Biomaterials 31 (2010) 6069e6074
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Artificial oxygen carrier based on polysaccharidesepoly(alkylcyanoacrylates) nanoparticle templates Cédric Chauvierre a,1, *, Romila Manchanda b,1, Denis Labarre c, Christine Vauthier c, Michael C. Marden a, Liliane Leclerc a a
Institut National de la Santé et de la Recherche Médicale, INSERM U779, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre, France Biomedical engineering Department EC, 10555 West Flagler Street, Florida International University, Miami, FL 33174, USA Laboratoire de Physico-Chimie, Pharmacotechnie et Biopharmacie, UMR CNRS 8612, Faculté de Pharmacie, Université Paris XI, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cedex, France
b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2010 Accepted 21 April 2010 Available online 20 May 2010
Biomimetic nanoparticles based on polysaccharidesepoly(alkylcyanoacrylates) copolymers were initially developed in view of drug delivery. Core-shell nanoparticles covered with a sufficiently long brush of polysaccharides were shown to be very low complement activators and have the potential for long circulation times in the bloodstream. Such nanoparticles bearing haemoglobin were envisaged as potential red cell substitutes. Different core-shell nanoparticles with a brush shell made of dextran, dextran-sulphate, or heparin were prepared and haemoglobin (Hb) could be adsorbed on their surface. Benzene tetracarboxylic acid (BTCA) was used as a coupling agent for Hb to dextran-coated nanoparticles; the Hb loading capacity of the dextran nanoparticles showed a 9.3 fold increased. The coupled Hb maintained the allosteric properties of free Hb. While modification of nanoparticles by BTCA slightly increased complement activation, the further addition of Hb totally reversed this effect providing Hbloaded nanoparticles with a very low level of complement activation. Such nanoparticles could be a suitable alternative to haemoglobin solutions in the development of a blood substitute. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Nanoparticles Poly(alkylcyanoacrylates) Polysaccharides Benzene-1,2,4,5-tetracarboxylic acid Haemoglobin Oxygen carrier
1. Introduction In the last decade, major improvements have been obtained in the in vivo drug targeting field with the appearance of new generations of synthetic vectors. Nanoparticles endowed with a prolonged lifetime in the bloodstream and targeting properties have been described [1,2]. This remarkable breakthrough is recognized as a consequence of a new design of the nanoparticle surfaces. Members of the polyethylene glycol (PEG) family were leaders in this new concept, but more recently polysaccharides have been used successfully in order to mimic certain properties of the living cell surfaces [3]. In parallel, synthetic vectors were developed from alkylcyanoacrylate monomers, initially used as surgical glue, taking advantage of the in vivo biodegradability of the polymer and of its good compatibility with living tissues [4]. Thus, a new generation of poly(alkylcyanoacrylates) (PACA) nanoparticle coated with a polysaccharide brush was obtained by using an original redox radical polymerization mechanism in aqueous medium [5]. With such
* Corresponding author. Tel.: þ33 1 49 59 56 60; fax: þ33 1 49 59 56 61. E-mail address:
[email protected] (C. Chauvierre). 1 Equal contribution. 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.04.039
nanoparticles a very weak activation of the complement system could be obtained [6,7]. Due to the lower complement activation, a lower uptake by macrophages of the mononuclear phagocyte system, and an extended blood circulation time, can be expected [8]. In the prospect of developing an artificial oxygen carrier, this last generation of nanoparticles should already fulfill the absolute requirement of a long lifetime in the blood circulation. In addition the external layer of polysaccharides allowed us to develop a new strategy based on the binding of haemoglobin (Hb) at the surface of the nanoparticles. The easy access to Hb could lead to a great improvement concerning its oxygen delivery function, compared to previous studies describing loading of Hb into the carriers [9]. In a first attempt, heparinepoly(isobutylcyanoacrylates) (HepePIBCA) copolymer nanoparticles were prepared and successfully loaded with Hb [10]. Interestingly, the tolerance of these nanoparticles towards a cell line was highly improved by the presence of Hb [11]. In addition, the anticoagulant properties of free heparin were preserved in HepePIBCA nanoparticles, even in the presence of Hb. This makes such Hb-loaded heparin-nanoparticles particularly promising against vascular occlusive pathologies. However the clinical use of such a blood substitute requires additional progress to obtain a higher quantity of functional Hb in order to design an oxygen carrier able to satisfy the general blood transfusion requirements.
