Chem Biol Drug Des 2014 Research Letter
Carboxylated Hydroxyethyl Starch: A novel Polysaccharide for the Delivery of Doxorubicin Constantinos M. Paleos*, Zili Sideratou, Theodossis A. Theodossiou and Dimitris Tsiourvas Department of Physical Chemistry, IAMPPNM, NCSR ‘Demokritos’, 15310 Aghia Paraskevi, Attiki, Greece *Corresponding author: Constantinos M. Paleos,
[email protected] Hydroxyethyl starch (HES) was interacted with succinic anhydride affording a carboxylated derivative which has proved to be a promising polymeric drug delivery system. Specifically, this polymer is conveniently prepared, is biodegradable, non-immunogenic, and can encapsulate doxorubicin due to the protonation of the primary amino group of doxorubicin by the carboxylic group located on the branched scaffold of the polysaccharide. In addition, due to the polyhydroxylated character of the polysaccharide, the latter can act as a protective coating in an analogous manner to the PEGchains ensuring prolonged circulation in vivo. In vitro experiments showed controlled release of doxorubicin to the nuclei of DU145 prostate cancer cells when the anticancer drug is incorporated in the carboxylated hydroxyethyl starch. Key words: doxorubicin, drug delivery, encapsulation, hydroxyethyl starch, hyperbranched polymer Received 26 June 2014, revised 22 September 2014 and accepted for publication 30 September 2014
Hydroxyethyl Starch, HES (Scheme 1), is a commercially available semisynthetic polysaccharide, usually prepared from starch and is rich in amylopectin. Specifically, HES is a branched biopolymer consisting of alpha (1,4)-glycosidic-linked anhydroglucose units. The chain branching are formed by (1,6)-glycosidic bonds. Hydroxyethyl starches with molar degrees of substitution (MS) from 0.5 to 0.7 have proven to be effective in clinical use (1). Its structure, schematically depicted in Scheme 1, is similar to glycogen which is the branched glucose storage polymer of humans. This similarity of HES to glycogen is possibly one of the reasons for its non-immunogenicity (2,3). In addition, HES exhibits a number of biologically advantageous features such as biocompatibility, biodegradability, and a multiplicity of functional groups is readily accessible to functionalization affording synthetic multifunctional systems ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12447
(4). In this connection, dextran polysaccharide, mainly composed of 1,6-linked D-glucopyranose residues, which is also biocompatible and inert, has been used as drug delivery system and specifically for doxorubicin, DOX (5). In addition to the above properties, HES is both more soluble and stable compared to starch and most importantly an increase in vivo half-life is observed (6) when applied as drug delivery system. The half-life which is of the order of only a few minutes for natural starch, due to its rapid degradation by serum amylases (7), is increased for HES due to the steric hindrance exercised by the hydroxyethyl moieties. Such favorable properties rendered HES as one of the first-line colloidal plasma volume expanders (7). Moreover, due to its polyhydroxylated nature, it is now investigated as a potential substitute for poly(ethylene glycol) (PEG) (8). The latter polymer is widely used to stabilize nanoparticles including liposomes, by imparting a ‘stealth’ character which prolongs blood circulation of nanoparticles (9– 11). However, despite improvements relative to unmodified counterparts, PEG may not be the best choice for improving circulatory stability (12) of drugs and/or drug delivery systems and investigations have been published according to which PEG was replaced by oligo- or polysaccharides (8,13,14). In this connection, HES being a polyhydroxylated compound can also be a promising candidate as a protective coating for drugs and drug delivery systems. In view of these properties of HES together with its facile functionalization, it was reacted with succinic anhydride for the introduction of easily accessible carboxylic groups that are suitable for further functionalization or non-covalent interaction. The carboxylic group can interact with compounds bearing primary or secondary amino moieties forming either ionic salts or amides. This is the case, for instance, with the anticancer drug doxorubicin (Scheme 2), the primary amino group of which can interact with the carboxylic group of carboxylated HES forming either salts or amides depending on the conditions. Doxorubicin is widely used in chemotherapy for various tumors, including hematological malignancies, many types of carcinoma, and soft tissue sarcomas. The exact mechanism of action of doxorubicin is complicated and still somewhat unclear, although it is considered to interact with DNA by intercalation (15,16), including inhibition of DNA helicases, topoisomerase II, and RNA polymerase. 1
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Scheme 1: Simplified chemical structure of HES.
