Bromoester functionalized acetal derivatives of methyl α-D-glucopyranoside²⁶ were used to initiate atom transfer radical copolymerization of methyl methacrylate and glycidyl methacrylate in the presence of CuCl/dNbpy/CuCl₂ catalyst system in anisole at 30 °C.

Ethylenediamine-functionalized poly(glycidyl methacrylate-co-methyl methacrylate)s (example for S5EDA). GMA-based star copolymer (0.25 g, containing 9.15 × 10⁻⁴ mol of GMA units) was dissolved in mixture of tetrahydrofuran–dimethylformamide (5 mL, 50/50 vol%). Subsequently, EDA (3 mL, 0.045 mol) and TEA (0.6 mL) were added and the reaction mixture was stirred at 55 °C for 72 h. Next, it was concentrated by rotary evaporation. The remaining solution was precipitated and washed with diethyl ether prior to being redissolved in deionized water (15 mL). The product was purified by ultrafiltration (6 kDa cutoff membrane) and its water solution was lyophilized (2 days) to obtain the crude product.

Fluorescein-functionalized poly{(methyl methacrylate)-co-(2-hydroxy-3-[(2-aminoethyl)amine]propyl methacrylate)}s (example for S5EDAF). Amine-based star copolymer (15 mg, containing 4.16 × 10⁻⁵ mol of EDA functionalized units) was dissolved in 10 mL of deionized water and mixed with 0.05 M borax and 0.4 M NaCl aqueous solution (1.4 mL) and with 7 mL of FITC solution in methanol (2.8 mg mL⁻¹, 5.11 × 10⁻⁵ mol). The reaction mixture was kept at 37 °C while stirring for 4 h. The dye-conjugated copolymer was ultrafiltrated using 3 kDa molecular weight cutoff membrane against PB/H₂O deionized (1/6 vol%), and then lyophilized to obtain the crude product.

Spectroscopic characterization

Fourier transform infrared (FT-IR) analysis was carried out with a NICOLET 6700 spectrophotometer at room temperature by attenuated total reflection (ATR) method. Spectra were recorded at 16 scans per spectrum and 4 cm⁻¹ resolution in the range of 4000–650 cm⁻¹.

¹H NMR spectra of the polymer solutions in D₂O or CDCl₃ were collected on Varian Inova 300 MHz and 600 MHz spectrometer at 25 °C using TMS (regarding to spectra made in CDCl₃) as well as TMSP-d4 (regarding to spectra made in D₂O) as an internal standard.

Size exclusion chromatography. Molecular weights and dispersities were determined by size exclusion chromatography (SEC) equipped with an 1100 Agilent 1260 Infinity isocratic pump, autosampler, degasser, thermostatic box for columns and differential refractometer MDS RI Detector. Addon Rev. B.01.02 data analysis software (Agilent Technologies) was used for data collecting and processing. The SEC calculated molecular weight was based on calibration using linear polystyrene standards. Pre-column guard 5 μm (50 × 7.5 mm) and PLGel 5 μm MIXED-C (300 × 7.5 mm) column were used for separation. The measurements were carried out in methylene chloride (HPLC grade) as the solvent at 30 °C with flow rate of 0.8 mL min⁻¹.

Isothermal titration calorimetry (ITC). The isothermal titration calorimetric experiments were carried out on a Nano ITC Standard Volume instrument from TA Instruments Corp. (Lindon, UT USA). The measurement set consists of a sample degassing unit and microcalorimeter with a reference and sample cell of 1 mL in volume. Both cells are insulated by an adiabatic shield. All samples were stirred under 37 °C during measurement. The reference cell was filled with buffer solution (125 mg borax and 159 mg of NaCl dissolved in 250 mL of H₂O : MeOH = 4 : 10 v/v). After system stabilization FITC solution (6.4–11.7 mM) was injected into the sample cell, containing polymer solution (11–23 μM). The titration was carried out as a sequence of 35 injections of 7 μL every 300 seconds at the constant temperature 37 °C. The heat effect due to mixing and dilution was corrected by means of control experiments in which FITC was injected into the buffer solution and the buffer was titrated to the polymer solution. The stoichiometry n, which is defined as molar ratio of FITC to the particular copolymer, was determined by non-linear least-squares fitting using Launch NanoAnalyze software version 2.3.6 (TA Instruments).