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Other dextran-based polysaccharides were investigated in order to increase Hb loading and to keep a low complement activation capacity without impairing Hb functionality. Dextrans are widely used in medical applications, for instance as constituents of plasma expanders. A large choice of sizes is available and dextrans can be modified by different adducts, as already shown in the case of soluble dextran [12]. As introduced above, nanoparticles endowed with a very low complement activating capacity could be spontaneously obtained from dextranePIBCA block copolymers, provided that dextran was present on the surface as a brush of sufficient length [7,13]. In the present paper, we report modification of dextranePIBCA nanoparticles by benzene tetracarboxylic acid (BTCA) in order to increase binding of Hb, while maintaining both a low activation of complement and correct functionality of Hb, all properties required for a useful oxygen carrier. 2. Experiment section 2.1. Materials Dextran of molecular weight 70 kDa and 20 kDa, 10 kDa and 40 kDa dextransulphate, heparin 170 USP, benzene-1,2,4,5-tetracarboxylic acid (BTCA), and NEthyl-N0 -(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) were purchased from Sigma (St Louis, USA). Other chemicals were of the highest purified grade. Haemoglobin was obtained from outdated red blood cells from Etablissement Français du Sang (Hôpital Bicêtre, Le Kremlin-Bicêtre, France). After haemolysis and purification according to the method of Williams and Tsay [14], the haemoglobin solution was kept frozen at 80 C until use. Isobutylcyanoacrylate (IBCA) monomer was a gift from LoctiteÒ (Dublin, Ireland). 2.2. Nanoparticle preparation The same amount by weight of 20 or 70 kDa dextran, 10 or 40 kDa dextransulphate, or heparin 170 USP was added to 500 ml of isobutylcyanoacrylate monomer and left for polymerization according to Chauvierre et al. [5]. 2.3. Post synthesis polysaccharide modification of nanoparticles BTCA was grafted to 70 kDa dextran-coated PIBCA nanoparticles according to the method of Prouchayret et al. which increases the surface negative charge by the linker [12]. 2 ml of the nanoparticle suspension in water were treated with BTCA stabilised at pH 9 by addition of 1 M NaOH and left 1 h at room temperature for optimal reaction. The reaction was stopped by restoring pH 7 by addition of 6 M HCl. BTCA treated nanoparticles were then dialysed twice for 1 h against 150 mM NaCl, and then overnight with water, and finally for 2 h with water, to remove free BTCA. 2.4. Quantification of BTCA bound to nanoparticles
batch. The concentration of the different samples of nanoparticles collected after elution from the chromatography column was estimated by turbidimetry at 700 nm, to avoid haemoglobin interference. For each sample, the initial suspension was used. 2.8. Physical characterization of the nanoparticles Size and surface charge (zeta potential) were measured as previously described [6], using a dynamic light scattering instrument (Nano ZS, Malvern, UK). 2.9. Evaluation of complement activation by 2D immunoelectrophoresis of C3 The percentage of activation of C3 complement component induced by different nanoparticles was evaluated in human serum by comparative measurements of C3 cleavage, as previously described [7,16]. Practically, human serum was obtained after calcifying plasma from healthy donors and stored at 80 C until use. Veronalbuffered saline (VBS) was prepared as previously described [7]. To ensure a valid comparison of the different nanoparticles, sample volumes with equal surface area of hydrated nanoparticles (1000 cm2) were incubated under gentle agitation for 1 h at 37 C with 100 ml of human serum diluted 1:4 in VBS containing 0.15 mM Ca2þ and 0.5 mM Mg2þ ions (VBS2þ). The nanoparticle surface area was calculated from the average hydrodynamic diameters according to Vittaz et al. [17], with a value of 1.1 g cm3 for copolymer density [18]. The two dimensional immunoelectrophoresis of the sample has been previously described [6]. Nanoparticles prepared by the same method but with 15 kDa dextran were used as a positive control [7]. Each nanoparticle type was analyzed 5 times across the whole process. The height, the area, and the weight of the peaks on the immunoelectrophoretic plate corresponding to C3 and C3b respectively were measured 10 times. The height of the peaks was determined by hand on the 800 scanned immunoelectrophoretic plate with Adobe Acrobat 9 pro software. The same method was done to evaluate the area of the peaks but using the area under curve function of the software. The weight of the peaks was measured manually by cutting the peaks on paper sheet and weighing by using a precision balance (Mettler AT261). The results of the complement activation given by each type of nanoparticle were expressed as a percentage of C3 and C3b detected on the plate and compared to the controls. 2.10. Functional analysis of haemoglobin Hb function was tested as described previously [19] using a laser flash photolysis method. Spectroscopic measurements performed on nanoparticle suspensions were adapted for the turbidity inherent to this specific material. With respect to a sufficient Hb concentration, the optical path could be reduced to 1 mm instead of the 1 cm cells length generally used. Results are the mean of a high number of iterative measurements on the same sample. Despite the increase in noise level, the kinetic signal is not perturbed, since it corresponds to a change in absorption.
