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Polyester Nanoparticles Presenting Mannose Residues: Toward the Development of New Vaccine Delivery Systems Combining Biodegradability and Targeting Properties Jutta Rieger,*,†,‡,§ He´le`ne Freichels,§ Anne Imberty,‡ Jean-Luc Putaux,‡ Thierry Delair,| Christine Je´roˆme,§ and Rachel Auze´ly-Velty*,‡ Centre de Recherches sur les Macromole´cules Ve´ge´tales (CERMAV-CNRS), BP53, 38041 Grenoble cedex 9, France, Center for Education and Research on Macromolecules (CERM), University of Lie`ge, Sart-Tilman B6, B-4000 Lie`ge, Belgium, and Laboratoire des Mate´riaux Polyme`res et Biomate´riaux (UMR 5223), Universite´ Claude Bernard Lyon 1, 15 Boulevard Latarjet, 69622 Villeurbanne, France Received December 23, 2008
We report the synthesis of fully biodegradable polymeric nanoparticles presenting mannose residues at their surface and their interaction with lectins. A simple and versatile method was used to reach the surface functionalization of poly(D,L-lactic acid) (PLA) nanoparticles by mannose moieties: It consists in using an amphiphilic mannosylated poly(ethylene oxide)-b-poly(-caprolactone) (PEO-b-PCL) diblock copolymer as a bioresorbable surface modifier in a simple nanoprecipitation-evaporation procedure. The size and zeta potential of the nanoparticles were found to depend on the molar copolymer/PLA ratio, demonstrating the influence of the copolymer on the formation of the nanoparticles. The bioavailability of the mannose residues as specific recognition sites on the nanoparticle surface could be demonstrated by a modified enzyme-linked lectin assay (ELLA) using biotin-labeled lectins which interact specifically with R-D-mannopyrannoside derivatives. Besides specific interaction by lectin-mannose complex formation, nonspecific adsorption of the proteins on the nanoparticle surface was observed. These results were fully supported by isothermal titration calorimetry experiments which suggested that the balance between specific and nonspecific interactions can be controlled by the amount of glycosylated polymer used for the preparation of the nanoparticles. Such nanoparticles are expected to be specifically recognized by mannose receptors, which are highly expressed in cells of the immune system. The targeting properties of these carrier systems combined with their potential adjuvant effects due to their size in the range of 200-300 nm make them attractive candidates as vaccine delivery systems.
Introduction Over the past decade, polymeric nanoparticles, which can be defined as colloidal particles with a size ranging from 50 to 1000 nm, have received increasing interest both for drug delivery purposes and as diagnostic tools.1,2 The application of nanoparticles (NPs) as drug carriers is of particular interest because of numerous advantages: they can be easily prepared and purified and present good stability during storage and administration.3 Among the materials used for the preparation of such colloidal carrier systems, poly(D,L-lactide) (PLA) and poly(-caprolactone) (PCL) are synthetic polymers of special interest due to their unique properties of biocompatibility and biodegradability. In fact, they are readily hydrolyzed to nontoxic metabolites in physiological conditions. Moreover, the surface properties of NPs can be tuned to achieve biological activity and compatibility.4 For instance, NPs can readily be coated with poly(ethylene oxide) (PEO) using copolymers containing PEO blocks. PEO coatings are known to reduce the uptake of NPs by the reticular endothelial system * To whom correspondence should be addressed. E-mail: rachel.auzely@ cermav.cnrs.fr (R.A.-V.);
[email protected] (J.R.). † Present address: Laboratoire de Chimie des Polyme`res, Universite´ Pierre et Marie Curie-Paris 6, CNRS-UMR 7610, 4 Place Jussieu, 75252 Paris Cedex 05, France. ‡ CERMAV-CNRS, affiliated with Universite´ Joseph Fourier, and member of the Institut de Chimie Mole´culaire de Grenoble. § CERM. | UMR 5223.