Cell culture. Human dermal fibroblasts (NHDF-Neo, Lonza, Poland) and human colon cancer cells (HCT116, ATCC, CCL-247) were cultured in Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 Ham medium (Sigma Aldrich) containing 10% fetal bovine serum (PAA Laboratories, Immuniq, Poland), and antibiotic/antimycotic solution (100×) containing 10 000 U mL⁻¹ of penicillin G, 10 000 μg mL⁻¹ of streptomycin, 25 μg mL⁻¹ of amphotericin B (Fungizone) (HyClone, Thermo Scientific).

Light scattering measurements. The hydrodynamic diameter (D_h) of particles was measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-S90 equipped with an 4 mW He–Ne ion laser operating at λ = 633 nm. Samples placed in PMMA cell after dilution with deionized water or PBS (0.4 g L⁻¹) were put in the thermostated cell compartment of the instrument at 25 ± 0.1 °C. All of the sample measurements were performed at a fixed scattering angle of 90°. At least 5 correlation functions were analyzed per sample in order to obtain an average value.

ζ (Zeta)-potential measurements. The surface charge of the polymer matrix was measured in deionized water and 0.01 M PBS (pH = 7.4). Diluted samples (0.04 g L⁻¹) were placed in disposable folded capillary cell and put in the thermostated cell compartment of the instrument (Malvern Zetasizer Nano-Z) at 25 and 36.6 ± 0.1 °C. Potential measurements were carried out on each sample from five independent runs. Error bars reported in the ζ-potential plot refer to the width of the ζ-potential distribution for each sample.

Cytotoxicity. The star polymers were tested for cell viability and cytotoxicity using the CellTiter® Aqueous One Solution Cell Proliferation Assay (MTS assay). A total of 10 × 10³ HCT116 or NHDF cells per well were seeded in a 96-well micro titer plates and incubated overnight at 37 °C to allow cell adherence. A stock of polymer solution (1 mg mL⁻¹) was prepared in sterile PBS and its appropriate volumes were added to DMEM media in wells resulting 200 μL of working solution. Cells with diamino-functionalized copolymer were incubated at 37 °C in CO₂ for 24 h. After that, the cell culture media were aspirated and washed with PBS. Solution of MTS (20 μL) was added per well and incubated at 37 °C for 1 h. The absorbance of the formazan product at 490 nm was measured directly from 96-well assay plates using microplate spectrophotometer (BioTekEpoch).

Cell internalization. A total of 500 μL per well of 10 × 10³ of HCT116 or NHDF cells were seeded directly onto a 4-well culture slides and incubated for 24 h. Thereafter, the media were aspired and 300 μL of S5EDAF star polymer solution in PBS (100 μg mL⁻¹) was added. The cells were incubated at 37 °C for 24 h, again. The cell culture media were aspirated and the remaining attached cells were washed with PBS and fixed with 70% ethanol for 10 min followed by washing with deionized H₂O. After staining with DAPI (10 μg), the slides with cells were examined by confocal laser scanning microscopy (CLSM, Olympus FV1000 microscope). All imaging conditions, including laser power, photomultiplier tube, and offset settings, were aligned with fluorescence intensity of sample. Fluorescent polymer domains and DAPI were excited using argon-ion laser 488 nm line and diode laser 405 nm line, respectively. A multitracking technique was used to reduce interchannel cross-talk. Image acquisition was performed using 40× 0.95 NA oil immersion objective lens. Images were stored in TIF format and analyzed using IMARIS software (OLYMPUS). 3D reconstructions of specified cells were generated to determine exact intracellular localization of FTIC dye.