3. Results and discussion 3.1. BTCA loading BTCA (insert of Fig. 1) is a highly reactive compound capable of binding to dextran and presents acidic residues able to react with
The amount of non reacted BTCA was estimated spectrophotometrically at 215 nm (e ¼ 2.44 104 M1 cm1) in the dialysis medium brought to pH 9 by addition of 1 M NaOH. The amount of BTCA bound to 70 kDa dextran nanoparticles was calculated by subtracting the non reacted BTCA from the initial amount added. 2.5. BTCA working concentration determination The optimum binding concentration of BTCA on 70 kDa dextran nanoparticles was determined by adding increasing amounts of BTCA to the nanoparticle suspension. The value of 14 mg BTCA/ml of 70 kDa dextran nanoparticles was chosen as a reference in all subsequent experiments. 2.6. Haemoglobin (Hb) loading and concentration determination As already reported an overload of Hb (5 mg) was added to 1 ml of the nanoparticle suspension in water and allowed to react for at least 3 h at 4 C [10]. When using the BTCA adduct with the nanoparticles, 2 mg of EDAC were added to the mixture. The coupling reaction mechanism between BTCA and Hb is based on the well known covalent linkage between amine groups of Hb and the carboxylic groups of BTCA via a water soluble carbodiimide (EDAC). Methods to isolate Hb loaded nanoparticles and to measure the amount of Hb bound were previously detailed [10,15]. 2.7. Nanoparticle concentration To estimate the initial nanoparticle concentration, three samples of 100 ml of the nanoparticle suspensions in water were freeze-dried and weighed. The mean of the three samples was used as a reference for all subsequent measurements with the same
Fig. 1. Effect of BTCA concentration on the zeta potential (solid line with black circles) and Hb loading (dotted line with white circles) values of 70 kDa dextranePIBCA nanoparticles. Hb loading expressed in mg of Hb per g of polymer nanoparticles. The mass concentration of the suspensions (mg/ml) was 16.3 5.4 (n ¼ 17). Insert: Chemical structure of benzene-1,2,4,5-tetracarboxylic acid (BTCA). Zeta potential and Hb loading measurements were done in triplicate.