However, due to severe side effects of doxorubicin such as cytotoxicity in normal tissue and pervasive cardiotoxic effects (17), its application is limited. In the present communication, extending our work on the development of multifunctional dendritic drug delivery systems described in our reviews (18,19), a novel dendritictype carrier for doxorubicin is introduced. The properties of doxorubicin, bound electrostatically to the polymeric scaffold of carboxylated HES, should be very possibly affected. Specifically, doxorubicin can also be located inside the nanocavities, which are formed by the branched HES derivative, in analogy with the non-covalent attachment of the drug inside the nanocavities of conventional hyperbranched polymers. In principle, as a consequence of these structural features, release of the drug could be modified as well as its transport through cell membrane.
Methods and Materials Hydroxyethyl starch (Molecular weight 115.000–140.000, MS 0.40–0.44) was kindly donated by Serumwerk Bernburg AG, Bernburg, Germany. Succinic anhydride was a product of Fluka AG, Buchs, Switzerland and Amberlite MB-3 of Merck KGaA, Darmstadt, Germany.
Dynamic light scattering studies were performed employing an AXIOS-150/EX (Triton Hellas) apparatus with a 30 mW laser source and an Avalanche photodiode detector at an angle of 90°. At least ten light scattering measurements were collected, and the results were averaged. Size differences were not observed upon dilution. f-potential measurements were conducted using a ZetaPlus of Brookhaven Instruments Corp., Holtsville, NY, USA. Ten f-potential measurements were collected, and the results were averaged.
Synthesis of carboxylated hydroxyethyl starch (C-HES) To 7 9 106 mol (0.91 g) of hydroxyethyl starch (MW 130 000 Da), dissolved in dry DMSO, 7 9 104 mol (0.070 g) of succinic anhydride also dissolved in dry DMSO was added. To the reaction mixture, 7 9 105 mol (0.085 g) of 4-dimethylaminopyridine was added and the reaction was allowed for 24 h at 40 °C. The reaction mixture was precipitated with 50 mL of ethanol:ether (1:1) mixture. The precipitated material was dissolved in water and dialyzed for 48 h to remove reaction by-products. Following the dialysis, the aqueous solution was treated with Amberlite MB-3. Following this treatment, the water was distilled-off under reduced pressure and a glassy material was obtained. The structure of C-HES was established by proton and carbon NMR spectroscopy using a Bruker Avance DRX spectrometer operating at 500 and 125.1 MHz, respectively. H NMR (500 MHz, D2O): d (ppm) = 2.60–2.75 (m, CH2CH2COOH), 3.2–4.2 (m, CH-O, CH2-O of HES), 5.2– 5.6 (H-1(1?4), H-1(1?6), H-1 terminal, H-1 (close to hydroxyethyl group). 1
C NMR (125.1 MHz, D2O): d (ppm) = 177 (C=O, carboxyl group), 174 (C=O, carbonyl group), 96–100 (C-1 substituted, unsubstituted, and terminal), 82 (C-3 substituted), 79 (C-2 substituted), 77 (C-4 substituted), 75 (C-5 substituted), 74 (C-4 unsubstituted), 73 (C-3 unsubstituted), 72 (C-2 unsubstituted), 71 (C-7 unsubstituted, C-5 13
Scheme 2: Chemical structure of DOX.
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Carboxylated Hydroxyethyl Starch as Drug Delivery System
unsubstituted), 70–69 (C-4 terminal, C-6 substituted), 64– 65 (C-7 substituted, C-8 substituted), 60–62 (C-8 unsubstituted, C-6 unsubstituted), 29 (CH2CH2COOH).
Formation of doxorubicin/carboxylated hydroxyethyl starch, C-HES-DOX To 105 mol (0.0188 g) of carboxylated hydroxyethyl starch (MW of the monomer unit was taken 188.43), dissolved in water, 105 mol (0.058 g) of doxorubicin hydrochloride and 1.5 9 105 mol (0.016 g) of sodium hydrogen carbonate, also dissolved in water, were added and remained under stirring for several hours. Subsequently, the solution was subjected to dialysis (molecular weight cut-off 1200 Da) against water for 24 h and the final product was received after lyophilization. The resulting doxorubicin/carboxylated hydroxyethyl starch salt, C-HESDOX, is water soluble exhibiting stability in aqueous media at room temperature.
Determination of DOX For the determination of DOX encapsulated in C-HES, a calibration curve for various DOX concentrations (10– 100 lM) in DMSO was constructed using a Cary 100 Conc UV-Visible spectrophotometer (Varian Inc., Mulgrave, Victoria, Australia). For every batch prepared, C-HES-DOX aliquots (50 lL) were diluted in 700 lL of DMSO and the absorbance at 561 nm was registered using the same experimental conditions as those applied for the calibration curve’s construction.