Flow cytometry. A total of 5 × 10⁴ HCT116 cells were suspended in 840 μL of DMEM media, and with 160 μL of diamine-functionalized star polymer PBS solution (2.5 g L⁻¹) in a 12-well culture plate were incubated for 24 h at 37 °C. The control group did not receive any polymer. The cells were then collected into 1.5 mL tubes and centrifuged at 1100 rpm for 3 min to pellet the cells. The supernatant was removed and the cells were washed with 200 μL of PBS followed by centrifugation at 1100 rpm for 3 min. The cells were resuspended in Annexin V binding buffer and transferred to a borosilicate tubes followed by 2.5 μL of FITC Annexin V addition. After 15 minutes the 250 μL of PBS and 10 μL of PI solution (100 μg mL⁻¹) were added. The cells were gently vortex and incubated for 15 min at room temperature in the dark. Each sample was analyzed using ARIA III flow cytometer (Becton Dickinson). Fluorescence was measured using the FITC configuration (488 nm laser line, LP mirror 503, BP filter 530/30) for Annexin V, and PI (488 nm laser line, LP mirror 566, BP filter 585/42).

Results and discussion

Synthesis of amphiphilic water-soluble star-shaped copolymers

Previously, the synthesis of P(MMA-co-GMA) star-like copolymers with acetal derivatives of methyl D-glucopyranoside in the core has been reported.^26,29 GMA and different ratios of MMA/GMA have been used to obtain polymers varying the content of pendant reactive epoxide groups (25, 50, 75 and 100 mol%), which might have an influence on the adjustment of carried molecules, as well as the efficiency of conjugation, and steric hindrance. Thereafter, aminolysis of reactive epoxide groups with the use of a nucleophilic agent EDA allowed to obtain a water-soluble star copolymers with different content of amine groups (27–100 mol%) (Scheme 1 and Table 1). To ensure complete functionalization of the epoxy groups and no intra- or intermolecular crosslinking the molar ratio of EDA to GMA units was more than 40. Introduction of amine groups in the arms of the star polymers was confirmed by NMR and FT-IR spectroscopy.

Scheme 1 Synthesis of star-shaped copolymers with methyl α-D-glucopyranoside cores and fluorescein-functionalized polymethacrylate arms.

Table 1 Characterization of 3- and 4-arm GMA based star copolymers before and after modification with EDA

Entry	Initial copolymer	Star-shaped copolymers						Star-shaped copolymers modified with EDA^a
		FGMA	f	DParm	Mn,NMR	Mn,GPC	Đ	ζ-potential [mV]				Dh [nm]
								H2Odei	PBS	H2Odei	PBS	H2Odei	PBS
								36.6 °C		25.0 °C
a FEDA = FGMA, due to complete ring opening epoxide reaction with EDA.
S1EDA	S1	1	3	57	25 100	18 200	1.32	46.0	17.2	46.9	19.2	288	10
S2EDA	S2	0.75	3	79	31 900	23 800	1.32	46.3	19.8	36.5	20.7	136	12
S3EDA	S3	0.56	3	30	11 700	9900	1.28	33.9	15.8	36.7	11.3	30	6
S4EDA	S4	0.27	3	64	22 400	19 200	1.30	30.4	17.2	29.6	15.0	12	8
S5EDA	S5	0.52	4	75	37 700	31 700	1.20	18.9	18.6	24.1	19.3	4	13

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¹H NMR spectrum of star copolymers before reaction with EDA (Fig. 1a) shows the signals at 3.23, 2.68, and 2.64 ppm corresponding to the methylidene (–CH(O)–CH₂–, e) and methylene (–CH(O)–CH₂–, f) protons of the epoxide ring, respectively. After the ring-opening reactions of GMA-based star copolymer with EDA the signals assigned to epoxy groups (e and f) disappeared completely (Fig. 1b), whereas the peaks at 4.3 and 3.8 ppm (–OCH₂–, d) are shifted to 4.07 ppm due to different neighborhood after modification. The new signal located at 3.31–2.74 ppm can be assigned to methylidene proton adjacent to hydroxyl group and methylene protons adjacent to the amine groups (e′, CH–OH, f′, CH₂–NH–, g, CH₂–NH₂). The spectrum in D₂O (Fig. 1b) can be compared to the spectrum made for the same copolymer in CDCl₃, shown in Fig. 1c. The absence of most of the peaks indicates limited mobility of copolymer in CDCl₃ which is a non-polar solvent and may suggest that copolymer tends to create self-assembled nanostructures.

