- Email: [email protected]
Design of biocompatible surface-modified polyurethane and polyurea nanoparticles
Polymer 53 (2012) 6072e6080
Contents lists available at SciVerse ScienceDirect
Polymer journal homepage: www.elsevier.com/locate/polymer
Design of biocompatible surface-modified polyurethane and polyurea nanoparticles G. Morral-Ruíz a, b, P. Melgar-Lesmes a, M.L. García c, C. Solans b, d, M.J. García-Celma a, b, * a
Departament de Farmàcia i Tecnologia Farmacèutica, Unitat RþD associada al CSIC, Facultat de Farmàcia, Universitat de Barcelona, Av Joan XXIII s/n, 08028 Barcelona, Spain Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, Barcelona, Spain c Departament de Fisicoquímica, Facultat de Farmàcia, Universitat de Barcelona, Av Joan XXIII s/n, 08028 Barcelona, Spain d Institut de Química Avançada de Catalunya (IQAC), CSIC, Jordi Girona 18-26, 08034, Barcelona, Spain b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 July 2012 Received in revised form 16 October 2012 Accepted 19 October 2012 Available online 26 October 2012
This study is focused to design systems of polyurethane and polyurea nanoparticles prepared via interfacial polycondensation in O/W nano-emulsions with biocompatible components suitable for drug delivery systems. Polyurethane and polyurea nanoparticles with a small diameter (50e90 nm) and high kinetic stability were developed from O/W nano-emulsions in aqueous solution/polysorbate 80/oil systems. The influence of several factors on the particle size and polydispersity index was studied. The monomer concentration, oil/surfactant weight (O/S) ratio, polymerisation temperature and components that can be located in the droplet interface and the dispersed phase of nano-emulsion played a key role in the formation of these nanoparticle systems. The biocompatibility of these nanoparticles was assessed using haemolysis and cell viability assays. No significant haemolysis and low effects on human endothelial cell viability were obtained after incubation with nanoparticles. Therefore, herein we provide useful information for the development of biocompatible drug carriers using polyurethanes and polyureas. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Polymeric nanoparticle Interfacial polycondensation Nano-emulsion
1. Introduction The formation of polymeric [1e3] and lipid [4,5] nanoparticles has aroused a growing interest in the last decades owing to their great potential as controlled drug delivery systems. In this regard, the choice of biocompatible polymers as well as methods of preparation plays a key role in the formation of suitable drug carriers for drug delivery [6e9]. Polyurethanes and polyurea polymers are emerging as useful biomaterials for biomedical applications because of their synthetic versatility, excellent mechanical properties and good biocompatibility [10e13]. In this context, the formation of polyurethanes and polyureas employing aliphatic diisocyanates with incorporated aminoacids such as lysine and biodegradable polyether has acquired greater relevance in last years [14,15]. It is well known that both polymers can be obtained from the chemical reaction between a diisocyanate and a polyol or
* Corresponding author. Departament de Farmàcia i Tecnologia Farmacèutica, Unitat RþD associada al CSIC, Facultat de Farmàcia, Universitat de Barcelona, Av Joan XXIII s/n, 08028 Barcelona, Spain. Tel.: þ34 934024548; fax: þ34 934035937. E-mail addresses: [email protected] (G. Morral-Ruíz), melgarpedro@ yahoo.com (P. Melgar-Lesmes), [email protected] (M.L. García), csmqci@ cid.csic.es (C. Solans), [email protected] (M.J. García-Celma). 0032-3861/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.polymer.2012.10.039
diamine by a polycondensation or polyaddition process [16]. Nevertheless, the high reactivity of isocyanate groups makes difficult the right formation of these polymers in aqueous media. In this regard, several efforts have been developed in order to avoid this collateral reaction with water [17e19]. Nano-emulsions constitute an attractive alternative to prepare polyurethane and polyurea nanoparticles in aqueous systems due to their small droplet size and high kinetic stability [20e23]. Moreover, nano-emulsions can be obtained not only using highenergy methods but also by low-energy methods, which take advantage of the physicochemical properties of the system leading to the formation of even smaller and more uniform droplets [24,25]. Thus, the formation of polyurethane nanoparticles from O/W nano-emulsions in a one-step procedure has already been described in numerous reports [17e20]. However, in these studies, nanodroplets used as template are almost exclusively prepared using high-energy methods [17e20]. In addition, chemical catalysts [18,26] and ionic surfactants such as sodium dodecyl sulphate [27] which are widely used to achieve the formation of nanoparticles with a small particle diameter could generate biocompatibility problems. On the other hand, when nanoparticles are obtained via low-energy methods, organic solvents such as acetone are usually employed to carry out the procedures [28]. Therefore, the goal of
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
the work reported has been to develop systems of polyurethane and polyurea nanoparticles obtained by polycondensation at the droplet interface of O/W nano-emulsions prepared by low-energy methods in aqueous solution/nonionic surfactant/oil systems and using biocompatible components. In this context, we have studied the involvement of surfactant in the polymerisation reaction that has rarely been analysed to date [18,19,29]. Thus, the present study was aimed to investigate different parameters involved in the polymerisation process, especially focussing on the influence of polysorbate 80 as nonionic surfactant, and the biocompatibility of these formulations to optimise polyurethane and polyurea nanoparticles for future biomedical purposes. 2. Experimental 2.1. Materials The nonionic technical grade surfactant, polyoxyethylene 20-sorbitan monooleate (polysorbate 80, P80) was supplied from Fagron (Barcelona, Spain). Components used as highly hydrophilic materials such as polyethylene glycol 400 (PEG 400), polyethylene glycol 200 (PEG 200) and L-lysine (lys) as well as the monomer, isophorone diisocyanate (IPDI), were purchased from Sigmae Aldrich (Madrid, Spain). The oil components, saturated medium chain triglyceride (MCT) and soybean oil (SO) were obtained from Fagron and SigmaeAldrich, respectively. All chemicals were used without further purification. Deionised water was obtained from a Millipore-Milli-Q water purification system (Molsheim, France). Pig and human blood were purchased from AbD Serotec (Raleigh, NC, USA). All materials employed in biocompatibility studies, the primary human endothelial cells HUVECs (Human umbilical vein endothelial cells), Dulbecco’s Modified Eagle Medium (DMEM), Ham’s F-12 Nutrient Mix (F-12), foetal bovine serum (FBS), penicillin/ streptomycin antibiotic and endothelial cell growth supplement were supplied from Life Technologies Ltd. (Paisley, UK). 2.2. Methods 2.2.1. Preparation of polyurethane and polyurea nanoparticles Polyurethane and polyurea nanoparticles were prepared from O/W nano-emulsions by an interfacial polycondensation process in aqueous solution/polysorbate/diisocyanate/oil systems. The O/W nano-emulsions were prepared at 25 C by the Phase Inversion Composition (PIC) emulsification method as reported [30]. A defined amount of isophorone diisocyante was incorporated onto oil/surfactant mixtures. Then, aqueous component was dropwise added to these mixtures with continuous agitation with a vibromixer. Nanoparticles were formed by heating the nano-emulsion at temperatures between 55 and 80 C. The polymerisation reaction was considered as completed after 4 h and samples were kept at 25 C [30]. 2.2.2. Particle size and polydispersity index Measurements of the average particle size and polydispersity index were performed by Dynamic Light Scattering (DLS) at 25 C using a Zetasizer nano ZS (Malvern Instruments, UK). This instrument is equipped with a detector to analyse the intensity of the scattered light at a fix angle of 173 provided by a HellioneNeon laser (4 mW, l ¼ 633 nm). The values of Z-average and polydispersity index were calculated from the autocorrelation function of the scattered intensity by means of the cumulants analysis using DTS nano (Malvern Instruments) software. The nanoparticle suspensions were diluted 1/75 with ultra-purified water in order to avoid multiple scattering events.