Cell culture Cells used in this study were the human prostate carcinoma cell line DU145. The cells were grown in RPMI 1640 without phenol red, with 10% FBS, penicillin/streptomycin (100 IU/mL per 100 lg/mL) at 37 °C in a 5% CO2 humidified atmosphere. Cell incubation with C-HES or C-HESDOX (and consequently with DOX) was always performed in OPTIMEM (without phenol red).
Cell viability DU145 human prostate cancer cells were inoculated (20 9 103) into 96-well plates and allowed to incubate in complete media containing 10% FBS for 24 h. Cells were then treated with 1–10 lM control C-HES, free DOX, or CHES-DOX for 3 h in OPTIMEM. The mitochondrial redox function (translated as cell viability at t ≥ 24 h postincubation) of all cell groups was assessed by the MTT assay at selected time-points, namely 24 and 48 h postincubation. This was performed by replacing cell media with complete media containing 1 mg/mL MTT and incubating at 37 °C in a 5% CO2 humidified atmosphere for 2 h. MTT media were then removed from all cell groups and the produced formazan was solubilized with 100 lL DMSO per well. The plates were subsequently shaken for 10 min at 100 rpm in Chem Biol Drug Des 2014
a Stuart SI500 orbital shaker, and the end-point absorbance measurements at 562 nm were made in an Infinite €nnedorf, SwitzerM200 plate reader (Tecan group Ltd., Ma land). Blank values measured in wells with DMSO and no cells were in all cases subtracted. All experiments were repeated independently at least three times. MTT data are shown as means of at least six independent values with error bars representing one standard deviation. Student paired t-tests were performed on the MTT cytotoxicity data and the statistical significance follows the assignment: **p < 0.01, ***p < 0.001 and *****p < 0.00001, while no annotation implies no statistical significance, p > 0.05.
Cellular uptake DU145 cells were inoculated on 22 mm cover slips housed in 35 mm Petri dishes (10 9 105) and allowed to grow overnight in 2 mL of complete RPMI 1640. Subsequently, the cells were incubated with C-HES as a control, free DOX, and C-HES-DOX at various DOX concentrations (2–5 lM) for 3 h in OPTIMEM. After washing the cover slips with PBS, they were inverted onto microscope slides; the slides were placed under the Olympus UPLFLN40 9 objective (NA 0.75) of an Olympus BX-50 microscope coupled with an Olympus DP71 digital color camera, used to obtain epifluorescence microscopy images. Fluorescence excitation was facilitated with a Mercury USH 102D lamp (Ushio Inc., Tokyo, Japan) while fluorescence emission was imaged by employing a DAPI/FITC/TRITC filter, Chroma Technology Corp., Bellows Falls, VT, USA (excitation: 555 nm, emission > 580 nm).
Results and Discussion The backbone of HES consists of a-(1,4)-glycosidic-linked anhydroglucose units (AGU), which has branches formed by a-(1,6)-glycosidic bonds. Hydroxyethylation takes place predominantly at the C-2, C-3, and C-6 sites of the glucose rings. Substitution at C-2 position predominates over that at C-6, followed by C-3 position (20). In this study, carboxylation of HES was performed under mild experimental conditions. The introduction of carboxyl groups which can take place at the unsubstituted C-2, C-3, and C-6 positions of the glucose rings as well as at the hydroxyl groups of the substituted hydroxyethyl groups was established by 1 H NMR and 13C NMR (Figure S1). Specifically, the substitution of HES was confirmed by the appearance in the 1H NMR spectrum of a characteristic broad peak centered at 2.65 ppm, attributed to the a- and b-methylene protons adjacent to the carboxylic group. In addition, in the 13C NMR spectrum, the ester formation with primary and secondary hydroxyl groups of HES was confirmed by the appearance of the peak at 174 ppm. Furthermore, the introduction of carboxyl groups was confirmed by the peaks at 177 and 29 ppm attributed to the carbon atoms of (i) the carboxyl group and (ii) of the a- and b-methylenes relative to the carboxyl group, respectively. 3
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The molecular substitution (MS) of HES was calculated by the equation 1 using the integrals of the signals of the protons of carboxylic groups (I[CH2CH2COOH]) and the sum of the integrals of the signals from the anomeric protons I [H-1(HP)+(H-1(1?4)+H-1(1?6)+H-1 terminal] according to the literature (21): I[CH2 CH2 COOH=4 MS ¼ I [H - 1(HP) + (H - 1(1 ! 4) + H - 1(1 ! 