Fig. 1 ¹H NMR (300 MHz) spectra of S5 (four arm star) in CDCl₃ (a) and modified S5EDA in D₂O (b), and in CDCl₃ (c).

As shown in Fig. 2 the proton signals are broadened with the increase in contribution of amine-functionalized units. Since the intensity of an ¹H NMR signal is proportional to the number of proton nuclei giving rise to the signal, whereas the broadening of signals which is the greatest for S2EDA copolymer (F_EDA = 0.75 mol%) might indicate creation of aggregates in water solution. This concept was postulated by Collison et al.³⁰ as the protons in aggregated chains can not contribute to the NMR signal intensities due to steric limitation of the polymer backbone motions, which diminish the resonance intensity as a result of a change in the solution environment attributed to the formation of aggregates in the solution.

Fig. 2 ¹H NMR (300 MHz) spectra of EDA-functionalized star copolymers, S2EDA F_EDA = 75 mol% (a), S3EDA F_EDA = 56 mol% (b), and S4EDA F_EDA = 27 mol% (c).

Transformation of glycidyl groups into hydroxylamino groups was also confirmed by FTIR spectroscopy comparing spectra of before (Fig. 3a) and after modification (Fig. 3b). In the case of the unmodified copolymers (S2–S4) the characteristic absorption bands at 1720 cm⁻¹ and 903 cm⁻¹ ascribed to carbonyl group and epoxy ring were observed, respectively. In the spectra of diamine-functionalized copolymers (S2EDA–S4EDA) the broad absorbance band corresponding to the stretching vibration of hydroxyl group (ν_OH) appeared at 3570–3200 cm⁻¹. The deformation (δ_NH) and stretching (ν_NH) vibration absorptions of the primary and secondary amine groups (N–H, [double bond splayed left]N–H) appeared at 1555, 1640 and 3380 cm⁻¹, respectively. Additionally, the peaks at 905 and 841 cm⁻¹ which were assigned to the stretching vibration of epoxide groups almost completely diminished. These spectra variations showed that the epoxy groups had been successfully transformed into β-amino alcohol groups via ring opening reaction.

Fig. 3 FT-IR spectra of S2–S4 copolymers (a) and modified via ring opening reaction with EDA (b).

Apart from material, shape and composition, the main influencing factors for cytotoxicity are nanoparticle size and surface charge, including both charge density and charge polarity. Zeta potential measurements served to characterize the surface of star copolymers and helped to predict their behavior in different environments. As has been shown in Table 1, the positive values of ζ-potential for EDA-functionalized stars in deionized water (pH = 7.0) and PBS (pH = 7.4) were detected at 25 and 36.6 °C. The measurements revealed that the type of solvent and copolymer composition are obvious aspects which affect particle surface characteristics. Fig. 4a shows that zeta potential values are lower in PBS (19.8–15.8 mV) than that in deionized water (46.6–18.9 mV), whereas the change of temperature does not influence significantly on the electrokinetic properties of nanoparticle surface. The difference related to the solvent character can be explained by the fact that diamine-functionalized arms interact with salt components of PBS resulting in positive charge shielding. Taking into consideration the copolymer composition, Fig. 4b shows the increase of the zeta potential with EDA-modified GMA unit content in the arms, which is associated to increasing number of ionized amino groups. To compare star copolymers S3EDA and S5EDA with similar content of amino groups the zeta potential is lower for 4-armed copolymer, which means that the shape of star copolymer have influenced on the properties of particle surface. In our case unsymmetrical (one sugar unit in the core with three arms) vs. symmetrical (two sugar units in the core with four arms) topologies are designed for the studied polycations.