(RES) and thereby prolong their circulation in the blood stream.5-8 Similar to PLA and PCL, it has been approved by the Food and Drug Administration (FDA) for biomedical applications, as it is nontoxic and bioresorbable for a molecular weight lower than 20000 g/mol.9 Thus, fully biodegradable and stealth NPs have been prepared from diblock copolymers of poly(ethylene glycol) and poly(lactide), that is, PEO-b-PLA.10,11 However, the main drawback of such carriers is their nonspecific interaction with cells and proteins, leading to drug accumulation in nontarget tissues.11,12 To overcome this problem, recent studies aimed at functionalizing the surface of PLA NPs with specific ligands such as peptides, RNA-aptamers, antibodies, or carbohydrates to achieve specific targeting.13-15 In spite of the promising targeting properties of PLA NPs bearing monosaccharides as recognition signals, only few examples have been reported in the literature. Cho et al. prepared PLA NPs stabilized by a synthetic nondegradable polymer possessing galactose residues. These particles were shown to be specifically recognized and internalized by hepatocytes through the receptormediated mechanism.14,16,17 In this context, the aim of this work was to design fully degradable NPs coated by mannose ligands as targeting sites of mannose-receptors, which are highly expressed in cells of the immune system. Actually, mannosecoated submicron liposomes have been identified as potential vaccine carriers that may specifically target human blood dendritic cells (DCs), which express mannose receptors on their surface.18,19 These cells are effective in capturing, processing, and presenting antigens to native T cells, thus initiating cellular
10.1021/bm801492c CCC: $40.75 2009 American Chemical Society Published on Web 02/09/2009
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Figure 1. Scheme for the multistep reaction allowing the qualitative detection of particle-fixed mannose with biotin-labeled lectins.
immune response.20 They have as such been identified as a potent target for vaccine delivery to initiate adaptive immune responses.21 Besides liposomes, mannosylated polymeric NPs thus appear as interesting candidates for the development of new generations of vaccines.22 It has been shown that NPs exhibit “adjuvant” properties, that is, they act in a nonspecific manner to increase the specific immunity to an antigen.23,24 This enhanced immune response was clearly attributed to the “large” size of polymeric NPs and is not observed for soluble systems such as micelles.25 Based on these considerations, our approach for the synthesis of mannose-targeted NPs as potential vaccination vectors relied on the use of fully biocompatible and biodegradable materials, consisting of PLA and an amphiphilic mannosylated poly(ethylene oxide)-b-poly(-caprolactone) (PEOb-PCL) diblock copolymer. This copolymer was expected to simultaneously act as a stabilizer and a surface coating for the preparation of PLA nanoparticles. The synthesis of those mannosylated copolymers and their nonfunctionalized precursors has been reported recently, and due to their interfacial activity26 they seem suitable as biodegradable surface-active materials for the preparation of polymeric NPs by a simple coprecipitation procedure. To the best of our knowledge, such fully biodegradable/bioresorbable NPs coated with mannose residues have never been described before. In this paper, we show that the use of an amphiphilic mannosylated block copolymer may be a versatile approach to tune the surface properties of PLA NPs and especially their specific interaction with mannose-binding lectins, as demonstrated by a modified enzyme-linked lectin assay and isothermal titration calorimetry experiments.
Experimental Section Materials. R-N,N-Dimethylaminoethyl poly(ethylene oxide)-bpoly(-caprolactone) diblock copolymer (Me2N-PEO-b-PCL, referred to as copolymer 1 in Table 1) was synthesized by anionic polymerization of ethylene oxide initiated from N,N-dimethylaminoethyl alkoxide, followed by the ring opening polymerization of -caprolactone with the corresponding AlEt3 alkoxide. R-Diethylacetal-poly(ethylene oxide)b-poly(-caprolactone) (acetal-PEO-b-PCL, or copolymer 4 in Table 1) was obtained accordingly by anionic polymerization of ethylene oxide initiated from 3,3-diethoxy-1-propanyl alkoxide. The mannosylated amphiphilic PEO-b-PCL copolymer, Man-N+-PEO-b-PCL (copolymer 2 in Table 1) was synthesized from Me2N-PEO-b-PCL and 2-bromoethyl-R-D-mannopyranoside (ManOH-Br) as reported in the literature,26 where ManOH-Br was obtained in two steps from 1,2,3,4,6-penta-Oacetyl-D-mannopyranose by reaction with 2-bromoethanol. The endfunctionalization of the copolymer by mannose determined by 1H NMR spectroscopy was 80%. The number average molecular weight determined by 1H NMR, Mn, NMR was ) 8600 g/mol (with 5470 g/mol and 2830 g/mol for the PEO block and the PCL block, respectively), and the polydispersity index, Mw/Mn, determined by size exclusion chromatography, was 1.19. The nonmannosylated quarternized copolymer, Me3N+-PEO-b-PCL (copolymer 3 in Table 1), was prepared from Me2N-PEO-b-PCL by reaction with 5 mol equiv of iodomethane. Preparation of Nanoparticles. NPs were prepared by using a nanoprecipitation-evaporation procedure as previously described.27,28