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20 kDa dextran, or of negatively charged dextran sulphates, i.e., 10 kDa dextran-sulphate and 40 kDa dextran-sulphate, or of heparin (12 kDa), or of a 50e50 blend of heparin and 70 kDa dextran and finally 70 kDa DextraneBTCA. The size of the unmodified nanoparticles was not significantly changed by Hb adsorption according to the standard deviation of the size distribution. The hydrodynamic diameter varied from about 300 nm for 70 kDa dextran- to 100 nm for heparin-coated PIBCA nanoparticles. The size of the dextranePIBCA nanoparticles did not change significantly with the addition of BTCA alone (around 300 nm) as expected based on its low molecular weight (218 g/mol), whereas Hb binding resulted in a decreased size (around 24%) of the nanoparticles modified with BTCA. This phenomenon could be explained by a shrinking of the polysaccharide brush around the high quantity (see section 3.2.3) of bound Hb [20]. 3.2.2. Zeta potential effect As already reported the absolute value of the zeta potential of the unmodified nanoparticles increases with anionic polysaccharides [6] (Fig. 2B) but also if negatively charged chemical compounds such as BTCA were grafted onto the polysaccharidic brush of the nanoparticles (Fig. 1). The Zeta potential of the unmodified nanoparticles varied from 10 mV for nanoparticles bearing 70 kDa dextran to 47 mV for heparinePIBCA nanoparticles, and the zeta potential of BTCA modified dextran nanoparticles reached 31 mV. As expected, Hb binding resulted in a decreased absolute value of the zeta potential (Fig. 2B). This decrease expressed as a percentage of the initial charge (27%) was quite similar for unmodified nanoparticles; however, the decrease was much larger (2 fold) in the case of the nanoparticles modified with BTCA. This phenomenon can be explained by the higher quantity of bound Hb onto these nanoparticles (see next section). Fig. 2. Influence of the type of polysaccharides and of binding of BTCA on the size (A) and on the zeta potential values (B) of the polysaccharidesePIBCA nanoparticles, before (black columns) and after loading of haemoglobin (grey columns). All suspensions were homogeneous [polydispersity index (PI) < 0.1] except for those involving heparin (PI > 0.1). The number of samples varied from 5 to 20.
Hb. It has been extensively studied by Prouchayret et al. [12] in the prospect of Hb-based soluble blood substitute. Moreover, according to these authors, BTCA adduct may occupy the 2,3 diphosphoglycerate (2,3 DPG) crevice in the Hb molecule and play the same role as the natural affinity modulator of Hb. BTCA binding reaction with 70 kDa dextran nanoparticles (at 14 mg/ml) yield an effective association of 1.3 0.6 mg BTCA/ml of suspension (n ¼ 14). As BTCA nanoparticles weigh 16.3 5.2 mg/ml of suspension (n ¼ 17) the binding ratio approaches 1:13. Since the percentage of 70 kDa dextran is 22 1% (w/w) the BTCA:Dextran (w/w) binding reaction is approximately 1:3. It follows that one molecule of BTCA roughly corresponds to 4 glucose subunits, indicating a possible saturation of the exposed polysaccharides. Treatment of 70 kDa dextran nanoparticles with BTCA led to an increase in the magnitude of the zeta potential (more negative charge) as well as an increase of Hb binding (Fig. 1). Plateau values of zeta potential (31 mV) and Hb loading (214 mg of Hb/g of polymer nanoparticles) were reached corresponding, respectively, to a 3 and 9.3 fold increase relative to untreated nanoparticles (Figs. 2B and 3). 3.2. Hb binding 3.2.1. Size effect As already reported the size of the unmodified nanoparticles is dependent on the type and molecular weight of the polysaccharide used [6] (Fig. 2A). The shell of the nanoparticle was composed either of neutral dextran of different lengths, i.e. 70 kDa dextran,
3.2.3. Hb loading HeparinePIBCA nanoparticles have been shown to spontaneously bind Hb with a high efficiency [10]. Heparin is a very negatively charged polysaccharides, explaining the highly negative zeta potential measured for the surface of heparinePIBCA nanoparticles and surface charge was hypothesized to be the driving force to load Hb on nanoparticles. Zeta potentials of the nanoparticles and their Hb loading have been summarized in Fig. 3. As the various suspensions did not have the same mass of nanoparticles, it was of interest to consider the amount of Hb bound per g of polymer nanoparticles. Despite some discrepancies, the average amount of bound Hb (from 23 to 84 mg/g for 70 kDa dextran- and heparinePIBCA nanoparticles respectively) fits rather well with the charge surface. Our hypothesis is even further confirmed by the high degree of Hb binding (214 mg/g) on BTCA modified nanoparticles. So, the ratio of the quantity of Hb was increased 9.3 and 2.5 fold, compared to 70 kDa dextran and heparin-coated nanoparticles respectively. The difference in the mass fraction of Hb per nanoparticle is an important parameter for blood substitutes since Hb is the only active mass in terms of oxygen transport. Binding of Hb to the polysaccharides was strong enough to resist to physiological molarities like those of the elution buffer (150 mM Tris-acetate) and could not account for weak electrostatic or van der Waals bonds. Haemoglobin has long been known to bind glucose units leading to the genesis of glycated haemoglobin. This non enzymatic process, now well understood has been generalized to other proteins such as serum albumin or a-cristallin [21]. It consists of a two step mechanism. The first step is a fast and reversible formation of a Schiff base between a free amino group exposed at the surface of the protein (N-terminus valine of the b-globine chain for example) and a glucose residue, most likely the aldehyde
