In vitro biocompatibility evaluations of hyperbranched ...

J Mater Sci: Mater Med DOI 10.1007/s10856-013-5094-z

In vitro biocompatibility evaluations of hyperbranched polyglycerol hybrid nanostructure as a candidate for nanomedicine applications Ali Zarrabi • Mohammad Ali Shokrgozar Manouchehr Vossoughi • Mehdi Farokhi

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Received: 3 August 2013 / Accepted: 9 November 2013 Ó Springer Science+Business Media New York 2013

Abstract In the present study, a detailed biocompatibility testing of a novel class of hybrid nanostructure based on hyperbranched polyglycerol and b-cyclodextrin is conducted. This highly water soluble nanostructure with size of less than 10 nm, polydispersity of less than 1.3, chemical tenability and highly branched architecture with the control over branching structure could be potentially used as a carrier in drug delivery systems. To this end, extensive studies in vitro and in vivo conditions have to be demonstrated. The in vitro studies include in vitro cytotoxicity tests; MTT and Neutral Red assay as an indicator of mitochondrial and lysosomal function, and blood biocompatibility tests such as effects on coagulation cascade, and complement activation. The results show that these hybrid nanostructures, which can be prepared in a simple reaction, are considerably biocompatible. The in vivo studies showed that the hybrid nanostructure is well tolerated by

Electronic supplementary material The online version of this article (doi:10.1007/s10856-013-5094-z) contains supplementary material, which is available to authorized users. A. Zarrabi (&) Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran e-mail: [email protected] M. A. Shokrgozar National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran M. Vossoughi Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran, Iran M. Farokhi Department of Tissue Engineering and Cell Therapy, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran

rats even in high doses of 10 mg ml-1. After autopsy, the normal structure of liver tissue was observed; which divulges high biocompatibility and their potential applications as drug delivery and nanomedicine.

1 Introduction Recently, hyperbranched polymers have received enormous attention as carriers for drug delivery due to their intrinsic advantages such as high solubility, compact and three dimensional structure, flexibility, low viscosity, free of/rare chain entanglement, and abundant end-group functionality for suitable derivatizations [1–6]. Polyglycerol (PG) is one of the first focused hyperbranched polymers which has been synthesized by anionic ring opening multibranching polymerization of glycidol [7, 8]. PG is soluble in water freely and its usefulness for biological applications such as drug delivery has been proven several years ago [7, 9]. Several biocompatibility and cytotoxicity studies have been performed in recent years [7, 10–13]. Haag and co-workers have reported that hyperbranched PG are not only more protein resistant but also thermally and oxidatively more stable than polyethylene glycol (PEG) [14]. In addition to these advantages, large numbers of functional groups on the outer surface of Polyglycerol makes it appropriate for pharmaceutical and drug delivery purposes compared to linear PEG [15, 16]. Cyclodextrins (CDs) have been extensively studied in the supramolecular chemistry as host molecules due to their unique properties such as hydrophilic outside, hydrophobic inside, two types of hydroxyl functional groups, core cavity and biocompatibility [17, 18]. Incorporation of CDs into hyperbranched polymers leads to new highly branched threedimensional supramolecular structure having novel and

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useful properties arising from both CDs and hyperbranched. This hybrid nanostructure could act as a promising carrier for hydrophobic drugs as gust molecules of CD. Until now, only two types of this hybrid nanostructure have been investigated. Zhang and co-workers have developed a hybrid nanostructure containing a polyglycerol core and several grafted CDs [19], whereas we have developed a hybrid nanostructure containing a CD core with several polyglycerol branches [8]. It is of utmost importance in introducing a carrier for in vivo drug delivery applications that the carrier is biocompatible and nonimmunogenic [20]. Generally, polyglycerol is considered more biocompatible than CD. Therefore, attaching CD to peripheral of a polyglycerol makes the product less biocompatible than attaching polyglycerol to a CD core. Moreover, the first work requires several reactions for tosilation, amination and the related purification, which increases the risk of existing potential toxic solvent residues, whereas we have decreased the reaction step and reached to a one-pot reaction, like click chemistry. This again, increases the potential biocompatibility and its application as drug delivery system. In this study, we have evaluated the biocompatibility of our newly introduced hybrid nanostructure [8]. We selected the polymer of number average molecular weight (Mw) 4,000 g mol-1 for biocompatibility studies using a variety of techniques. Selection of this Mw was due to the fact that as the molecular weight increases, the polymeric material behaves more biocompatible. Therefore, the biocompatibility studies were conducted in this relatively low molecular weight [11]. Biocompatibility of a carrier must be assessed in animal models, but in vitro cell culture methods have become progressively more popular to reduce the amount of animal testing [21]. Additionally, in vitro biocompatibility tests have shown a higher sensitivity compared to in vivo biocompatibility tests [22]. Experiments such as MTT and Neutral Red (NR) were conducted to assess the effect of nanostructures on cell viability. Moreover, since this carrier was to deliver drug intravenously, the blood biocompatibility tests such as coagulation [prothrombin time (PT) and activated partial thromboplastin time (APTT)], and complement activation (C3 and C4 levels) were conducted in the first part as well as in vivo toxicity tests in rats in the second part. Evaluating the biological and physicochemical properties of this new hybrid nanostructure will result in valuable knowledge for future design of drug delivery systems.