6) + H - 1 terminal] (1) It was found that the MS is 0.15, that is, approximately 15 succinic acid moieties are attached per 100 glucose units. Thus, a novel anionic drug delivery system is obtained able to interact electrostatically with basic bioactive compounds, such as doxorubicin. Drug loading was calculated by determining the DOX concentration, employing UV–Vis spectroscopy, in the resulting DOX/C-HES solution obtained after the extensive dialysis described above. The DOX/C-HES mass ratio was found equal to ~0.176 g/g. Taking into consideration, the mean Mw of the C-HES repeating unit (~190.0) and the calculated MS of HES, it is estimated that there are ~0.4 DOX molecules per carboxylate group. The interaction of DOX with the carboxylic acid moieties is also exemplified by the f-potential values of the system. The f-potential of C-HES was found to be 15.0 mV (standard error 0.85), while the f-potential of DOX loaded C-HES was 6.2 mV (standard error 0.80) as a result of the (partial) charge neutralization upon salt formation. On the other hand, the particle size is about 25 3 nm in diameter as observed by dynamic light scattering spectroscopy and is not affected, within the experimental error, upon DOX encapsulation. Representative images of fluorescence microscopy on DU145 cells incubated with DOX and C-HES-DOX are shown in Figure 1. Cells incubated with control C-HES, that is, polysaccharide without DOX produced no measurable fluorescence and resulted in blank image acquisition. Cells incubated with free DOX (Figure 1A) clearly show DOX fluorescence in their nuclei, that is, the site of DOX action (22), while cells incubated with C-HES-DOX exhibit intense DOX fluorescence in their cytosol. This is apparA
ently due to DOX encapsulation in C-HES which has a different subcellular localization compared to free DOX. The toxicity of C-HES vs. free DOX is shown in Figure 2. DOX had a progressive toxicity by concentration and assessment time with a maximum cytocidal activity of c.a. 75% (25% survival) at 10 lM and at 48 h postincubation. C-HES-DOX also exhibited a progressive toxicity reaching a maximal cytocidal activity of c.a. 65% (35% survival) at 10 lM and at 48 h postincubation. Clearly in all cases, free DOX showed higher toxicity; however, at 48 h, C-HESDOX seemed to be almost equally effective with free DOX in terms of cytotoxicity. On the other hand, it should be noted that C-HES has no notable toxicity at all concentra-
Figure 2: Comparative cytotoxicity of C-HES, free DOX, and CHES-DOX following 3-h incubation. In all experiments, DOX concentration was 1, 5, and 10 lM while the corresponding CHES concentration was 3.4, 17.0, and 34.0 lg/mL, respectively. The cytotoxicity was assessed at 24 and 48 h postincubation by standard MTT assays. The survival rates are relative to media-only controls. MTT data are shown as means of at least six independent values with error bars representing one standard deviation. The statistical significance, as accrued by student paired t-tests follows the assignment: **p < 0.01, ***p < 0.001, ****p < 0.0001, and *****p < 0.00001 while no annotation implies no statistical significance, p > 0.05.
B
Figure 1: DU145 cells incubated with (A) free DOX and (B) C-HES-DOX for 3 h; DOX concentration 2 lM.
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tions studied, which were identical with those employed for C-HES-DOX experiments. Although C-HES-DOX is localized in cell cytosol and not in the nucleus, it becomes apparent that, over the timeperiod employed, there is a controlled DOX release from C-HES-DOX resulting in a rapidly escalating toxicity, which almost reaches that of the free DOX toxicity at 48 h. Moreover, the intense C-HES-DOX fluorescence which translates to higher DOX content, possibly due to enhanced cellular uptake properties of the proposed drug delivery system, is attributed to the polysaccharide nature of the carrier.
Conclusions Carboxylated HES is a promising polysaccharide-based hyperbranched drug delivery system which can be conveniently prepared, is biodegradable due to its polyhydroxylated structure, and can encapsulate active ingredients such as doxorubicin. The properties of doxorubicin, ionically bonded to the hyperbranched polysaccharide chain, were radically different as compared to free doxorubicin. In in vitro studies employing DU145 human prostate cancer cells, free DOX localization inside nuclei was drastically modified when the drug was encapsulated in carboxylated HES. In this case, C-HESDOX is preferentially localized in the cytosol and significant cytotoxicity was observed after ca. 48 h due to slow controlled release of DOX to the nucleus. This is attributed to the polysaccharide carrier which modifies doxorubicin’s transport through cell membranes and also drug’s controlled release because of its strong electrostatic attachment to the carbohydrate scaffold. Generalizing these results, we can assume that this behavior is not unique for doxorubicin and it can be observed with other weak basic drugs.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1 The 1H and are included.
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C NMR spectra of the C-HES
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