6073
2.2.3. Nanoparticle stability studies Stability studies were carried out by measuring the particle size of nanoparticle suspensions as a function of time during 6 months using Dynamic Light Scattering at 25 C. Samples were kept at 25 C during all the studied period (190 days). 2.2.4. Nanoparticle freeze-drying A fraction of 2 mL of nanoparticle suspension was freeze-dried. No cryoprotectant was added to the formulations. Samples were flash-frozen at 78 C in dried ice prior freeze-drying. Then, frozen samples were freeze-dried at 89 C under 0.01 mbar vacuum for 48 h in a glass chamber connected to a vacuum pump and a vapour condenser (Freeze-Dried Alpha 2-4 LD plus, Biobloc Scientific, Illkirch, France). 2.2.5. Transmission electron microscopy (TEM) characterisation Characterisation studies by TEM were carried out on a JEOL JEM 1010 (JEOL Ltd., Akishima, Japan) microscope. To prepare the samples, one droplet of nanoparticle suspension was placed on a carbon-coated copper grid and stained with an aqueous solution of uranyl acetate at 1% (w/v) prior observation. Average particle diameter of approximately 500 randomly selected nanoparticles from different TEM micrographs was determined using the morphometry software ImageJ v. 1.44. 2.2.6. In vitro red blood cell (RBC) haemolysis assay of the nanoparticles Haemolysis assay was performed using fresh pig and human blood as previously reported [31]. The erythrocytes were collected by centrifugation (Heraeus Megafuge 16R centrifuge, Thermo Scientific) at 3000 rpm for 10 min, and then washed three times with phosphate buffered saline (PBS) at pH 7.4. A stock dispersion was prepared by mixing 3 mL of centrifuged erythrocytes into 11 mL of PBS. Freeze-dried polyurethane and polyurea nanoparticles were resuspended in PBS and then slightly diluted in distilled water to reach isotonic values (ranging 280e300 mOsm/L). One hundred microlitre of stock erythrocyte dispersion was added to 1 mL of the nanoparticle dispersions. The tubes were then incubated for 10 min at 37 C in an Incubator Shaker. After incubation, the tubes were centrifuged (5 min at 13,000 rpm (IEC MicroCL 21R centrifuge, Thermo Scientific)) and the percentage of haemolysis was determined by comparing the absorbance (l ¼ 540 nm) of the supernatant with that of control samples totally haemolysed with 1 mL of distilled water as previously reported [32]. The supernatant absorbance of a blood sample not treated with nanoparticles and incubated with 1 mL of PBS was used as the negative control to obtain only the percentage particle-induced haemolysis. All experiments were reproduced five times with each type of formulation and data have been presented as the percentage of the complete haemolysis and Standard Error of the Mean (sem). 2.2.7. Cell culture and in vitro cell biocompatibility HUVECs were cultured in DMEM/F-12 medium supplemented with 10% foetal bovine serum (FBS), 100 mg/mL penicillin/streptomycin and 50 mg/mL endothelial cell growth supplement. Cells were grown at 37 C and 5% CO2. HUVECs were passaged when they reached 80% confluence and passages 2e5 were used for all experiments. To quantify the toxicity effect of nanoparticles in cell viability of human endothelial cells, we performed the MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt) assay (CellTiter96 Aqueous One Solution assay kit, Promega, Madison, WI, USA). HUVECs were seeded in 96-well plates at a cell density of 7.5 103 cells/well in complete media as
6074
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
described above. After 24 h, cells were serum-starved for 4 h (FBS at 0.1%) and incubated in media in the presence of 85 mg/mL naked polyurethane, pegylated polyurethane or lys-coated polyurea nanoparticles for 24 h at 37 C and 5% CO2. Cell viability was determined by adding 20 mL of MTS solution to each well. After 2 h, the absorbance was measured at 490 nm using an EPOCH microplate spectrophotometer (Biotek Instruments, Winooski, VT, USA). The cell viability was obtained by comparing the absorbance of nanoparticle-treated cells to that of control cells (treated with PBS). Each condition was performed in sextuplicate and reported as mean sem. 3. Results 3.1. Influence of composition parameters of O/W nano-emulsions in the formation of polyurethane and polyurea nanoparticles The description of the nano-emulsions used to prepare the nanoparticles is detailed in Table 1. In the first set of experiments, the effect of polyethylene glycol, lysine and monomer concentration of the nano-emulsion in the formation of nanoparticles was evaluated by analysing the particle size and polydispersity index by DLS at 25 C (Fig. 1). The samples were characterised just after preparation. A polymerisation temperature of 70 2 C was initially chosen according to previous studies [17,26]. Concentrations of IPDI ranging 0.01 mmol/ge 0.10 mmol/g were selected to prepare the nanoparticles. It should be noted, that these values of monomer concentration are lower than those reported in the literature [17e19]. The mean particle size (Fig. 1) increases with IPDI concentration and this occurs independently on the kind of particle. Nanoparticles with the biggest diameter corresponded to naked polyurethane compositions (Fig. 1). In contrast, the smallest particle sizes were obtained with lysine-coated polyurea samples. The minimum concentration of IPDI to form nanoparticles with a small particle size and low polydispersity (<0.15) was 0.02 mmol/g and 0.04 mmol/g for polyurethane and polyurea samples, respectively. Nanoparticles with a Z-average value below 85 nm were obtained with these monomer concentrations. It is important to mention that lys-coated polyurea nanoparticles showed an extremely small hydrodynamic diameter (around 50 nm) even using double of concentration of IPDI (Fig. 1). The influence of O/S ratio in the particle size of PEGpolyurethane and lys-coated polyurea samples was also studied. MCT/P80 ratios between 0/100 and 15/85 were selected according to the studies of phase behaviour at 25 C described in a previous
work [30]. The IPDI concentrations, 0.02 mmol/g and 0.04 mmol/g for PEG-polyurethane and lys-coated polyurea, respectively, were selected according to the results described above. Polymerisation was carried out by heating samples at 70 2 C. The nanoparticle size was determined by DLS at 25 C just after preparation and the results are shown in Fig. 2. The mean particle size of the PEG-polyurethane and lys-coated polyurea samples ranged 72e123 nm and 56e98 nm, respectively (Fig. 2). Surprisingly, the particle diameter decreased lineally at increasing O/S ratios [22,29]. Although the particle size decreases with the increase in O/S ratios, the polydispersity index tends to rise. Even so, all polydispersity indexes were lower than 0.20. Therefore, while the smallest diameter is achieved with O/S ratio of 15/85, we selected the O/S ratio of 10/90 for biocompatibility studies owing to a lower concentration of surfactant is required to obtain the nanoparticles. For this O/S ratio PEG-polyurethane and lys-coated polyurea nanoparticles with a particle diameter of 89.3 nm and 67.5 nm, respectively, and low polydispersity index were obtained. 3.2. Influence of temperature in the formation of polyurethane and polyurea nanoparticles To study the influence of temperature in the formation of polyurethane and polyurea nanoparticles, samples were prepared with the optimal concentration of IPDI, O/S, PEG 400 and lys. Thus, nano-emulsions with a 10/90 O/S ratio and 90 wt% aqueous solution containing IPDI and the highly hydrophilic components at suitable concentrations were heated at temperatures between 55 and 80 C to form nanoparticles. Polymerisation was not detected at lower temperatures than 55 C. The mean particle size and the polydispersity index of these compositions were analysed using DLS (Fig. 3). As observed in Fig. 3, the mean particle diameter increases with the temperature in all studied formulations. Regarding to the polydispersity index, low values (PDI 0.15) were only achieved when the temperature of polymerisation was higher than 65 C. In fact, temperatures equal or lower than 65 C in most of samples led to the formation of two populations detected by DLS intensity analysis, being the first one between 10 and 15 nm (corresponding to droplets of nano-emulsions) and a second one of polymeric nanoparticles (Supplementary data). After testing all these temperatures, it was found that the temperature corresponding to 70 C is the best choice to prepare polyurethane and polyurea nanoparticles, since for this
Table 1 Composition of polyurethane and lys-coated polyurea nanoparticles obtained from O/W nano-emulsions and micellar solutions. Nano-emulsions correspond to oil/surfactant weight (O/S) ratios 5/95 and micellar solutions to O/S ratios of 0/100. Samples were prepared with a constant concentration of 90 wt% aqueous solution in aqueous solution/ polysorbate 80/saturated medium chain triglycerides and aqueous solution/polysorbate 80/soybean oil systems. Composition of polyurethane and polyurea nanoparticles
Naked polyurethane PEG-polyurethane
PEG 200-polyurethanea SO-PEG-polyurethaneb Lys-coated polyurea
a b
O/S ratio (wt%)
IPDI (mmol/g)
10/90 0/100 5/95 10/90 15/85 10/90 10/90 0/100 5/95 10/90 15/85
0.02e0.08 0.02 0.02 0.01e0.10 0.02 0.02 0.02 0.04 0.04 0.04e0.08 0.04
Highly hydrophilic component (mmol/g)
Diisocyante/highly hydrophilic component molar ratio
Diisocyanate/molecules with OH or NH2 groups molar ratio
PEG PEG PEG PEG PEG PEG Lys: Lys: Lys: Lys:
e 1:1 1:1 0.5:0.5e5:5 1:1 1:1 1:1 2:1 2:1 2:1e4:2 2:1
1:3.4e4:3.4 1:4.8 1:4.6 0.5:3.9e5:8.4 1:4.2 1:4.4 1:5 2:4.8 2e4.6 2:4.4e4:5.4 2:4.2
400: 0.02 400: 0.02 400: 0.01e0.10 400: 0.02 200: 0.02 400: 0.02 0.02 0.02 0.02e0.04 0.02
Polyethylene glycol 200 was used instead of polyethylene glycol 400. Soybean oil was used as oily component instead of saturated medium chain triglycerides.
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
6075
Fig. 1. Influence of IPDI concentration in the formation of polyurethane and polyurea nanoparticles. Mean particle diameter (Z-average) and polydispersity index (PDI) determined by DLS at 25 C of nanoparticles obtained from O/W nano-emulsions with 90 wt% aqueous solution and O/S ratio: 10/90 in water/polysorbate 80/saturated medium chain triglycerides and aqueous solution/polysorbate 80/medium chain triglycerides systems as a function of IPDI concentration.
temperature the smallest particle diameter with a low polydispersity index (PDI 0.15) is achieved.
3.3. Influence of PEG molecular weight and the composition of dispersed phase in the formation of pegylated polyurethane nanoparticles Eventually, the influence of both the molecular weight of PEG and the oily component in the formation of PEG-polyurethane nanoparticles was also evaluated. To study both factors, samples with PEG 200 and soybean oil (oily component with hydroxyl groups) were prepared. The composition of these nanoparticles is given in Table 1 (see PEG 200-polyurethane and SO-PEG polyurethane samples). Characterisation studies of the obtained nanoparticles were performed by TEM as illustrated in Fig. 4. TEM micrographs showed the formation of a single population of well-defined PEG-polyurethane particles with a narrow size distribution and a mean particle diameter of 48 5 and 54 5 nm for PEG 200-polyurethane and SO-PEG-polyurethane samples, respectively (Fig. 4).
3.4. Stability studies of PEG-polyurethane and lys-coated polyurea nanoparticles The stability of PEG-polyurethane and lys-coated polyurea nanoparticles was determined measuring the particle diameter as a function of time by DLS at 25 C (Fig. 5). Nanoparticles were prepared at the optimal temperature, PEG 400, lys and monomer concentration. As observed in Fig. 5a, the aqueous dispersions of nanoparticles showed a high kinetic stability since the increase in the particle size did not exceed 50 nm during 6 months or beyond. Moreover, no significant changes in the appearance of the samples (such as increase of turbidity and sedimentation) were observed within the studied period. The best stability results were obtained with the 5/95 O/S ratio in both, PEG-polyurethane and lys-coated polyurea nanoparticles. In these compositions, the mean particle size remained constant during all the stability study. Regarding to the other O/S ratios, a slight increase of the diameter was observed during the first month and after that the particle size did not vary at all. Two representative TEM images of PEG-polyurethane nanoparticles taken at 0 and 30 days after preparation are shown in
Fig. 2. Influence of O/S ratio in the formation of polyurethane and polyurea nanoparticles. Mean particle diameter (Z-average) and polydispersity index determined by DLS at 25 C of nanoparticles obtained from O/W nano-emulsions at 90 wt% of aqueous solution at different O/S ratios in aqueous solution/polysorbate 80/saturated medium chain triglyceride systems.
6076
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
Fig. 3. Influence of temperature in the formation of polyurethane and polyurea nanoparticles. Mean particle size and polydipsersity index measured by DLS at 25 C for compositions with 90 wt% aqueous solution and 10/90 wt% O/S ratio corresponding to pegylated polyurethane and lys-coated polyurea nanoparticles as a function of polymerisation temperature.