Table 1. List of PEO-b-PCL Diblock Copolymers Used for the Preparation of NPs
Briefly, 0.2 g of PLA (BioMe´rieux, Mn ) 32650 g/mol, Mw/Mn ∼ 1.5) and increasing amounts of amphiphilic copolymer (0.09, 0.46, 1, 1.37, and 10 mol equiv with respect to PLA) were dissolved in 10 mL of acetone (Elvetec, 99%). The mixtures were stirred for 1 h to ensure complete dissolution of the polymers and then added dropwise to 7 mL of MilliQ water under slight stirring (250 rpm). Acetone was removed by evaporation under vacuum at room temperature to yield NP dispersions with final solids contents in the range of 3-5% (0.3-0.5 g of polymer in 10 g of NPs dispersion). The nanodispersions were stored at 4 °C. For the recognition assays with biotin-labeled lectins (referred to as modified enzyme-linked lectin assay) and isothermal titration calorimetry experiments, the NPs dispersions were diluted with MilliQ water and Tris-(hydroxymethyl) aminomethane buffer (1 M TrisHCl, 0.3 mM CaCl2, pH ) 7.5) to reach final concentration of 0.1 M Tris and 0.03 mM CaCl2. Knowing the NP concentration and their composition, the mannose content in the different experiments could be calculated. Dynamic Light Scattering (DLS). The hydrodynamic diameter (DH) and the particle size distribution (PDI) of the nanoparticles were determined at 25 °C by quasi-elastic light scattering measurements using the cumulant method and a Malvern Zetasizer NanoZS from Malvern instruments (U.K.). Each value was the average of at least five measurements. A polydispersity index of 0.05 denotes a monodisperse colloid, whereas above 0.5, the dispersion is broadly distributed.29 Zeta Potential Measurements. Particle surface characterization was performed by measuring the electrophoretic mobilities (3 × 5 measurements) by laser Doppler anemometry using a Malvern Zetasizer NanoZS (Malvern Instrument, U.K.). The conversion to zeta potentials uses the Smoluchowski relation ζ ) µe × η/ε0εr, where µe is the electrophoretic mobility, 0 and r are, respectively, the permittivity of the vacuum and the relative permittivity of the medium, η is the viscosity of the medium, and ζ is the zeta potential. Cryo-Transmission Electron Microscopy (cryo-TEM). The morphology and size of the polymer NPs were determined from cryo-TEM
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images. According to protocols reported elsewhere,30-32 thin liquid films of NPs dispersions (0.1 wt % solids content) were prepared on NetMesh (Pelco, U.S.A.) “lacey” carbon membranes and quench-frozen in liquid ethane. Once mounted in a Gatan 626 cryo-holder cooled with liquid nitrogen, the samples were transferred in the microscope and observed at low temperature (-180 °C). Images were recorded on Kodak SO163 films, using a Philips CM200 “Cryo” electron microscope operating at 80 kV. The negatives were digitized off-line using a Kodak Megaplus CCD camera and size measurements were carried out using the ImageJ software. Number-average, weight-average, and Z-average mean diameters (DN, DW, and DZ, respectively) were calculated using the expressions
∑ND i
DN )
i
∑N i
i
i
∑ND i
; DW )
4 i
i
∑
NiD3i
i
∑ND i
; DZ )
6 i
i
∑ND i
5 i
(1)
i
th
where Ni is the number of particles at the i class in the size-distribution histogram. A polydispersity index was also calculated as PTEM ) DW/ DN . Biological Lectin Recognition Assay. The mannose-specific lectin “BclA” from Burkholderia cenocepacia, which has been recently characterized,33 was used for all interaction studies. Recombinant BclA was produced in E. coli as previously described33 and labeled with biotin following the manufacturer protocol (Sigma). The lectin recognition ability of nanoparticles was examined by a modified procedure of the so-called enzyme-linked lectin assay (ELLA)34 that allows determination of the presence of mannose-moieties at the NP surface. After a multistep reaction (Figure 1), the interaction of the NPs with biotin-labeled BclA lectin was revealed by measuring the absorbance of the colored end-product. NPs were first incubated with the biotinylated lectin, then with streptavidin-phosphatase, and finally with p-nitrophenyl phosphate (pNPP), the