2 Materials and methods Materials, characterization methods and hybrid nanostructure’s synthesis procedure are discussed in supplementary information.

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2.1 Biocompatibility testing 2.1.1 In vitro Cell culture studies are generally the first step of evaluating the biocompatibility of a material due to the fact that they provide an investigation of toxicity in a simplified system that minimizes the effect of confounding variables [23]. In this study L929 and BT-20 cell lines were cultured in RPMI 1,640 (Biowhittaker, Belgium) with 10 % fetal bovine serum (FBS; Sigma-Aldrich, USA), 100 U ml-1 penicillin and 100 lg ml-1 streptomycin (Sigma, USA) at 37 °C in 5 % CO2. 2.1.1.1 MTT assay The viability of cells was quantified by a colorimetric assay for cellular growth based on the cleavage of a yellow tetrazolium salt (MTT) to purple formazan crystals by mitochondrial active cells [24]. Briefly, cells were seeded in 96-well plates (Sarstedt) at a concentration of 20,000 for L929 and BT-20 per well in 100 ll of medium. The plates were incubated at 37 °C and 5 % CO2 in air for 24 h. After 24 h, the culture medium was replaced with 100 ll of serial dilutions of our hybrid nanostructure solutions with different concentrations of 0.5, 1, and 5 mg ml-1 in each well and the cells were further incubated for additional periods ranging from 24 to 72 h. Control cells were only incubated with fresh culture medium. All nanostructure containing media and controls were seeded in eight separate wells. After 24, 48 and 72 h of incubation, 100 ll of medium was replaced by 100 ll MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl) tetrazolium bromide, at a concentration of 0.5 mg ml-1 in PBS(1x)). The reagent was incubated in dark for 3 h at 37 °C. The MTT was reduced to an insoluble formazan precipitate by mitochondrial succinic dehydrogenase of viable cells. Afterwards formazan crystals were solubilized by incubating in 100 ll of DMSO for 30 min. The absorbance of each well, identifying the quantity of viable cells, was read at 545 nm on a microplate reader (Stat Fax-2100, AWARENESS, Palm City, USA). 2.1.1.2 NR assay All stages were identical to the stages mentioned in MTT assay instead of the third stage. In this assay, after removing the culture media, 100 ll/well NR solution was added to each well. After 4 h, NR solution was replaced with solution containing: 1 % glacial acetic acid, 50 % ethanol, and 49 % distilled water and then, plates were incubated in 37 °C and 5 % CO2 for 15 min. The absorbance was measured at 570 nm using ELISA reader. 2.1.1.3 Blood biocompatibility Coagulation: Blood coagulation involves a series of proteolytic reactions resulting


in the formation of a fibrin clot. Primarily, the hybrid nanostructures were tested for their probable effects on blood coagulation time [25]. PT and APTT, as parameters of blood biocompatibility for biomaterials in contact with blood, were measured by means of a coagulation analyzer, using mechanical end point determination (ST4, Diagnostica Stago). For the PT determination, the extrinsic and common coagulation was activated by InnovinÒ reagent when incubated with plasma, and then the clotting time was measured. InnovinÒ is a lyophilized reagent consisting of recombinant human tissue factor, synthetic phospholipids (thromboplastin), calcium ions, a heparin-neutralizing compound, buffers and stabilizers (bovine serum albumin) [26]. For the APTT determination, the intrinsic and common coagulation pathways were activated by adding actin as partial thromboplastin reagent and calcium chloride to plasma, and then the clotting time was measured. The hybrid nanostructure effect on coagulation behavior was studied by adding plasma to different concentrations of polymer solution in isotonic saline (9:1 dilution plasma:polymer solution) to the final concentrations of 0.5, 1 and 5 mg ml-1, and mixing at 37 °C for 5 min before applying the coagulation reagents. For control samples, equal volumes of isotonic saline instead of polymer solution were added to plasma. Each experiment was conducted in triplicate. Complement activation: To evaluate the complement activation, the cleavage of complement components C3 and C4 were monitored using a commercial single radial immuno-diffusion (SRID) immunoassay kit. Activation studies were performed using anticoagulated plasma (plasma in 3.8 % sodium citrate) isolated by centrifugation (3,000xg for 15 min at room temperature) from whole blood donations from a single donor. The plasma was chosen as test medium owing to the fact that it is an appropriate reflection of proteins that contact polymer in vivo. The plasma was added to polymer solutions in isotonic saline to the final concentrations of 0.5, 1 and 5 mg ml-1. As control sample, plasma was added to saline without any polymer traces inside. The mixtures were then incubated for 2 h at 37 °C before samples were withdrawn. Briefly, each sample was applied to one well of an SRID plate according to manufacturer’s instructions. The SRID principle is based on the interaction of C3/C4 in the sample and their specific antibodies coated on the plate leading to a visible precipitate relative to their concentration. Zone diameters were measured at 48 h after sample application in accordance with the manufacturer’s suggestion. The differences between C3/C4 concentrations in samples and control were taken into account as bioincompatibility index. The values reported are the means of triplicate measurements.