Fig. 5b. The micrographs observed at 30 days showed a population of nanoparticles with a higher diameter as compared to the samples analysed just after preparation. The samples were also freeze-dried to improve the long-term stability of PEG-polyurethane and lys-coated polyurea nanoparticles during the storage. Results are shown in Table 2. The mean particle size and polydispersity of the samples were measured by DLS before, following freeze-drying process and after storing the freeze-dried at 25 C during 2 years. The samples were
reconstituted in 30 s by mechanic stirring after the addition of 2 mL of water to the freeze-dried sample. The stability at 25 C of the reconstituted nanoparticle suspensions was also studied (Fig. 6). As shown in Table 2, the mean particle diameter for PEGpolyurethane nanoparticles was around 70 nm and for lys-coated polyurea samples ranging between 55 and 60 nm. Thus no significant differences in the particle size were observed between the original suspension of nanoparticles and the reconstituted samples even after being stored for 2 years. Nevertheless the polydispersity
Fig. 4. Transmission electron micrographs (TEM) and histograms of the particle size of polyurethane nanoparticles. a) PEG 200-polyurethane and b) SO-PEG-polyurethane nanoparticles. Both samples were prepared at an IPDI/PEG molar ratio 1/1 with 90 wt% aqueous phase and O/S ratio of 10/90 in water/PEG 200/polysorbate 80/IDI/saturated medium chain triglyceride and water/PEG 400/polysorbate 80/IPDI/soybean oil systems, respectively.
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
6077
Fig. 5. Stability studies of polyurethane and polyurea nanoparticles and TEM micrographs at representative periods of time. a) Mean particle size as a function of time determined by DLS at 25 C for nanoparticles prepared with 90 wt% aqueous solution and different O/S ratios in the aqueous solution/polysorbate 80/medium chain triglyceride systems. b) TEM images corresponding to PEG-polyurethane nanoparticles (90 wt% aqueous component, O/S ratio: 10/90) observed at different periods of time.
index slightly increased, especially in lys-coated polyurea nanoparticles. However, the values of polydispersity indexes in both nanoparticle systems were lower than 0.20. The determinations of the particle size by DLS in reconstituted samples as a function of time (Fig. 6) showed similar results to the stability studies. Thus, PEG-polyurethane and lys-coated polyurea nanoparticles underwent a slight increase of the particle diameter during the first days and after that the particle size remained constant. In these samples, an increase of 25e35% in the particle diameter was quantified during the first days.
potential to damage red blood cells (haemolysis). Haemolysis, the abnormal breakdown of RBC, can lead to anaemia or other pathological conditions. As shown in Table 3, naked polyurethane and lys-coated polyurea nanoparticles did not show haemolysis in pig and human RBC at all. On the other hand, PEG-polyurethane nanoparticles produced a 0.5% of haemolysis when tested in pig RBC. However, this outcome does not exceed the permissible limits of haemolysis for biomedical purposes which are 0.8%, as per the Council of Europe guidelines [33] and 1% as per the US FDA guidelines [34].
3.5. Haemocompatibility study of naked polyurethane, PEGpolyurethane and lys-coated polyurea nanoparticles in red blood cells
3.6. In vitro endothelial cell biocompatibility studies of naked polyurethane, PEG-polyurethane and lys-coated polyurea nanoparticles
Nanotechnology applications and biomaterials designed for systemic administration have to pass previous biocompatibility tests. These tests include an in vitro assay to establish the material’s
The cytotoxicity of naked polyurethane, PEG-polyurethane and lys-coated polyurea nanoparticles was evaluated in HUVEC cells using the MTS assay. Fig. 7 shows the viability results obtained after
Table 2 Z-average and polydispersity index (PDI) determined by DLS at 25 C of the nanoparticle suspension and the reconstituted suspension from freeze-drying just preparation and after storing samples at 25 C for 2 years. PEG-polyurethane and lys-coated nanoparticles were prepared in nano-emulsions with a 90 wt% aqueous component and an O/S ratio of 10/90 in the aqueous solution/polysorbate 80/saturated medium chain triglycerides 812 systems. Suspension of nanoparticles before freeze-drying
Re-suspension of freeze-dried t¼0
PEG-polyurethane Lys-coated polyurea
t ¼ 2 years
Z-average (nm)
PDI
Z-average (nm)
PDI
Z-average (nm)
PDI
68.90 55.73
0.14 0.11
66.93 59.40
0.16 0.17
67.90 56.50
0.15 0.16
6078
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
Fig. 6. Mean particle size as a function of time determined by DLS at 25 C for the nanoparticle suspensions reconstituted after a freeze-drying process. Samples correspond to compositions prepared with 90 wt% aqueous solution and an O/S ratio of 10/ 90 in the aqueous solution/polysorbate 80/medium chain triglycerides systems.
24 h of incubation with the different types of nanoparticles. Both naked polyurethane and PEG-polyurethane nanoparticles only produced around 8% decrease in viability of human endothelial cells. On the other hand lys-coated polyurea nanoparticles promoted around 14% reduction in endothelial cell viability after 24 h of incubation.
4. Discussion Two main chemical species are implicated in the formation of polyurethane and polyurea polymers by polycondensation reaction: Isocyanates and alcohols or amines [16]. Thereby, the polymerisation reaction takes place at the nano-emulsion droplet interface through the nucleophilic attack of hydroxyl or amine groups on carbonyl group of diisocyanate, leading to the disappearance of the characteristic linkage NCO of IPDI and the formation of new carbamate and urea linkages thus indicating that polyurethane and polyurea polymers are obtained [19,28,30]. In this polymerisation process, only one diisocyante group is consumed as a consequence of the reaction with the hydroxyl group to form the polyurethane [18,28,30] while two molecules of IPDI are usually required for each group of amine to obtain polyurea [30]. Nevertheless, a secondary reaction may occur in aqueous dispersions which leads to the loss of the stoichiometry during the process and to the formation of hydrolysis products, urea and CO2 [17e19,28]. For this reason, a diisocyanate monomer (IPDI) with a slow reactivity in water, and biocompatible components with hydroxyl or amine groups (polysorbate 80, PEG 200, PEG 400, lys
Table 3 Percentage of haemolysis of naked polyurethane, lys-coated polyurea and PEGpolyurethane nanoparticles. All experiments were reproduced five times with each type of formulation and data are presented as the mean of percentage of the complete haemolysis Standard Error of the Mean (sem). Formulation
Naked polyurethane PEG-polyurethane Lys-coated polyurea
Osmolality (mOsm/Kg)
Range of haemolysis (% sem) Pig red blood cells
Human red blood cells
295 291 298
0 0.50 0.11 0
0 0 0
Fig. 7. Endothelial cell biocompatibility studies of polyurethane and polyurea nanoparticles. Cell viability was evaluated by the MTS assay on HUVEC cells after 24 h of incubation with naked polyurethane, PEG-polyurethane and lys-coated polyurea nanoparticles at a concentration of 85 mg/mL. A total cell number of 7.5 103 HUVECcells/well was seeded. Samples labelled as control correspond to HUVEC cells treated with PBS. ***: p < 0.001.
and soybean oil) were carefully selected to prepare the polyurethane and polyurea nanoparticles described in this study. In this regard, it is especially important to select the composition variables and polymerisation temperature to obtain polymeric nanoparticles with a suitable particle size and polydispersity properties. Full conversion of monomer and, consequently, the sign of the completion of polycondensation reaction was corroborated by FT-IR analysis, as described in a previous study [30]. Considering the stoichiometry of the polymerisation reaction, any increment in the monomer concentration must be followed by the corresponding increase of PEG or lys concentration. In our studies, we observed that the presence of additional component with hydroxyl groups (PEG) in the formulation was not strictly necessary to obtain polyurethane nanoparticles. As a matter of fact, polyurethane nanoparticles were also obtained with the only incorporation of IPDI to the water/polysorbate 80/medium chain triglyceride system. This can be explained by the reaction between IPDI and free hydroxyl groups of polysorbate 80. Moreover, no reaction was observed among IPDI and medium chain triglyceride because this oily compound does not have reactive groups in its chemical structure [35]. Furthermore, it can be assumed that IPDI preferably reacts with surfactant hydroxyl groups rather than with water hydroxyls due to a minor energy intake is required for the chemical reaction [16]. Therefore, the surfactant would significantly contribute to the polymerisation process as previously reported [30,36]. The decrease of particle diameter (naked polyurethane < pegylatedpolyurethane (PEG-polyurethane) < lys-coated polyurea), was associated to the involvement of PEG and lys in the polymerisation reaction at the droplet interfaces since all parameters such as O/S ratio, water concentration and temperature were constant. Moreover, the chemical reaction between isocyanate groups of IPDI and hydroxyl or amine groups of PEG and lys, respectively, is enhanced due to the structural location of the reactive groups in these molecules. Altogether, these results also indicate that the molecules located at the interface determine the characteristics of nanoparticles as previously reported [18,29]. Polydispersity indexes higher than 0.20 were associated with the coexistence of two different populations of particles. For low concentrations of IPDI, the quantity of monomer is not enough to allow the polymerisation process in all droplets of nano-emulsion. Therefore, nano-emulsions and nanoparticles can be detected simultaneously by DLS. In contrast, an excess of monomer would lead to the formation of nanoparticles and large aggregates.