substrate of phosphatase. Because BclA interacts with RMan through calcium ions, experiments were performed at 25 °C in 0.1 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer (TBT, pH 7.5, containing 0.03 mM CaCl2). At this pH value, BclA is negatively charged, as its isoelectric point is 5.6. Typically, 100 µL of a NP dispersion (2% of solids content) was added to 950 µL of TBT (pH 7.5) containing 0.03 mM CaCl2, 0.05% (w/v) Tween 20, and 3% (w/v) bovine serum albumin (BSA, Sigma) to achieve passivation of the surface and reduce nonspecific interactions. The particles were centrifuged at 4000 g for 9 min, 850 µL were removed and the residual 200 µL homogenized in 700 µL of TBT containing 3% (w/v) BSA, and incubated with 100 µL of biotin-labeled BclA solution in TBT [c ) 5 µL/mL or 10 µL/mL] for 1 h at 37 °C. The particles were washed twice by centrifugation at 4000 g for another 9 min and redispersed in TBT containing 3% (w/v) BSA, centrifuged again, and redispersed in TBT containing 3% (w/v) BSA to a final volume of 900 µL. After this washing step, 100 µL of streptavidin-phosphatase was added (diluted in TBT with 3% (w/v) BSA to 1/1500). After 30 min of incubation at 37 °C, the samples were washed twice by centrifugation as described previously and then 850 µL of the supernatant were removed. The 150 µL were homogenized with a pipet and then 200 µL of pNPP substrate (1 tablet in 5 mL) were added. After 15 min of incubation at room temperature, addition of 100 µL of 1 N sulfuric acid to quench the reaction and centrifugation, the absorbance was measured at 490 nm with a µQuant reader apparatus (Bio Tek instruments). Isothermal Titration Calorimetry (ITC). ITC experiments were carried out using the BclA lectin on a Microcal VP-ITC titration microcalorimeter (Northampton, U.S.A.). All titrations were made in 0.1 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer pH 7.5 with 0.03 mM CaCl2 at 25 °C. The reaction cell (V ) 1.4478 mL) contained either a NP dispersion (total solids content ) 2-3%) prepared from a mixture of PLA with PEO-b-PCL block copolymer 3 (1 equiv) or 2 (1 equiv) or NPs (total solids content ) 2-3%) prepared from a mixture of PLA with PEO-b-PCL block copolymer 1 (0.5 equiv) and copolymer 2 (0.5 equiv). Under such conditions, the theoretical concentration of mannose was 0.25 or 0.5 mM. A series of 30 injections of 10 µL from the
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computer-controlled 300 µL microsyringe at an interval of 5 or 10 min of the solution of BclA ([monomer] ) 1.139 mM) were performed into the receptor solution while stirring at 300 rpm at 25 °C. The raw experimental data were reported as the amount of heat produced after each injection of ligand as a function of time.
Results and Discussion Characterization of the Nanoparticle Size, Charge, and Shape. It has been reported that amphiphilic copolymers could be used as stabilizers and surface-modifiers of PLA nanoparticles using various preparation techniques.6,8,10 As reported previously, the PEO-b-PCL copolymers 1 and 2 listed in Table 1 show interfacial activity.26 The mannosylated PEO-b-PCL copolymer 2 is thus a promising candidate for the surface-modification of polymeric NPs by mannose. In a first step of this study, we compared the features of the NPs as a function of the nature of the amphiphilic PEO-b-PCL copolymer used (mannosylated copolymer 2 or its precursor 1) and the copolymer/PLA molar ratio. The NPs were prepared according to a procedure similar to the nanoprecipitation-evaporation technique:28 the water-insoluble polymer is first dissolved in a water-miscible volatile organic solvent. Then the addition of this organic polymer solution to a large volume of water will induce the precipitation of the polymer to form polymeric NPs. PLA and mixtures of PLA with increasing amounts of surface-active PEO-b-PCL copolymers 1 and 2 (0.09, 0.46, 1.37 molar equiv with respect to PLA) were dissolved in acetone and “(co)-nanoprecipitated” in water. The organic solvent is finally removed by evaporation under reduced pressure, leading to dispersions of NPs at final solids contents in the range of 3% (0.3 g of polymer in 10 g of NPs dispersion). Due to their interfacial activity, the copolymers were expected to be mainly localized at the surface of the NPs. It should be noted first that the precipitation of PLA or coprecipitation of PLA with a copolymer led in all cases to the formation of NPs. The “self-stabilization” of the PLA NPs prepared without PEO-b-PCL copolymer, called “pure PLA NPs”, might be explained by electrostatic repulsion of the carboxylic acid endgroups of the PLA chains synthesized by polyaddition. The solid content of the dispersions was determined gravimetrically