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VBS2þ VBSeEDTA Dextran 15 Dextran 70 Dextran 70 Dextran 70 Dextran 70
kDa kDa kDa þ Hb kDa þ BTCA kDa þ BTCA þ Hb
C3b/(C3þC3b) ratios Height
Area
Weight
0.14 0.18 0.89 0.21 0.25 0.38 0.16
0.18 0.18 0.93 0.20 0.27 0.38 0.17
0.17 0.20 0.93 0.22 0.28 0.39 0.16
Strength of activation
Spontaneous Negative (control) Strong (reference) Weak Weak Low Near spontaneous
C3 activation after 1 h incubation of 1000 cm2 samples with 100 ml of human serum diluted 1:4 in VBS2þ at 37 C, as measured by 2-D immunoelectrophoresis and expressed as C3b/(C3 þ C3b) ratios (n ¼ 5). The height, area and weight of the C3 and C3b peaks were determined 10 times on each nanoparticle type. The average of these values was used to determine the corresponding C3b/(C3 þ C3b) ratios, which have a typical error of 0.10.
Fig. 3. Influence of the type of polysaccharides and BTCA binding on zeta potential values of the nanoparticles (grey columns) and on Hb loading (black points). Hb loading expressed in mg of Hb per g of polymer nanoparticles. The mass concentration of the suspensions (mg/ml) were: 16 5 for 70 kDa DextraneBTCA coated nanoparticles, 30 7 for Heparin coated nanoparticles and 35 5 for the other polysaccharide coated nanoparticles. The number of samples varied from 5 to 20.
function in C1. It is followed by a second step which consists of an irreversible Amadori rearrangement of the sugar leading to the formation of a ketoamine residue [22]. Even though we cannot strictly relate this mechanism as the only one leading to the binding of Hb to the nanoparticles it could be assumed that a few sites of the polysaccharides would be free for reacting with the amine residues of the protein. Dextran used in the present work was extracted from Leuconostoc mesenteriodes and, according to the supplier, is mainly a linear a-D-1,6-glucose-linked glucan. The degree of branching is low, approximately 5%, with short branches of mostly 1e2 glucose units long and gives 10 nmoles and 34 nmoles of side chains for respectively 70 kDa and 20 kDa dextrans fitting very well with the 12.5 nmoles and 30.7 nmoles of Hb bond respectively to the corresponding polysaccharides. 1 ml of 70 kDa dextran nanoparticles (16 mg) accommodated 5.7 mmole of BTCA and 53 nmole of Hb. Expressed as binding sites, around 1% of the BTCA sites are occupied with Hb; there is apparently some room for improvement, which probably depends on the steric hindrance of the Hb binding. Actually a simple increase in the nanoparticle concentration (pseudo haematocrit), from 1.5% to the physiological red blood cell level of 45%, would bring the Hb level to about 10 g % which is in the physiological range. It is still of interest to increase the amount of Hb relative to the other components, especially the poly(alkylcyanoacrylates), in the purpose of applying these nanoparticles as an artificial oxygen transporter.
Binding of BTCA led to a slight increase of the level of complement activation. This could be due to a slight crosslinking of the dextran chains in the nanoparticle corona. In fact, crosslinked dextran, i.e., SephadexÒ, has been shown to be a strong activator of complement [24]. In contrast, the binding of Hb on the BTCAmodified nanoparticles reduced the complement activation capacity to a very low level similar to that obtained with the VBS2þ reference evaluating the level of spontaneous activation in the experimental conditions used in this work. Apparently an exterior layer of Hb provides one of the most inert surfaces for the nanoparticles. These results were unexpected regarding possible effects related to the molar weights of Hb and BTCA, assuming a larger modification of surface of 70 kDa dextran nanoparticles by the Hb
3.3. Complement activation It was previously shown that complement activation was very low when nanoparticles covered with a brush of 70 kDa dextran were dispersed in serum in the presence of divalent cations [6,7,23]. As complement activation is an amplified process, small variations in the surface structure of nanoparticles can result in striking differences in complement activation capacity. Thus it was very important to assess the complement activating capacities of the modified nanoparticles. The degree of complement activation induced by the nanoparticles after incubation in diluted serum containing calcium and magnesium ions was evaluated by conversion of C3 into C3b and evidenced by 2-D immunoelectrophoresis. The results are given in Table 1 and illustrated in Fig. 4. The presence of Hb on these nanoparticles did not modify the low level of complement activation.