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
Regarding to the influence of O/S ratio, the differences observed in the particle size can only be attributed to the reaction at the droplet interfaces between IPDI and polysorbate 80, since all tested parameters involved in the polymerisation process were maintained constant and only O/S ratio was modified. Thus, a decrease in surfactant concentration is reflected with a decrease in the particle diameter. These results support the hypothesis [30] that the highly hydrophilic component and the surfactant compete for the monomer in the polymerisation process. As expected, the particle size of lys-coated polyurea systems was smaller than PEGpolyurethane samples for all O/S ratios although a higher concentration of monomer was required for their preparation, and both type of nanoparticles were smaller than naked polyurethane nanoparticles. This fact agrees with the involvement of PEG and lys in the formation of the polymeric matrix. The effect of polymerisation temperature on nanoparticle size can be explained as follows: A decrease of temperature implies a delay in the polymerisation process and consequently, the formation of polymeric matrix progressively occurs at the droplet interfaces through the reaction between IPDI and hydroxyl or amine groups of both, the surfactant and PEG 400 or lys. Since the reaction with water requires higher activation energy than the reaction with hydroxyl and amine groups, almost all of the monomer concentration would be involved in this polymerisation and a minimum reaction with water should be observed [16]. Conversely, polymerisation process takes place very quickly at high temperatures. On the one hand, the reaction between diisocyanate and water is promoted and on the other hand the collisions among droplets are increased as a consequence of temperature. Subsequently, a high number of droplets can be involved in the formation of each nanoparticle and hence, the particle diameter would rise in a short period of time. Apart from the composition parameters and temperature described above, both the use of PEG with a lower molecular weight and the incorporation of an oily component with free hydroxyl groups in their chemical structure were also found to have an effect in the particle size. Thereby, as expected and in comparison to equivalent samples prepared with PEG 400 and saturated medium chain triglycerides (PEG-polyurethane nanoparticles [30]), lower particle sizes were obtained with PEG 200 (PEG 200-polyurethane). In this regard, the measured diameter of PEG-polyurethane nanoparticles was around 70 nm [30]. Therefore, it seems that the particle size tends to decrease in agreement with the molecular weight of polyethylene glycol as previously reported [28]. On the other hand, the decrease of the particle size in the SO-PEG-polyurethane sample (soybean oil as dispersed phase) was attributed to the involvement of the hydroxyl groups of soybean oil in the polymerisation reaction. It should be noted that the replacement of the medium chain triglyceride with soybean oil did not mean any change in the mean droplet diameter of the O/W nano-emulsions before being heated at polymerisation temperature. Consequently, a polymeric matrix constituted by copolymers derived from the reaction between IPDI and hydroxyl groups of surfactant, PEG 400 and soybean oil should be taken into consideration. The stability studies have shown a slight growth in the nanoparticle size during the first 30 days that can be attributed to a swelling phenomenon. Thus, the water embedded into the polymeric matrix led to a raise in the mean particle diameter around of a 28e30% for 10/90 O/S ratios and around of a 48e60% for higher O/S ratios. This increase in the particle size was also followed by little fluctuations in the polydispersity values as detected by DLS during the first days. However, the polydispersity indexes never exceeded 0.20. Moreover, TEM images taken at 0 and 30 days allowed to corroborate the swelling phenomenon produced when nanoparticles are kept in aqueous suspension. To prevent the flocculation
6079
and the swelling phenomena produced during the storage, PEGpolyurethane and lys-coated polyurea nanoparticles were freezedried. In this regard, it has been reported that suitable concentrations of nonionic surfactant such as polysorbate 80 provide a high degree of protection against the freezing stress [37]. Therefore, no cryoprotectant which might modify the particle size, polydispersity and morphologic properties was incorporated to the formulations. The freeze-dried studies demonstrated that the particle diameter practically remained unmodified even after storing the samples for a long period. Likewise, nanoparticles showed again a swelling process when were dispersed in the aqueous medium. Therefore, the freeze-drying could be considered an effective method to preserve the integrity of nanoparticles and to avoid aggregation and the swelling phenomena during the storage. We also evaluated the haemolytic and cytotoxic effects of polymeric nanoparticles using pig and human erythrocytes and human endothelial cells, respectively. Both RBC and endothelial cells are the main cell types in contact with any pharmaceutical formulation that could be intravenously administered. Therefore the in vitro haemolysis and MTS assay in normal primary cells can adequately reflect the biological effects of nanoparticles. The results of the haemolysis assay showed that naked polyurethane, PEGpolyurethane and lys-coated polyurea nanoparticles are hemocompatible and equally suitable to be intravenously administered. Moreover naked polyurethane and PEG-polyurethane nanoparticles showed very low effects on human endothelial cell viability after 24 h of incubation thus showing a high biocompatibility as potential drug carriers. On the other hand lys-coated polyurea nanoparticles showed a significantly higher toxicity than naked and pegylated nanoparticles likely due to the cationic nature of lysine as basic amino acid. Furthermore, it is important to emphasise that the tested dispersions of naked polyurethane, PEG-polyurethane and lys-coated polyurea nanoparticles were used in non tumoral cells at a concentration similar than habitually described for other biocompatible nanoparticles [38,39] and for a period of time much higher than the residence time of any intravenous formulation thus testing the maximum toxicity effects that these nanoparticles could promote. 5. Conclusion In the present study we have designed polyurethane and polyurea nanoparticles that can be potential drug carriers owing to their small particle diameter, their formulation with biocompatible components and the low concentration of aliphatic diisocyanate required in the preparation of these nanoparticles in comparison to the rates of IPDI monomer typically used in other reports [17e19]. This means an important approach for the design of drug delivery systems in order to prevent possible toxicity problems. Thus, naked polyurethane, PEG-polyurethane and lys-coated polyurea nanoparticles with a diameter in the range of 50e90 nm, low polydispersity indexes and high kinetic stability have been obtained from selected O/W nanoemulsions systems by an interfacial polycondensation process. Composition variables such as monomer concentration, O/S ratio, the components that can be located at the droplet interface and the dispersed phase as well as the polymerisation temperature were found as critical parameters to control the formation, particle size and polydispersity properties of polyurethane and polyurea nanoparticles. In addition, these nanoparticles possesses a polymeric matrix constituted by copolymers obtained from the chemical reaction between the isocyanate groups of the monomer and the nucleophile groups of the surfactant, PEG 400 or lysine, as previously described [30]. Furthermore, although the involvement of the surfactant molecules in the polymerisation reactions occurring in nanoparticles obtained from nano-emulsion systems had been
6080
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
already reported [36], for the first time, we have described the formation of polyurethane particles exclusively from the reaction between a diisocyanate and a surfactant with hydroxyl groups. This represents the first step in the formulation of new polymeric matrix with more biocompatible components to be employed as safe drug delivery systems for pharmaceutical purposes. Indeed, since we have demonstrated that these nanoparticles are biocompatible, this means that these formulations have interesting features for future design of blood-contacting applications (such as intravenous administration or endovascular devices) with a very attractive safety profile. All these data indicate that naked polyurethane, PEG-polyurethane and lys-coated polyurea nanoparticles can serve as promising biomaterials suitable for applications in complex biological media. Acknowledgement The authors wish to acknowledge the sponsorship of the Spanish Ministry of Education and Science, DGI (CTQ 2008-06892C03-02/PPQ and CTQ 2011-29336-C03/PPQ), “Generalitat de Catalunya” DURSI (Grant 2009 SGR-961), Asociación Española para el Estudio del Hígado (AEEH) and CIBER-BBN. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008e2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fundation. The authors also acknowledge Dr. Montserrat Rigol from Institut Clinic del Torax, Hospital Clinic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) for her help with the handling of blood samples. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.polymer.2012.10.039. References [1] Nagarwal RC, Kant Sh, Singh PN, Maiti P, Pandit JK. J Control Release 2009;136: 2e13. [2] Kumari A, Yadav SK, Yadav SC. Colloid Surf B 2010;75:1e18. [3] Parveen S, Misra R, Sahoo SK. Nanomed-Nanotechnol 2012;8:147e66. [4] Delmas Th, Fraichard A, Bayle PA, Teixier I, Bardet M, Baudry J, et al. J Colloid Sci Biotechnol 2012;1(1):16e25. [5] Poletto FS, Fiel LA, Lopes MV, Schaab G, Gomes AMO, Guterres SS, et al. J Colloid Sci Biotechnol 2012;1(1):89e98. [6] Soppimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE. J Control Release 2001;70:1e20.
[7] Chaubal MV. Drug Discov Today 2004;9:603e9. [8] Mora-Huertas CE, Fessi H, Elaissari A. Int J Pharm 2010;385:113e42. [9] Aboubakar M, Puisieux F, Couvreur P, Vauthier C. Int J Pharm 1999;183(1): 63e6. [10] Nair LS, Laurencin CT. Prog Polym Sci 2007;32:762e98. [11] Bonzani IC, Adhikari R, Houshyar Sh, Mayadunne R, Gunatillake P, Stevens MM. Biomaterials 2007;28(3):423e33. [12] Sarkar D, Ch. Yang J, Gupta AS, Lopina ST. J Biomed Mater Res A 2009;90(1): 263e71. [13] Laschke MW, Strohe A, Scheuer C, Eglin D, Verrier S, Alini M, et al. Acta Biomater 2009;5:1991e2001. [14] Marcos-Fernández A, Abraham GA, Valentín JL, Román JS. Polymer 2006;47: 785e98. [15] Hafeman AE, Zienkiewicz KJ, Zachman AL, Sung HJ, Nanney LB, Davidson JM, et al. Biomaterials 2011;32:419e29. [16] Ionescu M. Polyols and basic chemistry of polyurethanes. In: Ionescu M, editor. Chemistry and technology of polyols for polyurethanes. Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK: Rapra Technology; 2005. p. 1e15. [17] Tiarks F, Landfester K, Antonietti M. J Polym Sci Chem 2001;39:2520e4. [18] Barrère M, Landfester K. Macromolecules 2003;36:5119e25. [19] Zanetti-Ramos BG, Lemos-Senna E, Soldi V, Borsali R, Cloutet E, Cramail H. Polymer 2006;47:8080e7. [20] Gaudin F, Sintes-Zydowicz N. Colloid Surf A 2008;331:133e42. [21] Landfester K. Adv Mater 2001;13:765e8. [22] Landfester K, Tiarks F, Hentze HP, Antonietti M. Macromol Chem Phys 2000; 201:1e5. [23] Anton N, Benoit JP, Saulnier P. J Control Release 2008;128:185e99. [24] Solans C, Izquierdo P, Nolla J, Azemar N, Garcia-celma MJ. Curr Opin Colloid Interfase Sci 2005;10:102e11. [25] Calderó G, García-Celma MJ, Solans CJ. Colloid Interface Sci 2011;353:406e11. [26] Johnsen HMS, Schmid RB. J Microencapsul 2007;24:731e42. [27] Torini L, Argillier JF, Zydowicz N. Macromolecules 2005;38:3225e36. [28] Bouchemal K, Briançon S, Perrier E, Fessi H, Bonnet I, Zydowicz N. Int J Pharm 2004;269:89e100. [29] Zanetti-Ramos BG, Lemos-Senna E, Cramail H, Cloutet E, Borsali R, Soldi V. Mat Sci Eng C 2008;28(4):526e31. [30] Morral-Ruíz G, Solans C, García ML, García-Celma MJ. Langmuir 2012;28(15): 6256e64. [31] Chen C, Cheng YC, Yu CH, Chan SW, Cheung MK, Yu PHF. J Biomed Mater Res A 2008;87:290e8. [32] Aparicio RM, García-Celma MJ, Vinardell MP, Mitjans M. J Pharm Biomed 2005;39:1063e7. [33] Guide to the preparation, use and quality assurance of blood components. 6th ed. Strasbourg Cedex Germany: Council of Europe Publishing; 2000. [34] US License NumberFDA summary basis of approval of red blood cells frozen and red blood cells deglycerolized, reference no. 86-0335, applicant. Bethesda, MD: Department of the Navy, Naval Hospital; 1986. p. 635e10. [35] Rowe RC, Sheskey PJ, Weller P, editors. Handbook of pharmaceutical excipients. 6th ed. London, UK, Washington, USA: J. Pharmaceutical Press: & American Pharmaceutical Association; 2009. p. 429. [36] Rosenbauer EM, Landfester K, Musyanovych A. Langmuir 2009;25(20): 12084e91. [37] Hillgren A, Lindgren J, Aldén M. Int J Pharm 2002;237:57e69. [38] Wadajkar AS, Bhavsar Z, Ko Ch Y, Koppolu B, Cui W, Tang L, et al. Acta Biomater 2012;8(8):2996e3004. [39] Kim Se, Seong K, Kim O, Kim S, Seo H, Lee M, et al. Biomacromolecules 2010; 11:555e60.