and the NPs were analyzed by DLS and cryo-TEM. Table 2 summarizes the features of the NPs. One can notice that for all conditions, the conversion of the polymer initially dissolved in acetone into polymeric NPs was very high (80-90%). Furthermore, while the polydispersity indices were always about 0.1 independently of the sample, the amount and nature of copolymer clearly had an effect on the size of the NPs. Indeed, the addition of surface-active Me2N-PEO-b-PCL 1 copolymer decreases the NPs average hydrodynamic diameter DH (DH < 180 nm) compared to pure PLA NPs (DH ) 191 nm). In contrast, the coprecipitation of PLA with the quarternized positively charged Man-N+-PEO-b-PCL 2 copolymer results in larger NPs. Hence, the higher the copolymer/PLA ratio, the stronger those effects. The properties (charge, size) of the end-group seem to have a great impact on the NPs formation. To better understand the role of the (co)polymers, the zeta potential of the NPs was measured at pH 6 and constant ionic force. The zeta potential of the pure PLA NPs is negative, which can be explained by the carboxylic acid end-group on PLA chains, contributing to their stability. Upon addition of copolymer 1 endcapped by a tertiary amine (Me2N-PEO-b-PCL), the negative charge diminished with increasing copolymer amount and became positively charged for the highest copolymer/PLA molar ratio (1.37), while preserving the stability of the NPs. Indeed, Me2NPEO-b-PCL is partially protonated at pH 6 due to its high pKa
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Table 2. Characteristics of Polymer Nanoparticles Prepared from Acetone Solutions Containing Different Amounts of Copolymers with Respect to PLA copolymer
copo/PLAtheor [molar equiv]
conva [%]
tSb [%]
DHc [nm]
PDId
ζe [mV]
Me2N-PEO-b-PCL (1) Me2N-PEO-b-PCL (1) Me2N-PEO-b-PCL (1) Man-N+-PEO-b-PCL (2) Man-N+-PEO-b-PCL (2) Man-N+-PEO-b-PCL (2)
PLA 0.09 0.46 1.37 0.09 0.46 1.37
82 82 88 86 86 89 83
2.7 3.0 3.2 4.2 2.7 3.0 3.4
191 180 156 140 199 225 258
0.13 0.13 0.12 0.08 0.10 0.15 0.14
-56 -49 -13 15 -41 7 21
No. A B C D E F G
f
a Conversion of polymer initially dissolved in acetone into nanoparticles: mass of solids transformed in nanoparticles [g]/mass of polymer [g] initially dissolved in acetone × 100. b Solids content in g/100 g. c Hydrodynamic diameter averaged on 5 × 10 measurements performed in MilliQ water. d PDI ) polydispersity index. e Zeta potential obtained from electrophoretic mobility µe at pH 6. f Particles without copolymer.
Figure 2. Top: Cryo-TEM images of NPs made of PLA (a), PLA with 0.46 equiv of Me2N-PEO-b-PCL copolymer (b) and PLA with 0.46 equiv of Man-N+-PEO-b-PCL copolymer (c). Bottom: corresponding size-distribution histograms.
(pKa ∼ 9.3). One can also notice that, unsurprisingly, the zeta potential increased to a greater extent for NPs prepared with the quarternized copolymer 2. These measurements thus provided a first evidence of the ability of amphiphilic polymers to modify the surface properties of NPs when used as “co-polymers” (in addition to PLA) in a nanoprecipitation procedure. At this stage, it is difficult to give a clear explanation for the discrepancy in the variation of particle size when PLA is coprecipitated with increasing amounts of either copolymer 1 or 2. Actually, it has already been reported that the coprecipitation of PLA with surface-active copolymers leads to a decrease of the NPs size, due to the diminution of the interfacial tension.35 The present case seems more complicated; as described above, the zeta potential tends to positive values for both types of copolymers, but to a more pronounced extent for the quarternized copolymer 2. Interaction between oppositely charged end-groups of the copolymers and PLA during the nanoprecipitation-evaporation procedure may be responsible of the increased average diameter of NPs. The NPs size will thus be function of several parameters such as the interfacial activity and the charge of the copolymers. In addition to zeta potential measurements, which suggested that the surface properties of the NPs were modified, we evaluated the percentage of PEO at the NP surface (with respect to the total amount of PEO in the NP) by 1H NMR spectroscopy following a procedure described by Vila et al.36 Experiments performed with particles prepared under conditions given in Table 2 (entries B-G) revealed that the percentages of detectable PEO chains were in
Table 3. Number-Average (DN), Weight-Average (DW) and Z-Average (DZ) Mean Diameters Calculated from Cryo-TEM Images sample
DN (nm) [std (nm)]
DW (nm)
DZ (nm)
PTEMa
PLA Me2N-PEO-b-PCL (1) Man-N+-PEO-b-PCL (2)
101 [44] 63 [30] 93 [46]
172 121 190
235 176 284
1.7 1.9 2.0
a
PTEM is a polydispersity index defined as DW/DN.