2.1.2 In vivo 2.1.2.1 Animal study Wistar male rats (8 weeks) were injected intravenously via lateral tail vein with the polymer solution of concentration 10 mg ml-1 in isotonic saline (three rats). The same amounts of rats were treated with saline alone as control group. The injected volume was 200 ll/100 g rat. All animals were observed post administration twice a day during the treatment periods for morbidity or mortality. The tracing signs of clinical ill health such as body weight loss, dry eye, changes in appetite, and behavioral changes such as altered gait, and lethargy were deeply controlled [15]. In addition, the skin at injection sites was monitored suspiciously for any probable inflammation. 2.1.2.2 Histology and pathology assay Upon termination after 7 days, the animals were sacrificed through cervical dislocation and following incision, the livers were removed and fixed in buffered 10 % formaldehyde solution for 24 h. Subsequently, samples were processed in tissue processing device (Shandoncitadel 1000). The samples were embedded in melt paraffin and the slices with diameter of 5 lm sections were produced by rotator microtome (Shandon-AS 325). The section were then stained with eosin and hematoxylin and examined under a light microscope (Zeiss, Germany). The aforementioned protocol was reviewed and approved by the Pasteur Animal Care Group in Pasteur Institute of Iran prior to conducting the studies. 2.2 Statistical analysis All quantitative data were analyzed using SPSS software (version 15). Data are reported as mean values ± standard deviation (SD) and value were considered significant at P \ 0.05. Statistical comparisons were performed using parametric analysis of variance [ANOVA (Tukey)].

3 Results The results of hybrid nanostructure synthesis as well as NMR, GPC and DLS characterization results are discussed in the supplementary information. 3.1 Biocompatibility testing 3.1.1 In vitro Here, we report the cytotoxicity studies of b-CD-g-PG hybrid nanostructure as well as their interactions with blood.

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3.1.1.1 MTT assay Generally, b-CD and aliphatic polyethers like PG are biocompatible [27]. This behavior is the consequent of their similarities to polysaccharides and polyethylene glycol. Consequently, it is expected that PG grafted b-CD also displays biocompatible properties. Figure 1 illustrates the MTT results for the 24, 48 and 72 h incubation of two cell models, Mouse connective tissue fibroblasts L929 as normal cell line and human breast cancer cell BT-20 as cancerous cell line, in contact with our hybrid nanostructures. The results of the MTT assay, as an indicator of mitochondrial function, showed no sign of toxicity toward normal and cancerous cell lines even at the high concentration of 10 mg ml-1 after 72 h incubation. It is observed that for L929 cell, viability significantly increased following treatment with b-CD-g-PG0.33(5) hybrid nanostructures from 0.1 to 10 mg ml-1 (in b-CD-gPG0.33(5) the ratio of initiator to hydroxyl group (P/OH) is [7/21] = 0.33 and the ratio of monomer to initiator (G/P) is 5). The maximum proliferation response was at 0.5 mg ml-1 hybrid nanostructures, which significantly increased the optical density and therefore relative viability. The optical microscopy was also used to explore the probable cell morphology changes due to the exposure of cells to our hybrid nanostructures in the monolayer culture. The results showed that the morphology of L929 and BT20 cells due to the presence of b-CD-g-PG0.33(5) hybrid nanostructures was not affected even at high concentration of 10 mg ml-1, which confirms the hybrid nanostructure’s high level biocompatibility (Data not shown). 3.1.1.2 NR assay Similar to MTT assay, the biocompatibility of b-CD-g-PG0.33(5) hybrid nanostructures was confirmed with NR study (Fig. 2). However, no significant proliferation rate was observed among samples to control group. The samples 0.5 mg ml-1 hybrid nanostructure had a higher proliferation than other samples and control group. According to obtained data, the biocompatibility of b-CDg-PG0.33(5) hybrid nanostructures were decreased after incubation time increased. It is observed that L929 and BT20 cells had similar pattern of growth rate in comparison to each other. 3.1.1.3 Blood biocompatibility This biocompatibility for the newly developed hybrid nanostructure was assesses by evaluating the coagulation (PT and APTT) cascade and complement activation under in vitro conditions. All blood biocompatibility experiments were conducted through adding hybrid nanostructure solutions in control buffer to plasma solution. The probable dilution effects were compensated by deducing the results of addition of equal volume of control buffer to plasma solution from the main experiment results.