Contents lists available at SciVerse ScienceDirect
Polymer journal homepage: www.elsevier.com/locate/polymer
Design of biocompatible surface-modified polyurethane and polyurea nanoparticles G. Morral-Ruíz a, b, P. Melgar-Lesmes a, M.L. García c, C. Solans b, d, M.J. García-Celma a, b, * a
Departament de Farmàcia i Tecnologia Farmacèutica, Unitat RþD associada al CSIC, Facultat de Farmàcia, Universitat de Barcelona, Av Joan XXIII s/n, 08028 Barcelona, Spain Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, Barcelona, Spain c Departament de Fisicoquímica, Facultat de Farmàcia, Universitat de Barcelona, Av Joan XXIII s/n, 08028 Barcelona, Spain d Institut de Química Avançada de Catalunya (IQAC), CSIC, Jordi Girona 18-26, 08034, Barcelona, Spain b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 July 2012 Received in revised form 16 October 2012 Accepted 19 October 2012 Available online 26 October 2012
This study is focused to design systems of polyurethane and polyurea nanoparticles prepared via interfacial polycondensation in O/W nano-emulsions with biocompatible components suitable for drug delivery systems. Polyurethane and polyurea nanoparticles with a small diameter (50e90 nm) and high kinetic stability were developed from O/W nano-emulsions in aqueous solution/polysorbate 80/oil systems. The influence of several factors on the particle size and polydispersity index was studied. The monomer concentration, oil/surfactant weight (O/S) ratio, polymerisation temperature and components that can be located in the droplet interface and the dispersed phase of nano-emulsion played a key role in the formation of these nanoparticle systems. The biocompatibility of these nanoparticles was assessed using haemolysis and cell viability assays. No significant haemolysis and low effects on human endothelial cell viability were obtained after incubation with nanoparticles. Therefore, herein we provide useful information for the development of biocompatible drug carriers using polyurethanes and polyureas. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Polymeric nanoparticle Interfacial polycondensation Nano-emulsion
1. Introduction The formation of polymeric [1e3] and lipid [4,5] nanoparticles has aroused a growing interest in the last decades owing to their great potential as controlled drug delivery systems. In this regard, the choice of biocompatible polymers as well as methods of preparation plays a key role in the formation of suitable drug carriers for drug delivery [6e9]. Polyurethanes and polyurea polymers are emerging as useful biomaterials for biomedical applications because of their synthetic versatility, excellent mechanical properties and good biocompatibility [10e13]. In this context, the formation of polyurethanes and polyureas employing aliphatic diisocyanates with incorporated aminoacids such as lysine and biodegradable polyether has acquired greater relevance in last years [14,15]. It is well known that both polymers can be obtained from the chemical reaction between a diisocyanate and a polyol or
* Corresponding author. Departament de Farmàcia i Tecnologia Farmacèutica, Unitat RþD associada al CSIC, Facultat de Farmàcia, Universitat de Barcelona, Av Joan XXIII s/n, 08028 Barcelona, Spain. Tel.: þ34 934024548; fax: þ34 934035937. E-mail addresses: [email protected] (G. Morral-Ruíz), melgarpedro@ yahoo.com (P. Melgar-Lesmes), [email protected] (M.L. García), csmqci@ cid.csic.es (C. Solans), [email protected] (M.J. García-Celma). 0032-3861/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.polymer.2012.10.039
diamine by a polycondensation or polyaddition process [16]. Nevertheless, the high reactivity of isocyanate groups makes difficult the right formation of these polymers in aqueous media. In this regard, several efforts have been developed in order to avoid this collateral reaction with water [17e19]. Nano-emulsions constitute an attractive alternative to prepare polyurethane and polyurea nanoparticles in aqueous systems due to their small droplet size and high kinetic stability [20e23]. Moreover, nano-emulsions can be obtained not only using highenergy methods but also by low-energy methods, which take advantage of the physicochemical properties of the system leading to the formation of even smaller and more uniform droplets [24,25]. Thus, the formation of polyurethane nanoparticles from O/W nano-emulsions in a one-step procedure has already been described in numerous reports [17e20]. However, in these studies, nanodroplets used as template are almost exclusively prepared using high-energy methods [17e20]. In addition, chemical catalysts [18,26] and ionic surfactants such as sodium dodecyl sulphate [27] which are widely used to achieve the formation of nanoparticles with a small particle diameter could generate biocompatibility problems. On the other hand, when nanoparticles are obtained via low-energy methods, organic solvents such as acetone are usually employed to carry out the procedures [28]. Therefore, the goal of
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
the work reported has been to develop systems of polyurethane and polyurea nanoparticles obtained by polycondensation at the droplet interface of O/W nano-emulsions prepared by low-energy methods in aqueous solution/nonionic surfactant/oil systems and using biocompatible components. In this context, we have studied the involvement of surfactant in the polymerisation reaction that has rarely been analysed to date [18,19,29]. Thus, the present study was aimed to investigate different parameters involved in the polymerisation process, especially focussing on the influence of polysorbate 80 as nonionic surfactant, and the biocompatibility of these formulations to optimise polyurethane and polyurea nanoparticles for future biomedical purposes. 2. Experimental 2.1. Materials The nonionic technical grade surfactant, polyoxyethylene 20-sorbitan monooleate (polysorbate 80, P80) was supplied from Fagron (Barcelona, Spain). Components used as highly hydrophilic materials such as polyethylene glycol 400 (PEG 400), polyethylene glycol 200 (PEG 200) and L-lysine (lys) as well as the monomer, isophorone diisocyanate (IPDI), were purchased from Sigmae Aldrich (Madrid, Spain). The oil components, saturated medium chain triglyceride (MCT) and soybean oil (SO) were obtained from Fagron and SigmaeAldrich, respectively. All chemicals were used without further purification. Deionised water was obtained from a Millipore-Milli-Q water purification system (Molsheim, France). Pig and human blood were purchased from AbD Serotec (Raleigh, NC, USA). All materials employed in biocompatibility studies, the primary human endothelial cells HUVECs (Human umbilical vein endothelial cells), Dulbecco’s Modified Eagle Medium (DMEM), Ham’s F-12 Nutrient Mix (F-12), foetal bovine serum (FBS), penicillin/ streptomycin antibiotic and endothelial cell growth supplement were supplied from Life Technologies Ltd. (Paisley, UK). 2.2. Methods 2.2.1. Preparation of polyurethane and polyurea nanoparticles Polyurethane and polyurea nanoparticles were prepared from O/W nano-emulsions by an interfacial polycondensation process in aqueous solution/polysorbate/diisocyanate/oil systems. The O/W nano-emulsions were prepared at 25 C by the Phase Inversion Composition (PIC) emulsification method as reported [30]. A defined amount of isophorone diisocyante was incorporated onto oil/surfactant mixtures. Then, aqueous component was dropwise added to these mixtures with continuous agitation with a vibromixer. Nanoparticles were formed by heating the nano-emulsion at temperatures between 55 and 80 C. The polymerisation reaction was considered as completed after 4 h and samples were kept at 25 C [30]. 2.2.2. Particle size and polydispersity index Measurements of the average particle size and polydispersity index were performed by Dynamic Light Scattering (DLS) at 25 C using a Zetasizer nano ZS (Malvern Instruments, UK). This instrument is equipped with a detector to analyse the intensity of the scattered light at a fix angle of 173 provided by a HellioneNeon laser (4 mW, l ¼ 633 nm). The values of Z-average and polydispersity index were calculated from the autocorrelation function of the scattered intensity by means of the cumulants analysis using DTS nano (Malvern Instruments) software. The nanoparticle suspensions were diluted 1/75 with ultra-purified water in order to avoid multiple scattering events.