the range of 65-100%. Consequently, mannose moieties (that are fixed at the PEO chain ends) should be available for interaction with lectins. As mentioned above, the NPs were also characterized by cryoTEM. Typical images of NPs prepared from PLA solutions, and mixtures of PLA with copolymer Me2N-PEO-b-PCL (0.46 equiv) or Man-N+-PEO-b-PCL (0.46 equiv) embedded in a thin film of vitreous ice are shown in Figure 2. In the three cases, the NPs appear to be spherical and no aggregation was observed. The size-distribution histograms of each sample are also shown in Figure 2. The diameter of 800 NPs was measured from a series of cryo-TEM images and the number-average mean diameter DN was calculated. Again, NPs prepared in the presence of copolymer 1 (DN ) 63 nm) were smaller than pure PLA NPs (DN ) 101 nm) or NPs prepared in the presence of copolymer 2 (DN ) 93 nm). In the three cases, the standard deviation (std) is rather high and close to 50% of DN. The weight-average (DW) and Z-average (DZ) mean diameters and polydispersity index PTEM ) DW/DN were also calculated and
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Table 4. Characteristics of Polymer Nanoparticles Used for the Biological Lectin Recognition Assay (Modified ELLA) and ITC No. A G H I J K L
copolymer
copo/PLA theora [molar equiv]
Me2N-PEO-b-PCL (1) Man-N+-PEO-b-PCL (2) Me3N+-PEO-b-PCL (3) Me3N+-PEO-b-PCL (3) acetal-PEO-b-PCL (4) Man-N+-PEO-b-PCL (2) + Me2N-PEO-b-PCL (1)
PLAf 1 1 1 10 10 0.5 0.5
DHb [nm]
PDIc
ζ [mV]
abs (“ELLA”)
191 212 314 263 271 289 206
0.13 0.11 0.32 0.12 0.13 0.25 0.15
-56 -23d +11d +22d n.d.e n.d. n.d.
0.96 0.90 2.04 0.94 0.95 0.96 n.d.
a Copolymer/PLA ratio in the acetone used for the nanoprecipitation procedure. b Hydrodynamic diameter averaged on 5 × 10 measurements performed in diluted tris buffer for all sample except for sample A (pure PLA NP) that was measured in MilliQ water. c PDI ) polydispersity index. d Zeta potential obtained from electrophoretic mobility µe measured at pH 7.6. e n.d. ) not determined. f Particles prepared without copolymer. The deviation of the NP sizes in Tables 2 and 4 can be attributed to the different solvents in which the measurements were performed. As reported before higher NP sizes were observed in the presence of salts (cf. NPs G, H, I, J, K, L in buffer solution).10
Figure 3. Absorbance measured from the interaction between the BclA lectin labeled with biotin and nanoparticles prepared from PLA and copolymers 1-4, in the presence of a streptavidin-phosphatase conjugate and its substrate, p-nitrophenyl phosphate.
are summarized in Table 3. The high polydispersity of the particles may explain the difference of the average diameters determined by DLS and cryo-TEM. Despite this discrepancy, the order of magnitude was similar and the same tendency for the three different types of particles was observed. Recognition of Surface-Exposed Mannose by Lectins. In the next step, we investigated the ability of the mannosefunctionalized NPs to be recognized by BclA lectin which specifically binds terminal R-D-mannopyranosyl (RMan) residues.33 This dimeric lectin binds methyl R-D-mannopyrannose (RMeMan) with high affinity (Ka (BclA/RMeMan) ) 3.6 × 105 M-1), that is, much more strongly than other commercially available mannose-specific lectins such as Galanthus Nivalis Agglutinin (GNA; Ka (GNA/RMeMan < 100 M-1) or Concanavalin A (ConA; Ka (ConA/RMeMan) ) 8.2 × 103 M-1).37 The affinity constant of BclA was derived from isothermal titration calorimetry (ITC) experiments performed at 25 °C which also provided the values of the enthalpy of complexation (∆H ) -23 kJ/mol) and stoichiometry of the interaction (number of binding sites per monomer receptor n ) 0.83).33 To investigate the interaction of the NPs with BclA, two different studies were performed. The first one relies on a modified enzyme-linked lectin assay using biotin-labeled BclA, of which the complex with a streptavidin-phosphatase conjugate is spectrometrically evidenced, as detailed in the Experimental Section (Figure 1). Table 4 and Figure 3 summarize the absorbance values (Abs) resulting from the interaction between biotinylated BclA with pure PLA NP (entry A) or with particles prepared from mixtures of PLA with mannosylated (2) or nonmannosylated (1, 3) copolymers at the same copolymer/PLA molar ratio of 1 (entries G, H, and I). It clearly reveals that the absorbance for NPs containing the mannosylated copolymer (entry H) is significantly