Fig. 4. Quantification of the activation of complement. Sample volumes with equivalent surface area (1000 cm2) of nanoparticles were incubated for 1 h at 37 C with human serum diluted 1:4 in VBS2þ. Then each sample was subjected to 2-D immunoelectrophoresis of C3 antigens. The start of migration is indicated by the arrows. The relative amounts of C3 and C3b are determined from the first and the second peak respectively. Nanoparticle samples are Dextran 70 kDa (I), Dextran 70 kDa þ HbA (II), Dextran 70 kDa þ BTCA (III) and Dextran 70 kDa þ BTCA þ HbA (IV). The number of samples was from 4 to 6.
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nanoparticle coated with heparin, the amount of Hb loaded is increased 2.7 fold [10]. Modification of the shell by BTCA increased the complement activating capacity relative to unmodified nanoparticles; however, the presence of Hb on the BTCA-modified nanoparticles reversed the effect on the complement activation, which returned to the level of spontaneous activation evaluated in the absence of nanoparticles. While the surface charge may be the driving force in the adsorption of Hb to polysaccharides, use of BTCA as linker offers specific binding sites to greatly increase the overall Hb loading. Finally, Hb bound to the nanoparticles via BTCA behaved like free Hb indicating a well preserved function for exchanging oxygen. Such nanoparticles meet the different aspects of the necessary requirements to be further developed as a blood substitute. Acknowledgements
Fig. 5. Flash kinetics at different laser energies, to modify the fraction of hemes photodissociated. The fraction dissociated is approximately 40, 20, 10, and 5% (top to bottom); curves are normalized to correct for the observed change in absorbance. As for reference Hb in solution (not shown), at higher energies, there is a higher amplitude for the slow kinetic phase, which corresponds to the deoxy (T-state) conformation of Hb tetramers. This indicates that the Hb attached to the nanoparticles is capable of switching between the classical R and T states. Flash photolysis measurements were done at least 10 times.
larger molecule (64 kDa). As Hb is immunologically neutral regarding complement activation, its presence on the nanoparticle surface could mask the undesirable effects caused by BTCA modifications. 3.4. Hb function A typical flash photolysis experiment of Hb bound to BTCA modified nanoparticles is shown in Fig. 5. After photo-dissociation, the CO recombination displayed two kinetic phases, characteristic of the R and T allosteric states. Note that the slow phase is unique to Hb tetramers in the deoxy (T-state) conformation. The fraction of slow phase is dependent on the level of photo-dissociation (Fig. 5), as for reference Hb samples (not shown), indicating that the Hb within the nanoparticles is capable of switching between the R and T state conformations. Previous results for nanoparticles without linkers [10] showed less variation versus laser energy, indicating some difficulty for the bound Hb to make the allosteric transition. This was not the case for the present results, and Hb bound to the nanoparticles via BTCA behaved like free Hb indicating a well preserved function for binding and releasing oxygen. Generally the equilibrium and kinetic (flash photolysis) methods are in agreement for modifications of Hb function considering a large number of mutations or effectors [25]. The improvement of Hb function, relative to non-linked Hb, is possibly due to a structural effect where the linker allows the protein to protrude out of the polysaccharide chains. 4. Conclusions A new type of potential oxygen carrier based on nanoparticle coated with a brush shell of dextran has been obtained; modification of the shell by BTCA enhances haemoglobin binding and improves the allosteric ligand binding properties of the bound Hb. When compared to unmodified nanoparticle coated with dextran, the amount of Hb loaded is increased 9.3 fold. Moreover, when compared to our previous “best” loading obtained with
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