6073
2.2.3. Nanoparticle stability studies Stability studies were carried out by measuring the particle size of nanoparticle suspensions as a function of time during 6 months using Dynamic Light Scattering at 25 C. Samples were kept at 25 C during all the studied period (190 days). 2.2.4. Nanoparticle freeze-drying A fraction of 2 mL of nanoparticle suspension was freeze-dried. No cryoprotectant was added to the formulations. Samples were flash-frozen at 78 C in dried ice prior freeze-drying. Then, frozen samples were freeze-dried at 89 C under 0.01 mbar vacuum for 48 h in a glass chamber connected to a vacuum pump and a vapour condenser (Freeze-Dried Alpha 2-4 LD plus, Biobloc Scientific, Illkirch, France). 2.2.5. Transmission electron microscopy (TEM) characterisation Characterisation studies by TEM were carried out on a JEOL JEM 1010 (JEOL Ltd., Akishima, Japan) microscope. To prepare the samples, one droplet of nanoparticle suspension was placed on a carbon-coated copper grid and stained with an aqueous solution of uranyl acetate at 1% (w/v) prior observation. Average particle diameter of approximately 500 randomly selected nanoparticles from different TEM micrographs was determined using the morphometry software ImageJ v. 1.44. 2.2.6. In vitro red blood cell (RBC) haemolysis assay of the nanoparticles Haemolysis assay was performed using fresh pig and human blood as previously reported [31]. The erythrocytes were collected by centrifugation (Heraeus Megafuge 16R centrifuge, Thermo Scientific) at 3000 rpm for 10 min, and then washed three times with phosphate buffered saline (PBS) at pH 7.4. A stock dispersion was prepared by mixing 3 mL of centrifuged erythrocytes into 11 mL of PBS. Freeze-dried polyurethane and polyurea nanoparticles were resuspended in PBS and then slightly diluted in distilled water to reach isotonic values (ranging 280e300 mOsm/L). One hundred microlitre of stock erythrocyte dispersion was added to 1 mL of the nanoparticle dispersions. The tubes were then incubated for 10 min at 37 C in an Incubator Shaker. After incubation, the tubes were centrifuged (5 min at 13,000 rpm (IEC MicroCL 21R centrifuge, Thermo Scientific)) and the percentage of haemolysis was determined by comparing the absorbance (l ¼ 540 nm) of the supernatant with that of control samples totally haemolysed with 1 mL of distilled water as previously reported [32]. The supernatant absorbance of a blood sample not treated with nanoparticles and incubated with 1 mL of PBS was used as the negative control to obtain only the percentage particle-induced haemolysis. All experiments were reproduced five times with each type of formulation and data have been presented as the percentage of the complete haemolysis and Standard Error of the Mean (sem). 2.2.7. Cell culture and in vitro cell biocompatibility HUVECs were cultured in DMEM/F-12 medium supplemented with 10% foetal bovine serum (FBS), 100 mg/mL penicillin/streptomycin and 50 mg/mL endothelial cell growth supplement. Cells were grown at 37 C and 5% CO2. HUVECs were passaged when they reached 80% confluence and passages 2e5 were used for all experiments. To quantify the toxicity effect of nanoparticles in cell viability of human endothelial cells, we performed the MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt) assay (CellTiter96 Aqueous One Solution assay kit, Promega, Madison, WI, USA). HUVECs were seeded in 96-well plates at a cell density of 7.5 103 cells/well in complete media as
6074
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
described above. After 24 h, cells were serum-starved for 4 h (FBS at 0.1%) and incubated in media in the presence of 85 mg/mL naked polyurethane, pegylated polyurethane or lys-coated polyurea nanoparticles for 24 h at 37 C and 5% CO2. Cell viability was determined by adding 20 mL of MTS solution to each well. After 2 h, the absorbance was measured at 490 nm using an EPOCH microplate spectrophotometer (Biotek Instruments, Winooski, VT, USA). The cell viability was obtained by comparing the absorbance of nanoparticle-treated cells to that of control cells (treated with PBS). Each condition was performed in sextuplicate and reported as mean sem. 3. Results 3.1. Influence of composition parameters of O/W nano-emulsions in the formation of polyurethane and polyurea nanoparticles The description of the nano-emulsions used to prepare the nanoparticles is detailed in Table 1. In the first set of experiments, the effect of polyethylene glycol, lysine and monomer concentration of the nano-emulsion in the formation of nanoparticles was evaluated by analysing the particle size and polydispersity index by DLS at 25 C (Fig. 1). The samples were characterised just after preparation. A polymerisation temperature of 70 2 C was initially chosen according to previous studies [17,26]. Concentrations of IPDI ranging 0.01 mmol/ge 0.10 mmol/g were selected to prepare the nanoparticles. It should be noted, that these values of monomer concentration are lower than those reported in the literature [17e19]. The mean particle size (Fig. 1) increases with IPDI concentration and this occurs independently on the kind of particle. Nanoparticles with the biggest diameter corresponded to naked polyurethane compositions (Fig. 1). In contrast, the smallest particle sizes were obtained with lysine-coated polyurea samples. The minimum concentration of IPDI to form nanoparticles with a small particle size and low polydispersity (<0.15) was 0.02 mmol/g and 0.04 mmol/g for polyurethane and polyurea samples, respectively. Nanoparticles with a Z-average value below 85 nm were obtained with these monomer concentrations. It is important to mention that lys-coated polyurea nanoparticles showed an extremely small hydrodynamic diameter (around 50 nm) even using double of concentration of IPDI (Fig. 1). The influence of O/S ratio in the particle size of PEGpolyurethane and lys-coated polyurea samples was also studied. MCT/P80 ratios between 0/100 and 15/85 were selected according to the studies of phase behaviour at 25 C described in a previous
work [30]. The IPDI concentrations, 0.02 mmol/g and 0.04 mmol/g for PEG-polyurethane and lys-coated polyurea, respectively, were selected according to the results described above. Polymerisation was carried out by heating samples at 70 2 C. The nanoparticle size was determined by DLS at 25 C just after preparation and the results are shown in Fig. 2. The mean particle size of the PEG-polyurethane and lys-coated polyurea samples ranged 72e123 nm and 56e98 nm, respectively (Fig. 2). Surprisingly, the particle diameter decreased lineally at increasing O/S ratios [22,29]. Although the particle size decreases with the increase in O/S ratios, the polydispersity index tends to rise. Even so, all polydispersity indexes were lower than 0.20. Therefore, while the smallest diameter is achieved with O/S ratio of 15/85, we selected the O/S ratio of 10/90 for biocompatibility studies owing to a lower concentration of surfactant is required to obtain the nanoparticles. For this O/S ratio PEG-polyurethane and lys-coated polyurea nanoparticles with a particle diameter of 89.3 nm and 67.5 nm, respectively, and low polydispersity index were obtained. 3.2. Influence of temperature in the formation of polyurethane and polyurea nanoparticles To study the influence of temperature in the formation of polyurethane and polyurea nanoparticles, samples were prepared with the optimal concentration of IPDI, O/S, PEG 400 and lys. Thus, nano-emulsions with a 10/90 O/S ratio and 90 wt% aqueous solution containing IPDI and the highly hydrophilic components at suitable concentrations were heated at temperatures between 55 and 80 C to form nanoparticles. Polymerisation was not detected at lower temperatures than 55 C. The mean particle size and the polydispersity index of these compositions were analysed using DLS (Fig. 3). As observed in Fig. 3, the mean particle diameter increases with the temperature in all studied formulations. Regarding to the polydispersity index, low values (PDI 0.15) were only achieved when the temperature of polymerisation was higher than 65 C. In fact, temperatures equal or lower than 65 C in most of samples led to the formation of two populations detected by DLS intensity analysis, being the first one between 10 and 15 nm (corresponding to droplets of nano-emulsions) and a second one of polymeric nanoparticles (Supplementary data). After testing all these temperatures, it was found that the temperature corresponding to 70 C is the best choice to prepare polyurethane and polyurea nanoparticles, since for this
Table 1 Composition of polyurethane and lys-coated polyurea nanoparticles obtained from O/W nano-emulsions and micellar solutions. Nano-emulsions correspond to oil/surfactant weight (O/S) ratios 5/95 and micellar solutions to O/S ratios of 0/100. Samples were prepared with a constant concentration of 90 wt% aqueous solution in aqueous solution/ polysorbate 80/saturated medium chain triglycerides and aqueous solution/polysorbate 80/soybean oil systems. Composition of polyurethane and polyurea nanoparticles
Naked polyurethane PEG-polyurethane
PEG 200-polyurethanea SO-PEG-polyurethaneb Lys-coated polyurea
a b
O/S ratio (wt%)
IPDI (mmol/g)
10/90 0/100 5/95 10/90 15/85 10/90 10/90 0/100 5/95 10/90 15/85
0.02e0.08 0.02 0.02 0.01e0.10 0.02 0.02 0.02 0.04 0.04 0.04e0.08 0.04
Highly hydrophilic component (mmol/g)
Diisocyante/highly hydrophilic component molar ratio
Diisocyanate/molecules with OH or NH2 groups molar ratio
PEG PEG PEG PEG PEG PEG Lys: Lys: Lys: Lys:
e 1:1 1:1 0.5:0.5e5:5 1:1 1:1 1:1 2:1 2:1 2:1e4:2 2:1
1:3.4e4:3.4 1:4.8 1:4.6 0.5:3.9e5:8.4 1:4.2 1:4.4 1:5 2:4.8 2e4.6 2:4.4e4:5.4 2:4.2
400: 0.02 400: 0.02 400: 0.01e0.10 400: 0.02 200: 0.02 400: 0.02 0.02 0.02 0.02e0.04 0.02
Polyethylene glycol 200 was used instead of polyethylene glycol 400. Soybean oil was used as oily component instead of saturated medium chain triglycerides.
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
6075
Fig. 1. Influence of IPDI concentration in the formation of polyurethane and polyurea nanoparticles. Mean particle diameter (Z-average) and polydispersity index (PDI) determined by DLS at 25 C of nanoparticles obtained from O/W nano-emulsions with 90 wt% aqueous solution and O/S ratio: 10/90 in water/polysorbate 80/saturated medium chain triglycerides and aqueous solution/polysorbate 80/medium chain triglycerides systems as a function of IPDI concentration.
temperature the smallest particle diameter with a low polydispersity index (PDI 0.15) is achieved.
3.3. Influence of PEG molecular weight and the composition of dispersed phase in the formation of pegylated polyurethane nanoparticles Eventually, the influence of both the molecular weight of PEG and the oily component in the formation of PEG-polyurethane nanoparticles was also evaluated. To study both factors, samples with PEG 200 and soybean oil (oily component with hydroxyl groups) were prepared. The composition of these nanoparticles is given in Table 1 (see PEG 200-polyurethane and SO-PEG polyurethane samples). Characterisation studies of the obtained nanoparticles were performed by TEM as illustrated in Fig. 4. TEM micrographs showed the formation of a single population of well-defined PEG-polyurethane particles with a narrow size distribution and a mean particle diameter of 48 5 and 54 5 nm for PEG 200-polyurethane and SO-PEG-polyurethane samples, respectively (Fig. 4).
3.4. Stability studies of PEG-polyurethane and lys-coated polyurea nanoparticles The stability of PEG-polyurethane and lys-coated polyurea nanoparticles was determined measuring the particle diameter as a function of time by DLS at 25 C (Fig. 5). Nanoparticles were prepared at the optimal temperature, PEG 400, lys and monomer concentration. As observed in Fig. 5a, the aqueous dispersions of nanoparticles showed a high kinetic stability since the increase in the particle size did not exceed 50 nm during 6 months or beyond. Moreover, no significant changes in the appearance of the samples (such as increase of turbidity and sedimentation) were observed within the studied period. The best stability results were obtained with the 5/95 O/S ratio in both, PEG-polyurethane and lys-coated polyurea nanoparticles. In these compositions, the mean particle size remained constant during all the stability study. Regarding to the other O/S ratios, a slight increase of the diameter was observed during the first month and after that the particle size did not vary at all. Two representative TEM images of PEG-polyurethane nanoparticles taken at 0 and 30 days after preparation are shown in
Fig. 2. Influence of O/S ratio in the formation of polyurethane and polyurea nanoparticles. Mean particle diameter (Z-average) and polydispersity index determined by DLS at 25 C of nanoparticles obtained from O/W nano-emulsions at 90 wt% of aqueous solution at different O/S ratios in aqueous solution/polysorbate 80/saturated medium chain triglyceride systems.
6076
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
Fig. 3. Influence of temperature in the formation of polyurethane and polyurea nanoparticles. Mean particle size and polydipsersity index measured by DLS at 25 C for compositions with 90 wt% aqueous solution and 10/90 wt% O/S ratio corresponding to pegylated polyurethane and lys-coated polyurea nanoparticles as a function of polymerisation temperature.
Fig. 5b. The micrographs observed at 30 days showed a population of nanoparticles with a higher diameter as compared to the samples analysed just after preparation. The samples were also freeze-dried to improve the long-term stability of PEG-polyurethane and lys-coated polyurea nanoparticles during the storage. Results are shown in Table 2. The mean particle size and polydispersity of the samples were measured by DLS before, following freeze-drying process and after storing the freeze-dried at 25 C during 2 years. The samples were
reconstituted in 30 s by mechanic stirring after the addition of 2 mL of water to the freeze-dried sample. The stability at 25 C of the reconstituted nanoparticle suspensions was also studied (Fig. 6). As shown in Table 2, the mean particle diameter for PEGpolyurethane nanoparticles was around 70 nm and for lys-coated polyurea samples ranging between 55 and 60 nm. Thus no significant differences in the particle size were observed between the original suspension of nanoparticles and the reconstituted samples even after being stored for 2 years. Nevertheless the polydispersity
Fig. 4. Transmission electron micrographs (TEM) and histograms of the particle size of polyurethane nanoparticles. a) PEG 200-polyurethane and b) SO-PEG-polyurethane nanoparticles. Both samples were prepared at an IPDI/PEG molar ratio 1/1 with 90 wt% aqueous phase and O/S ratio of 10/90 in water/PEG 200/polysorbate 80/IDI/saturated medium chain triglyceride and water/PEG 400/polysorbate 80/IPDI/soybean oil systems, respectively.