higher than that measured for NPs without mannose (entries A, G, and I), used as references (2.0 vs 0.9). The positive response of the reference samples might be explained by nonspecific adsorption of the lectin or the streptavidin-phosphatase conjugate. When the assay was performed with another mannose-specific lectin, biotinylated GNA, similar results were obtained (Supporting Information, Figure SI-1). The absorbance for mannosylated NPs was again significantly higher than for the nonmannosylated reference sample, which reveals the interaction between mannose residues and the lectin. A second type of control experiments was also carried out, performing the assays according to standard conditions but in the absence of GNA lectin. The response for these samples was in the very same order of magnitude as for the nonmannosylated (see Figure SI-I). This means that both mannosylated and nonmannosylated NPs tend to nonspecifically adsorb proteins, such as streptavidin or lectins. Only the mannosylated NPs showed significantly enhanced affinity to lectins due to additional specific mannose-lectin interaction. To gain insight into the origin of nonspecific adsorption (electrostatic interaction, nonspecific adsorption through hydrophobic interactions, etc.), we compared the absorbance resulting from the interaction of labeled BclA with negatively charged PLA NPs (Table 4, entry A), positively charged PEO-coated PLA particles prepared with a large excess (10 equiv) of copolymer 3 (Table 4, entry J) and negatively charged PEOcoated particles prepared from a mixture of PLA and a large excess (10 equiv) of neutral copolymer 4 (Table 4, entry K). As can be observed from Table 4 and Figure 3, the values of absorbance measured are in the same order of magnitude whatever the nature of the surface of NPs without mannose. It seems that the charge of the NPs has no significant influence on the interaction with the BclA lectin (which is negatively charged at pH 7.5) and, hence, that the observed nonspecific adsorption is not governed by electrostatic interactions. Moreover, experimental data published previously38 have shown that the adsorption behavior of a protein is related to its structure stability. Thus, conformationally flexible proteins may adsorb on hydrophobic and hydrophilic surfaces under attractive as well as repulsive electrostatic conditions. These proteins contain an extra driving force for adsorption, related to structural rearrangements in the molecule, that can outweigh unfavorable contributions.38 In conclusion, these results clearly evidence the ability of mannosylated NPs to be selectively recognized by mannose-binding lectins. However, this specific interaction is associated with nonspecific adsorption of the lectin on the NP surface in spite of the PEO coating. Isothermal titration calorimetry (ITC) was then used as a complementary technique to study more deeply the NP-lectin
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Figure 4. Thermograms for the calorimetric titration of different NPs dispersions by BclA lectin ([monomer] ) 1.139 mM): (A) NPs prepared from a mixture of PLA with 1 molar equiv of copolymer 3 (entry I in Table 4), (B) NPs prepared from a solution containing PLA with 1 molar equiv of copolymer 2 (entry H in Table 4), (C) same dispersion of NPs as (B) but diluted twice, and (D) NPs prepared from a mixture of PLA with 0.5 molar equiv of each copolymer 1 and 2 (entry L in Table 4). Illustrations represent NPs coated with PEG chains and/or PEG chains terminated by a mannose residue.