Fig. 4 Zeta potentials of EDA-modified star copolymers presented as a mean ± sd (a); dependence of diamine unit content (F_EDA) on zeta potential and hydrodynamic diameter values for particles based on 3-arm star-shaped copolymers S1EDA–S4EDA (b); and zeta potential distribution by volume plots of S1EDA–S4EDA in deionized water at 25 °C (c).

The results of hydrodynamic diameter (D_h) measurements, which were performed at 25 °C in PBS and deionized water, are summarized in Table 1. The influence of type of solvent on the star-shaped particles interactions can be inferred from the change in D_h values. In the PBS environment the particles with D_h ∼ 6–13 nm are observed, where their size changes proportionally to DP_arm, whereas in water the particles based on 3-armed star copolymers gradually become larger with the increase of EDA group content (Fig. 4b) indicating aggregation. The biggest aggregates reaching the size above 250 nm are formed in the case of S1EDA with homogeneous composition of arms (without MMA units). The smallest particles of 4-armed copolymer S5EDA in water seems to be irregular behavior in comparison to 3-armed S3EDA with similar EDA content (50 mol%), but lower DP of EDA in arm (37 units in S5EDA vs. 15 units in S3EDA). These parameters let to assume that the particle size of S5EDA in water should be at least the same or even larger, but these lack of correlation is well related to the results of zeta potential. In PBS S5EDA behaves similarly to the other stars, although particles are 3 times larger than that ones in water, but still small in the size of 13 nm.

Synthesis of polymer conjugates with fluorescein isothiocyanate

Fluorescein isothiocyanate (FITC) is amine-reactive derivative of fluorescein dye that has wide-ranging application as antibody and other probe labels for use in fluorescence microscopy, flow cytometry and immunofluorescence-based assays.³¹ Our intention was chemical conjugation of fluorescein derivative to the star copolymer via amino groups to generate fluorescent marker of biological evaluation in vitro. After liofilization the fluorescein-labeled star copolymer S2EDAF was not soluble in water and other polar solvents. However, the solutions of other polymers can be attained using the appropriate proportions of deionized water and DMSO (98/2 vol% for S3EDAF and S5EDAF; 50/50 vol% for S4EDAF). Regardless, NMR analysis of labeled star copolymers in D₂O/DMSO-d₆ was insufficient for good quality spectra, therefore, they were obtained in DMSO-d₆. Fig. 5 presents ¹H NMR spectra of S4EDAF with chemically conjugated fluorescein isothiocyanate by means of thiocarbamide bonding. The region from 6–11 ppm, which represents characteristic proton signals belonging to fluorescein linked to amine groups in arms of star copolymer, is enlarged and outlined with the dotted frame. The broad signal around 10 ppm corresponds to the proton of the carboxyl group in the attached fluorescein units. Next, in the region of 7.00–8.50 ppm signals of hydrogen atoms in benzene ring with acidic one in –COOH group can be seen. The most pronounced peaks in the range of 6.40–6.80 ppm are assigned to protons in xanthene group.

Fig. 5 ¹H NMR (600 MHz) spectra of S4EDAF conjugate of star copolymer with fluorescent dye.

The amount of fluorescein molecules attached to the selected star copolymers was calculated from ¹H NMR spectra. In addition, the ITC method was applied in order to determine the maximum amount of FITC that could be linked to the macromolecule. Data from 35 injection sequence shown in Fig. 6 were analyzed on base of a independent model, which assumes one type of active centers that do not interact with each other, and excludes any molecular interactions. Each peak in the calorimetric titration plot represents a single injection of FITC solution. The positive deviation of the heat signal from the baseline within addition of FITC indicates that thiourea bond formation is exothermic. Whereas from the shape of the isotherm, which goes to zero, it is apparent that the actual binding saturation is reached. In contrast to the reactions in conventional flask, that were carried out with the molar ratio 1 : 1 (S2EDA, S3EDA, S5EDA) or 1 : 2 (S4EDA) of amino groups in the copolymer to FITC and characterized by NMR, the ITC experiments illustrate the maximum amount of FITC molecules that can create thiourea bonds as the result of chemical reaction between isothiocyanate group in FITC and amino group in copolymer arms. The reaction efficiency and the number of conjugated molecules calculated by NMR analysis (E, n_FITC) and the ITC experiments (E_b, n_b,FITC) are presented in Table 2. The ITC results suggest that an excess of MMA or EDA-functionalized units is associated with reduced conjugation effect (∼50%). In case of MMA excess lower efficiency may be caused by hydrophobic interactions between the arms shielding some of the amine groups, whereas EDA-functionalized unit excess makes that FITC with steric hindrance can be attached to half of the amine groups in the star copolymer. The conjugation efficiency is also limited for S5EDAF and lower than for analogous S3EDAF (four-arm vs. three-arm star copolymer), which means that number of arms and their distribution related to type of sugar core is additional crucial parameter in polymer modification by dye. NMR results for the latter pair of polymers are in a good correlation with ITC data, whereas significantly larger conjugation efficiency of S4EDA with excess of FITC in reaction with EDA groups suggests the association of FITC molecules, what could explain overestimated value.