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
6077
Fig. 5. Stability studies of polyurethane and polyurea nanoparticles and TEM micrographs at representative periods of time. a) Mean particle size as a function of time determined by DLS at 25 C for nanoparticles prepared with 90 wt% aqueous solution and different O/S ratios in the aqueous solution/polysorbate 80/medium chain triglyceride systems. b) TEM images corresponding to PEG-polyurethane nanoparticles (90 wt% aqueous component, O/S ratio: 10/90) observed at different periods of time.
index slightly increased, especially in lys-coated polyurea nanoparticles. However, the values of polydispersity indexes in both nanoparticle systems were lower than 0.20. The determinations of the particle size by DLS in reconstituted samples as a function of time (Fig. 6) showed similar results to the stability studies. Thus, PEG-polyurethane and lys-coated polyurea nanoparticles underwent a slight increase of the particle diameter during the first days and after that the particle size remained constant. In these samples, an increase of 25e35% in the particle diameter was quantified during the first days.
potential to damage red blood cells (haemolysis). Haemolysis, the abnormal breakdown of RBC, can lead to anaemia or other pathological conditions. As shown in Table 3, naked polyurethane and lys-coated polyurea nanoparticles did not show haemolysis in pig and human RBC at all. On the other hand, PEG-polyurethane nanoparticles produced a 0.5% of haemolysis when tested in pig RBC. However, this outcome does not exceed the permissible limits of haemolysis for biomedical purposes which are 0.8%, as per the Council of Europe guidelines [33] and 1% as per the US FDA guidelines [34].
3.5. Haemocompatibility study of naked polyurethane, PEGpolyurethane and lys-coated polyurea nanoparticles in red blood cells
3.6. In vitro endothelial cell biocompatibility studies of naked polyurethane, PEG-polyurethane and lys-coated polyurea nanoparticles
Nanotechnology applications and biomaterials designed for systemic administration have to pass previous biocompatibility tests. These tests include an in vitro assay to establish the material’s
The cytotoxicity of naked polyurethane, PEG-polyurethane and lys-coated polyurea nanoparticles was evaluated in HUVEC cells using the MTS assay. Fig. 7 shows the viability results obtained after
Table 2 Z-average and polydispersity index (PDI) determined by DLS at 25 C of the nanoparticle suspension and the reconstituted suspension from freeze-drying just preparation and after storing samples at 25 C for 2 years. PEG-polyurethane and lys-coated nanoparticles were prepared in nano-emulsions with a 90 wt% aqueous component and an O/S ratio of 10/90 in the aqueous solution/polysorbate 80/saturated medium chain triglycerides 812 systems. Suspension of nanoparticles before freeze-drying
Re-suspension of freeze-dried t¼0
PEG-polyurethane Lys-coated polyurea
t ¼ 2 years
Z-average (nm)
PDI
Z-average (nm)
PDI
Z-average (nm)
PDI
68.90 55.73
0.14 0.11
66.93 59.40
0.16 0.17
67.90 56.50
0.15 0.16
6078
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
Fig. 6. Mean particle size as a function of time determined by DLS at 25 C for the nanoparticle suspensions reconstituted after a freeze-drying process. Samples correspond to compositions prepared with 90 wt% aqueous solution and an O/S ratio of 10/ 90 in the aqueous solution/polysorbate 80/medium chain triglycerides systems.
24 h of incubation with the different types of nanoparticles. Both naked polyurethane and PEG-polyurethane nanoparticles only produced around 8% decrease in viability of human endothelial cells. On the other hand lys-coated polyurea nanoparticles promoted around 14% reduction in endothelial cell viability after 24 h of incubation.
4. Discussion Two main chemical species are implicated in the formation of polyurethane and polyurea polymers by polycondensation reaction: Isocyanates and alcohols or amines [16]. Thereby, the polymerisation reaction takes place at the nano-emulsion droplet interface through the nucleophilic attack of hydroxyl or amine groups on carbonyl group of diisocyanate, leading to the disappearance of the characteristic linkage NCO of IPDI and the formation of new carbamate and urea linkages thus indicating that polyurethane and polyurea polymers are obtained [19,28,30]. In this polymerisation process, only one diisocyante group is consumed as a consequence of the reaction with the hydroxyl group to form the polyurethane [18,28,30] while two molecules of IPDI are usually required for each group of amine to obtain polyurea [30]. Nevertheless, a secondary reaction may occur in aqueous dispersions which leads to the loss of the stoichiometry during the process and to the formation of hydrolysis products, urea and CO2 [17e19,28]. For this reason, a diisocyanate monomer (IPDI) with a slow reactivity in water, and biocompatible components with hydroxyl or amine groups (polysorbate 80, PEG 200, PEG 400, lys
Table 3 Percentage of haemolysis of naked polyurethane, lys-coated polyurea and PEGpolyurethane nanoparticles. All experiments were reproduced five times with each type of formulation and data are presented as the mean of percentage of the complete haemolysis Standard Error of the Mean (sem). Formulation
Naked polyurethane PEG-polyurethane Lys-coated polyurea
Osmolality (mOsm/Kg)
Range of haemolysis (% sem) Pig red blood cells
Human red blood cells
295 291 298
0 0.50 0.11 0
0 0 0
Fig. 7. Endothelial cell biocompatibility studies of polyurethane and polyurea nanoparticles. Cell viability was evaluated by the MTS assay on HUVEC cells after 24 h of incubation with naked polyurethane, PEG-polyurethane and lys-coated polyurea nanoparticles at a concentration of 85 mg/mL. A total cell number of 7.5 103 HUVECcells/well was seeded. Samples labelled as control correspond to HUVEC cells treated with PBS. ***: p < 0.001.
and soybean oil) were carefully selected to prepare the polyurethane and polyurea nanoparticles described in this study. In this regard, it is especially important to select the composition variables and polymerisation temperature to obtain polymeric nanoparticles with a suitable particle size and polydispersity properties. Full conversion of monomer and, consequently, the sign of the completion of polycondensation reaction was corroborated by FT-IR analysis, as described in a previous study [30]. Considering the stoichiometry of the polymerisation reaction, any increment in the monomer concentration must be followed by the corresponding increase of PEG or lys concentration. In our studies, we observed that the presence of additional component with hydroxyl groups (PEG) in the formulation was not strictly necessary to obtain polyurethane nanoparticles. As a matter of fact, polyurethane nanoparticles were also obtained with the only incorporation of IPDI to the water/polysorbate 80/medium chain triglyceride system. This can be explained by the reaction between IPDI and free hydroxyl groups of polysorbate 80. Moreover, no reaction was observed among IPDI and medium chain triglyceride because this oily compound does not have reactive groups in its chemical structure [35]. Furthermore, it can be assumed that IPDI preferably reacts with surfactant hydroxyl groups rather than with water hydroxyls due to a minor energy intake is required for the chemical reaction [16]. Therefore, the surfactant would significantly contribute to the polymerisation process as previously reported [30,36]. The decrease of particle diameter (naked polyurethane < pegylatedpolyurethane (PEG-polyurethane) < lys-coated polyurea), was associated to the involvement of PEG and lys in the polymerisation reaction at the droplet interfaces since all parameters such as O/S ratio, water concentration and temperature were constant. Moreover, the chemical reaction between isocyanate groups of IPDI and hydroxyl or amine groups of PEG and lys, respectively, is enhanced due to the structural location of the reactive groups in these molecules. Altogether, these results also indicate that the molecules located at the interface determine the characteristics of nanoparticles as previously reported [18,29]. Polydispersity indexes higher than 0.20 were associated with the coexistence of two different populations of particles. For low concentrations of IPDI, the quantity of monomer is not enough to allow the polymerisation process in all droplets of nano-emulsion. Therefore, nano-emulsions and nanoparticles can be detected simultaneously by DLS. In contrast, an excess of monomer would lead to the formation of nanoparticles and large aggregates.