interactions. Recently, we successfully applied this technique to the characterization of interactions between micelles of copolymer 2 and BclA in terms of apparent binding constant, Ka, enthalpy of binding, ∆H, and stoichiometry, n, of the interaction.26 One of the advantages of ITC is that it directly measures the enthalpy of interaction between the lectin and its carbohydrate ligand, avoiding the use of intermediate complexation agents as in the modified ELLA. The ITC experiments were performed by adding the BclA lectin to the NPs dispersion in the microcalorimeter cell. Figure 4 displays the thermograms for the calorimetric titration of NPs prepared from PLA and the quarternized copolymer 3 (entry I in Table 4) used as a control, NPs prepared from PLA and mannosylated copolymer 2 (entry H in Table 4), and NPs obtained with both copolymer 1 and copolymer 2 (entry L in Table 4). First of all, we could notice that the thermograms were completely different from those obtained with the micelles composed of the corresponding copolymers. It can be observed that the addition of BclA to positively charged NPs without mannose (thermogram A) results in the production of exclusively endothermic peaks. The magnitude of the absorbed heat progressively decreases until the last injection, thus suggesting the presence of nonspecific interactions between the lectin and NPs that have to be entropy-driven. Actually, dehydration, deionization, structural rearrangement of the adsorbate, and electrostatic repulsion between the adsorbate and the adsorbent have been considered as endothermic processes.39 In this study, the contribution of electrostatic repulsion can be discarded as evidenced by modified ELLA. Therefore, the observed positive enthalpies for the adsorption of BclA on the NP surface imply an entropy-driven process that might be attributed to hydrophobic
interactions or to structural changes in the lectin. Moreover, as can be seen from thermogram A, the ∆H values decrease with increasing coverage of the surface with BclA, which may be due to saturation of titration. This decrease might also be related to the inhomogeneous character of the NP surface and dependence of the conformation and orientation of the adsorbed lectin on the surface coverage.38 In contrast, in the case of NPs prepared from a mixture of PLA and mannosylated copolymer 2 (1 equiv, thermogram B), heat is produced after each injection of BclA, which can be attributed to the exothermic complexation with mannose present at the NP surface.26 However, at the end of the titration, endothermic peaks appear in addition to exothermic ones which might again be related to nonspecific interactions between PLA and BclA. As shown by thermogram C, the second event appears earlier in the titration when the theoretical concentration of mannose was half-decreased (dispersion of NPs (entry H in Table 4) diluted twice), thus confirming the sugar-lectin nature of the interactions as long as substantial amounts of mannose are available for complex formation with BclA. Finally, a more complex heat response was observed in the case of the titration of NPs prepared from a mixture of PLA, copolymer 1 and mannosylated copolymer 2 (0.5 equiv of each copolymer, entry L in Table 4). Nonspecific interactions seem to precede mannose-BclA complexation, which can be related to the decreased number of mannose moieties on the NP surface. It can also be assumed that accessibility of mannose is hampered by the presence of nonmannosylated copolymer having a hydrophilic PEO block of same length.40 However, it can be noticed that the magnitude of released heat attributed to mannose complexation is similar to that of thermogram C, demonstrating
Polyester Nanoparticles Presenting Mannose Residues
the possibility to control the magnitude of mannose-lectin interactions as a function of the mannosylated copolymer/PLA molar ratio. To conclude, state-of-the-art characterization techniques (ELLA, a spectrometric sandwich assay, and ITC) allowed us to characterize the interaction between biodegradable nanoparticles and lectins. It was found that all types of NPs studied tend to nonspecifically adsorb proteins. For mannosylated NPs the specific interaction with mannose-specific lectins clearly dominates, which demonstrates the bioavailability of mannose at the NPs surface.
Conclusion We report here a very simple and versatile method to tune the surface properties of polymeric nanoparticles. Thus, novel fully biodegradable nanoparticles, which present mannose residues at their surface were obtained. The presence and bioavailability of surface-exposed mannose could be evidenced by state-of-the-art recognition studies, namely, a modified enzyme-linked lectin assay and isothermal titration calorimetry. The interaction between sugar residues on the NP surface and the lectin was found to be largely affected by the surface density of the mannosylated copolymer. In addition, the interaction was shown to be accompanied by nonspecific adsorption of the lectin on the NP surface. Although no quantitative data could be obtained from isothermal titration calorimetry, this technique provided crucial information about the mechanism of NP-lectin interaction. The nonspecific adsorption of the lectin was shown to be an entropy-driven process and tends to occur after the specific interaction with mannose. These data thus clearly demonstrate the ability of such NPs to specifically interact with mannose-receptors, making them interesting candidates as vaccine delivery systems to dendritic cells. Finally, we believe that this work will be helpful in the design of biodegradable nanoparticles for site-specific drug delivery. Acknowledgment. J.R., H.F., and C.J. (CERM) are grateful to the “Interuniversity Attraction Poles Program (PAI 6/27) Functional Supramolecular Systems” and “Region Wallonne” in the framework of the project VACCINOR for financial support of this research. J.R. was a recipient of a Marie Curie fellowship of the European Community’s Sixth Framework Programme under contract number MEST-CT-2004-503322 at CERMAV and acknowledges the CNRS for the Research Associate position. The authors thank the CGRI-FNRS-CNRS program for financial support of this research in the framework of bilateral cooperation. The authors of this paper would like to thank Marie-He´le`ne Charles (CNRS-bioMe´rieux) and Catherine Gautier (CERMAV) for their technical support. Supporting Information Available. Information on the modified enzyme-linked lectin assay with GNA. This material is available free of charge via the Internet at http://pubs.acs.org.
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