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Fig. 1 MTT assay values for a L929 and b BT-20 cells exposed to different hybrid nanostructure concentrations of 0.5, 1 and 5 mg ml-1. Values are mean ± SD

Coagulation: Biocompatibility of biomaterials contacting blood relates mainly to the thrombotic response induced by those biomaterials [28]. Since any variations of blood coagulation properties and behavior as a result of its contact with biomaterial may result in thrombosis, it could be considered as bioincompatibility of the studied biomaterial. The blood coagulation cascade consists of three pathways; intrinsic, extrinsic and common pathways. The extrinsic and common coagulation pathways are evaluated by PT, whereas, the intrinsic coagulation pathway is evaluated by APTT [29]. The PT value, expressed in seconds, is time required for formation of fibrin clot in plasma after the addition of the tissue thromboplastin to the anticoagulated plasma, while, that of APTT, also expressed in seconds, is time required for formation of fibrin clot after partial thromboplastin reagent and calcium chloride were added to anticoagulated plasma. The hybrid nanostructure samples with different concentrations were tested for coagulation effects using conventional clinical coagulation assays. The results are shown in Figs. 3 and 4. It is evident that even at high concentrations of 5 mg ml-1, there is not any statistical difference between PT results of samples measured compared to control samples. This reveals that the hybrid nanostructure


Fig. 3 Effect of hybrid nanostructure concentrations on the PT

Fig. 2 NR assay values for a L929 and b BT-20 cells exposed to different hybrid nanostructure concentrations of 0.5, 1 and 5 mg ml-1. Values are mean ± SD

has not influenced the extrinsic coagulation pathway. On the other hand, the hybrid nanostructure increased the APTT slightly and insignificantly compared to control. These data suggest that the nanostructured biomaterials do not interfere with the coagulation cascade and are perfectly blood biocompatible. No statistical differences was observed in PT and APTT (p [ 0.05). Complement activation: Following intravenous administration, drug delivery systems are exposed to phagocytes of the reticuloendothelial system (RES) and will be removed from the circulation [30]. Elements of the RES in the liver will phagocytose the carriers by the adsorption of certain blood components on the particle surface through complement system [31]. The complement system is composed of more than 30 distinct plasma and membrane-bound proteins that function either as enzymes or as binding proteins. The foremost function of complement system is to serve as a primitive self–nonself discriminatory defense system. This is accomplished by coating a foreign material with complement fragments (opsonization) and recruiting phagocytic cells that attempt to destroy and digest the intruder. The opsonization process as immune response to a foreign biomaterial utilizes specific proteins such as C3 and C4.

Fig. 4 Effect of hybrid nanostructure concentrations on the APTT

The ability of this system to function in the opsonization of foreign elements is accomplished mostly by recognizing various C3 and C4 fragments bound to the foreign element. Recognition of biomaterial-bound C3 and C4 fragments by white blood cells results in cell adherence and further activation of these immune system cells leading to production of degradative enzymes and destructive oxygen metabolites which in turn lead to destruction of the invader [32]. Complement activation induced by various biomaterials has been already reported in the literature [33–35]. However, in contrast to polymers such as poly(propylene), poly(acrylamide), dextran and regenerated cellulose [36], there are some polymers such as poly(N-vinylpyrrolidone) (PVP) and phosphorylcholine-based polymers that are reported not to activate complement system [37, 38]. Therefore, any significant increase in the concentration of C3 and C4 in plasma after being in contact with the biomaterial in comparison to the control plasma reveals the cytotoxicity of the biomaterial as measure of the extent of complement activation. Polymer samples were incubated with anticoagulated plasma (plasma in 3.8 % sodium citrate) at different concentrations at 37 °C for 2 h and the amount of C3/C4