Fig. 6 ITC thermogram of FITC binding to copolymer S2EDA with integrated heat profile of calorimetric titration after correction in inset.

Table 2 Data relating the FITC conjugation efficiency^a

Entry	FEDA	Reaction time [h]	E^a [%]	nFITC^a	Eb^b [%]	nb,FITC^b
a nFITC – number of conjugated FITC; ^abased on ^1H NMR spectra; ^bbased on ITC thermograms; n.d. – not dissolved in typical deuterated solvent used in NMR analysis.
S2EDAF	0.75	4	n.d.	n.d.	47	83 ± 1
S3EDAF	0.56	4	68	34	100	58 ± 1
S4EDAF	0.27	4	85	45	45	24 ± 1
S5EDAF	0.52	17	58	90	56	88 ± 1

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Cytotoxicity of star polymers

It is generally considered that polycations can bind to the negatively charged plasma membrane and destabilize them. This is a main reason why a higher cytotoxicity of such materials has been reported.^32,33 Cationic nanoparticles cause more pronounced disruption of plasma-membrane integrity, stronger mitochondrial and lysosomal damage, and a higher number of autophagosomes than anionic ones.³

To study the interactions of star copolymers with HCT-116 and NHDF as a cellular models, the cytotoxicity of S5EDA was first investigated by measuring the cell proliferation as an index of cell viability by means of the MTS assay. The concentration effect on the proliferation of HCT-116 and NHDF cells was evaluated and the relative viability is shown in Fig. 7. The NHDF cell viabilities ranged between 81% to 108% of the control with increasing concentrations from 0.013 mg mL⁻¹ to 0.500 mg mL⁻¹ exhibited low cytotoxicity of polymers, which suggests that they have potential for successful drug delivery and would not interfere with the MTS test of loaded drug. Another effect was observed for the proliferation of HCT 116 cells (columns with stripes in Fig. 7) showing the highest values at low star copolymer concentrations. However, when the concentration was increased up to 0.125 mg mL⁻¹ the cell viability decreased gradually reaching 58% and further at higher concentrations slightly increased to 70%. This indicates the properties of EDA-functionalized star copolymer are suitable to inhibit cell proliferation in the selected tumor cell model.

Fig. 7 Relative cell viability: 10 × 10³ of NHDF and HCT116 cells in respective medium incubated with star copolymer S5EDA PBS solution in 96 well micro titer plates in a humidified atmosphere containing 5% CO₂ at 37 °C for 24 h. MTS assay is based on bioreduction of tetrazolium salt into formazan changing solution color from yellow to orange. The results of cell proliferation are presented as a mean ± sd.