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
Regarding to the influence of O/S ratio, the differences observed in the particle size can only be attributed to the reaction at the droplet interfaces between IPDI and polysorbate 80, since all tested parameters involved in the polymerisation process were maintained constant and only O/S ratio was modified. Thus, a decrease in surfactant concentration is reflected with a decrease in the particle diameter. These results support the hypothesis [30] that the highly hydrophilic component and the surfactant compete for the monomer in the polymerisation process. As expected, the particle size of lys-coated polyurea systems was smaller than PEGpolyurethane samples for all O/S ratios although a higher concentration of monomer was required for their preparation, and both type of nanoparticles were smaller than naked polyurethane nanoparticles. This fact agrees with the involvement of PEG and lys in the formation of the polymeric matrix. The effect of polymerisation temperature on nanoparticle size can be explained as follows: A decrease of temperature implies a delay in the polymerisation process and consequently, the formation of polymeric matrix progressively occurs at the droplet interfaces through the reaction between IPDI and hydroxyl or amine groups of both, the surfactant and PEG 400 or lys. Since the reaction with water requires higher activation energy than the reaction with hydroxyl and amine groups, almost all of the monomer concentration would be involved in this polymerisation and a minimum reaction with water should be observed [16]. Conversely, polymerisation process takes place very quickly at high temperatures. On the one hand, the reaction between diisocyanate and water is promoted and on the other hand the collisions among droplets are increased as a consequence of temperature. Subsequently, a high number of droplets can be involved in the formation of each nanoparticle and hence, the particle diameter would rise in a short period of time. Apart from the composition parameters and temperature described above, both the use of PEG with a lower molecular weight and the incorporation of an oily component with free hydroxyl groups in their chemical structure were also found to have an effect in the particle size. Thereby, as expected and in comparison to equivalent samples prepared with PEG 400 and saturated medium chain triglycerides (PEG-polyurethane nanoparticles [30]), lower particle sizes were obtained with PEG 200 (PEG 200-polyurethane). In this regard, the measured diameter of PEG-polyurethane nanoparticles was around 70 nm [30]. Therefore, it seems that the particle size tends to decrease in agreement with the molecular weight of polyethylene glycol as previously reported [28]. On the other hand, the decrease of the particle size in the SO-PEG-polyurethane sample (soybean oil as dispersed phase) was attributed to the involvement of the hydroxyl groups of soybean oil in the polymerisation reaction. It should be noted that the replacement of the medium chain triglyceride with soybean oil did not mean any change in the mean droplet diameter of the O/W nano-emulsions before being heated at polymerisation temperature. Consequently, a polymeric matrix constituted by copolymers derived from the reaction between IPDI and hydroxyl groups of surfactant, PEG 400 and soybean oil should be taken into consideration. The stability studies have shown a slight growth in the nanoparticle size during the first 30 days that can be attributed to a swelling phenomenon. Thus, the water embedded into the polymeric matrix led to a raise in the mean particle diameter around of a 28e30% for 10/90 O/S ratios and around of a 48e60% for higher O/S ratios. This increase in the particle size was also followed by little fluctuations in the polydispersity values as detected by DLS during the first days. However, the polydispersity indexes never exceeded 0.20. Moreover, TEM images taken at 0 and 30 days allowed to corroborate the swelling phenomenon produced when nanoparticles are kept in aqueous suspension. To prevent the flocculation
6079
and the swelling phenomena produced during the storage, PEGpolyurethane and lys-coated polyurea nanoparticles were freezedried. In this regard, it has been reported that suitable concentrations of nonionic surfactant such as polysorbate 80 provide a high degree of protection against the freezing stress [37]. Therefore, no cryoprotectant which might modify the particle size, polydispersity and morphologic properties was incorporated to the formulations. The freeze-dried studies demonstrated that the particle diameter practically remained unmodified even after storing the samples for a long period. Likewise, nanoparticles showed again a swelling process when were dispersed in the aqueous medium. Therefore, the freeze-drying could be considered an effective method to preserve the integrity of nanoparticles and to avoid aggregation and the swelling phenomena during the storage. We also evaluated the haemolytic and cytotoxic effects of polymeric nanoparticles using pig and human erythrocytes and human endothelial cells, respectively. Both RBC and endothelial cells are the main cell types in contact with any pharmaceutical formulation that could be intravenously administered. Therefore the in vitro haemolysis and MTS assay in normal primary cells can adequately reflect the biological effects of nanoparticles. The results of the haemolysis assay showed that naked polyurethane, PEGpolyurethane and lys-coated polyurea nanoparticles are hemocompatible and equally suitable to be intravenously administered. Moreover naked polyurethane and PEG-polyurethane nanoparticles showed very low effects on human endothelial cell viability after 24 h of incubation thus showing a high biocompatibility as potential drug carriers. On the other hand lys-coated polyurea nanoparticles showed a significantly higher toxicity than naked and pegylated nanoparticles likely due to the cationic nature of lysine as basic amino acid. Furthermore, it is important to emphasise that the tested dispersions of naked polyurethane, PEG-polyurethane and lys-coated polyurea nanoparticles were used in non tumoral cells at a concentration similar than habitually described for other biocompatible nanoparticles [38,39] and for a period of time much higher than the residence time of any intravenous formulation thus testing the maximum toxicity effects that these nanoparticles could promote. 5. Conclusion In the present study we have designed polyurethane and polyurea nanoparticles that can be potential drug carriers owing to their small particle diameter, their formulation with biocompatible components and the low concentration of aliphatic diisocyanate required in the preparation of these nanoparticles in comparison to the rates of IPDI monomer typically used in other reports [17e19]. This means an important approach for the design of drug delivery systems in order to prevent possible toxicity problems. Thus, naked polyurethane, PEG-polyurethane and lys-coated polyurea nanoparticles with a diameter in the range of 50e90 nm, low polydispersity indexes and high kinetic stability have been obtained from selected O/W nanoemulsions systems by an interfacial polycondensation process. Composition variables such as monomer concentration, O/S ratio, the components that can be located at the droplet interface and the dispersed phase as well as the polymerisation temperature were found as critical parameters to control the formation, particle size and polydispersity properties of polyurethane and polyurea nanoparticles. In addition, these nanoparticles possesses a polymeric matrix constituted by copolymers obtained from the chemical reaction between the isocyanate groups of the monomer and the nucleophile groups of the surfactant, PEG 400 or lysine, as previously described [30]. Furthermore, although the involvement of the surfactant molecules in the polymerisation reactions occurring in nanoparticles obtained from nano-emulsion systems had been
6080
G. Morral-Ruíz et al. / Polymer 53 (2012) 6072e6080
already reported [36], for the first time, we have described the formation of polyurethane particles exclusively from the reaction between a diisocyanate and a surfactant with hydroxyl groups. This represents the first step in the formulation of new polymeric matrix with more biocompatible components to be employed as safe drug delivery systems for pharmaceutical purposes. Indeed, since we have demonstrated that these nanoparticles are biocompatible, this means that these formulations have interesting features for future design of blood-contacting applications (such as intravenous administration or endovascular devices) with a very attractive safety profile. All these data indicate that naked polyurethane, PEG-polyurethane and lys-coated polyurea nanoparticles can serve as promising biomaterials suitable for applications in complex biological media. Acknowledgement The authors wish to acknowledge the sponsorship of the Spanish Ministry of Education and Science, DGI (CTQ 2008-06892C03-02/PPQ and CTQ 2011-29336-C03/PPQ), “Generalitat de Catalunya” DURSI (Grant 2009 SGR-961), Asociación Española para el Estudio del Hígado (AEEH) and CIBER-BBN. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008e2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fundation. The authors also acknowledge Dr. Montserrat Rigol from Institut Clinic del Torax, Hospital Clinic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) for her help with the handling of blood samples. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.polymer.2012.10.039. References [1] Nagarwal RC, Kant Sh, Singh PN, Maiti P, Pandit JK. J Control Release 2009;136: 2e13. [2] Kumari A, Yadav SK, Yadav SC. Colloid Surf B 2010;75:1e18. [3] Parveen S, Misra R, Sahoo SK. Nanomed-Nanotechnol 2012;8:147e66. [4] Delmas Th, Fraichard A, Bayle PA, Teixier I, Bardet M, Baudry J, et al. J Colloid Sci Biotechnol 2012;1(1):16e25. [5] Poletto FS, Fiel LA, Lopes MV, Schaab G, Gomes AMO, Guterres SS, et al. J Colloid Sci Biotechnol 2012;1(1):89e98. [6] Soppimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE. J Control Release 2001;70:1e20.
[7] Chaubal MV. Drug Discov Today 2004;9:603e9. [8] Mora-Huertas CE, Fessi H, Elaissari A. Int J Pharm 2010;385:113e42. [9] Aboubakar M, Puisieux F, Couvreur P, Vauthier C. Int J Pharm 1999;183(1): 63e6. [10] Nair LS, Laurencin CT. Prog Polym Sci 2007;32:762e98. [11] Bonzani IC, Adhikari R, Houshyar Sh, Mayadunne R, Gunatillake P, Stevens MM. Biomaterials 2007;28(3):423e33. [12] Sarkar D, Ch. Yang J, Gupta AS, Lopina ST. J Biomed Mater Res A 2009;90(1): 263e71. [13] Laschke MW, Strohe A, Scheuer C, Eglin D, Verrier S, Alini M, et al. Acta Biomater 2009;5:1991e2001. [14] Marcos-Fernández A, Abraham GA, Valentín JL, Román JS. Polymer 2006;47: 785e98. [15] Hafeman AE, Zienkiewicz KJ, Zachman AL, Sung HJ, Nanney LB, Davidson JM, et al. Biomaterials 2011;32:419e29. [16] Ionescu M. Polyols and basic chemistry of polyurethanes. In: Ionescu M, editor. Chemistry and technology of polyols for polyurethanes. Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK: Rapra Technology; 2005. p. 1e15. [17] Tiarks F, Landfester K, Antonietti M. J Polym Sci Chem 2001;39:2520e4. [18] Barrère M, Landfester K. Macromolecules 2003;36:5119e25. [19] Zanetti-Ramos BG, Lemos-Senna E, Soldi V, Borsali R, Cloutet E, Cramail H. Polymer 2006;47:8080e7. [20] Gaudin F, Sintes-Zydowicz N. Colloid Surf A 2008;331:133e42. [21] Landfester K. Adv Mater 2001;13:765e8. [22] Landfester K, Tiarks F, Hentze HP, Antonietti M. Macromol Chem Phys 2000; 201:1e5. [23] Anton N, Benoit JP, Saulnier P. J Control Release 2008;128:185e99. [24] Solans C, Izquierdo P, Nolla J, Azemar N, Garcia-celma MJ. Curr Opin Colloid Interfase Sci 2005;10:102e11. [25] Calderó G, García-Celma MJ, Solans CJ. Colloid Interface Sci 2011;353:406e11. [26] Johnsen HMS, Schmid RB. J Microencapsul 2007;24:731e42. [27] Torini L, Argillier JF, Zydowicz N. Macromolecules 2005;38:3225e36. [28] Bouchemal K, Briançon S, Perrier E, Fessi H, Bonnet I, Zydowicz N. Int J Pharm 2004;269:89e100. [29] Zanetti-Ramos BG, Lemos-Senna E, Cramail H, Cloutet E, Borsali R, Soldi V. Mat Sci Eng C 2008;28(4):526e31. [30] Morral-Ruíz G, Solans C, García ML, García-Celma MJ. Langmuir 2012;28(15): 6256e64. [31] Chen C, Cheng YC, Yu CH, Chan SW, Cheung MK, Yu PHF. J Biomed Mater Res A 2008;87:290e8. [32] Aparicio RM, García-Celma MJ, Vinardell MP, Mitjans M. J Pharm Biomed 2005;39:1063e7. [33] Guide to the preparation, use and quality assurance of blood components. 6th ed. Strasbourg Cedex Germany: Council of Europe Publishing; 2000. [34] US License NumberFDA summary basis of approval of red blood cells frozen and red blood cells deglycerolized, reference no. 86-0335, applicant. Bethesda, MD: Department of the Navy, Naval Hospital; 1986. p. 635e10. [35] Rowe RC, Sheskey PJ, Weller P, editors. Handbook of pharmaceutical excipients. 6th ed. London, UK, Washington, USA: J. Pharmaceutical Press: & American Pharmaceutical Association; 2009. p. 429. [36] Rosenbauer EM, Landfester K, Musyanovych A. Langmuir 2009;25(20): 12084e91. [37] Hillgren A, Lindgren J, Aldén M. Int J Pharm 2002;237:57e69. [38] Wadajkar AS, Bhavsar Z, Ko Ch Y, Koppolu B, Cui W, Tang L, et al. Acta Biomater 2012;8(8):2996e3004. [39] Kim Se, Seong K, Kim O, Kim S, Seo H, Lee M, et al. Biomacromolecules 2010; 11:555e60.