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Fig. 5 Effect of hybrid nanostructure concentrations on the complement components C3 and C4 Table 1 Mean weight ± SD of rats after hybrid nanostructure administration Day

1

2

3

4

5

6

p value

Control

184.0 ± 1

192.6 ± 14

211.6 ± 20

199.0 ± 5

202.0 ± 2

205.5 ± 7

[0.05

Saline Polymer

178.4 ± 7 208.5 ± 15

173.6 ± 8 220.4 ± 13

193.6 ± 5 215.7 ± 7

198.5 ± 6 210.7 ± 8

201.3 ± 7 220.2 ± 6

212.7 ± 5 220.5 ± 7

[0.05 [0.05

Control: rats with no injection and same treatment Saline: rats injected with equal-volume of saline Polymer: rats injected with hybrid nanostructure solution of 10 mg ml-1 concentration

produced was measured (Fig. 5). The blood biocompatibility test by monitoring complement activation was performed under incubation condition lasting more than normal complement studies (2 h vs. 30 min) with the purpose of tracing any chances in accelerated complement activation [26]. The negative controls were plasma added to isotonic saline. The amount of C3/C4 generated by the effect of hybrid nanostructure even at high concentration of 5 mg ml-1 was not significantly different from that of the controls. Therefore, this hybrid nanostructure was found to be neutral to the complement system. Statistical analysis showed no significant differences between the values obtained from different concentrations of polymeric samples and controls (p [ 0.05).

3.1.2.2 Histology and pathology assay No pathological effects were observed in histological examination of animals after autopsy. The tissue structures (liver plate, sinusoidal and portal space) of livers were normal in comparison with control groups (Fig. 6). The number of immune cells, such as polymorph nuclear and lymphocyte cells were not increased comparing to control groups. In addition, fibroses tissue, a pathological marker of liver disease, was normal. Even high dose of b-CD-g-PG hybrid nanostructure did not have any harmful effect in liver tissues. This finding is very important concerning developing materials for drug delivery.

4 Discussion 3.1.2 In vivo 3.1.2.1 Animal studies Hybrid nanostructure solution with high concentration of 10 mg ml-1 polymer in isotonic saline was tested in rats. The animals were monitored for signs of toxicity. Weight gain was normal and indistinguishable from controls (Table 1). No kind of other unexpected and inconvenient indicators such as mortality, lethargy, dry eyes, change in appetite, injection site’s inflammation, and scruffy coats were seen in this period. Therefore, the hybrid nanostructure was well tolerated by rats and is biocompatible for any further in vivo applications.

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The use of high functional and biocompatible nanocarriers for drug delivery applications is a topic of considerable interest. For this purpose, we have designed and synthesized a hybrid nanostructure consisting of polyglycerol and cyclodextrin through facile anionic ring opening polymerization of glycidol by a deprotonated b-CD as core and initiator. This product exhibits several benefits such as high number of functional groups and excellent in vitro and in vivo biocompatibility with the possibility to tailor architecture and functionality. Beneficially, such hybrid nanostructures have a narrow size distribution and


Fig. 6 Histology of the liver of rats; a, b control rats; c, d rats fed with hybrid nanostructure; a, c 400x; b, d 1200x

polydispersities below 1.3 [8]. To be further exploited as drug delivery system, its biocompatibility was evaluated through different in vitro and in vivo tests. MTT assay showed no sign of toxicity toward normal and cancerous cell lines even at the high concentrations. However, the mechanism by which the viability of cells was increased after being in contact with the hybrid nanostructure is not yet established. We suggest that the increasing proliferation is due to consumption of nanostructure’s fundamental constituents including oligosaccharides by cells. The hybrid nanostructure is composed of oligosaccharide and glycerol, which are in the citric acid cycle of the cell and could be considered as the nutrient agent. NR test also confirmed the biocompatibility of hybrid nanostructure and no significant proliferation rate was observed among samples to control group. In addition to in vitro cytotoxicity tests, the blood biocompatibility is also important for drug delivery systems which are designed to deliver drugs intravenously. Blood biocompatibility tests of coagulation and complement activation were conducted and showed that the hybrid nanostructure does not have any interference with the coagulation cascade and complement system. Finally animal studies, in vivo, were performed and the autopsy results confirmed the profound biocompatibility of the hybrid nanostructure, which reveals the potential of this

hybrid nanostructure to act as a promising carrier in nanomedicine field. Acknowledgments We thank the funding by Iran Nanotechnology Initiative Council (INIC), Dr. Masoud Davanlou, MD, Danesh Pathobiology Laboratory for blood compatibility tests, and Ms. Somayeh Nazerian for her kind contribution in PT and APTT tests.

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