Flow cytometry is well known technique for the analyses of multiple parameters of individual cells, inter alia allows to distinguish viable cells from early and late stages of apoptosis and from necrotic cells, which could be quantitatively determined using appropriate assay. Since MTS assay has revealed that, ready after 24 h, 0.075 mg mL⁻¹ of star copolymer was sufficient to affect inhibition of tumor proliferation, Annexin-V specific assay was used to assess whether the observed effect of reduced proliferation was actually attributable to apoptotic effects. Annexin-V is a phospholipid binding protein that has a high affinity for phosphatidylserine, which is asymmetrically localized on the cytosolic side of cell membrane under normal conditions, but during the early stages of apoptosis it is reversed and exposed on the outer monolayer of the cell membrane. Taking advantage of high-affinity of Annexin-V for phosphatidylserine, the apoptotic cells could be quantitatively determined by using fluorescently labeled Annexin-V in combination with a vital dye such as PI (A/PI) to distinguish necrotic ones. In Fig. 8 the flow cytometry data are shown for untreated HCT-116 cells and with S5EDA after incubation for 24 h. The polymer assay results indicate that 35% of cells are in late apoptosis (A+/PI+) or already dead 7% (A−/PI+). The cell samples treated with the star copolymer showed higher rates of both apoptosis and necrosis in comparison with the untreated ones.

Fig. 8 Apoptosis assay. Cell death assayed by means of Annexin-V/PI (A/PI) double staining on HCT-116 cells exposed to 0.5 mg mL⁻¹ of star copolymer S5EDA for 24 h (right), and untreated sample as control (left). Viable cells (A−/PI−), late apoptotic (A+/PI+), early apoptotic (A+/PI−) and necrotic (A−/PI+).

Cellular uptake of star polymers

The successful internalization of star copolymers in cells after 24 h of incubation has been verified by confocal laser scanning microscopy. Fig. 9 shows fluorescence of S5EDAF (green) adjacent to the DAPI (blue) suggesting pancytoplasmic subcellular distribution of star polymer in both NHDF and HCT-116 cells. For drug delivery system, the localization of polymer inside the cell is an important factor to be effective. In both types of cell lines the localization of polymeric carrier is comparable. After 24 h its accumulation was stronger in the plasma membrane and in peripheral regions of the cytoplasm demonstrating the fluorescence mainly in organelles around the cell nucleus.

Fig. 9 Cell internalization of FITC conjugated star copolymers S5EDAF with NHDF cells (a) and HCT-116 cells (b) after 24 h. The confocal images were captured by means of sequential scanning mode under 40× (a) and 10× magnification (b). Green fluorescence by fluorescein units in star copolymer (excited with laser wavelength 488 nm), and blue fluorescence by DAPI dye (excited with laser wavelength 405 nm).

Conclusions

The new amphiphilic, water-soluble star copolymers with sugar cores and methacrylic arms bearing various content of hydroxylamino groups were synthesized and characterized toward DDS utilization. The positively charged copolymers were able to create aggregates in pH = 7.0. There has been shown that in deionized water the values of zeta potential can be varied widely with contribution of amine groups in polymer, which was well-correlated with the size of particles at maximum above 250 nm. The cell cultures exposed to the action of stars with increasing concentrations (0–0.5 mg mL⁻¹) in MTS assay demonstrated the promising for future DDS application behaviors with almost unchangeable proliferation of NHDF cells and significantly lower for HCT-116 cells. According Annexin-V assay results these copolymers affect HCT-116 cell cycle with higher toxicity and rate of apoptosis and cell death (reduced proliferation degree up to 60–70%). The amine-functionalized star copolymers were successfully conjugated with fluorescent FITC and characterized by ¹H NMR spectroscopy. Additionally, the ITC experiments were applied to determine the maximum number of FITC molecules that were able to create chemical bonds with each of the prepared star-shaped copolymers. The amount of chemically linked FITC was sufficient to confirm cellular internalization of copolymers into NHDF and HCT-116 cells by the use of confocal microscopy. However, the precise determination of polymer localization requires further studies involving typical identification as organelle markers.

Acknowledgements

This work was financially supported by the National Science Center (NCN, Poland) according to Decision no. DEC-2012/07/N/ST5/01875 (A.M. & D.N.). Biological experiments were performed in the Biotechnology Center of the Silesian University of Technology using equipment financed by the “Silesian Biofarma” program. M.S. was also supported by grant no. BKM/514/RAU-1/2013 t.26 from Silesian University of Technology.

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