Synthesis of Highly Functionalized Poly (alkyl cyanoacrylate)

Synthesis of Highly Functionalized Poly (alkyl cyanoacrylate)

(Parte 1 de 4)

Synthesis of Highly Functionalized Poly(alkyl cyanoacrylate) Nanoparticles by Means of Click Chemistry

Julien Nicolas,*,† Fethi Bensaid,† Didier Desmaele,‡ Mathurin Grogna,§ Christophe Detrembleur,§ Karine Andrieux,† and Patrick Couvreur†

Laboratoire de Physico-Chimie, Pharmacotechnie et Biopharmacie, UniV. Paris-Sud, UMR CNRS 8612, Faculte de Pharmacie, 5 rue Jean-Baptiste Clement, 92296 Chatenay-Malabry, France, Laboratoire Biocis, UniV. Paris-Sud, UMR CNRS 8076, Faculte de Pharmacie, 5 rue Jean-Baptiste Clement, 92296 Chatenay-Malabry, France, and Center for Education and Research on Macromolecules (CERM), UniVersity of Liege, Sart-Tilman, B6, 4000 Liege, Belgium

ReceiVed June 13, 2008; ReVised Manuscript ReceiVed September 9, 2008

ABSTRACT: A general methodology was proposed to prepare highly functionalized poly(alkyl cyanoacrylate) nanoparticles by means of Huisgen 1,3-dipolar cyclo-addition, the so-called click chemistry. To achieve this goal, different protocols were investigated to obtain azidopoly(ethylene glycol) cyanoacetate of variable molar mass, followedby a Knoevenagelcondensation-Michaeladditionreactionwith hexadecylcyanoacetateto produce a poly[(hexadecyl cyanoacrylate)-co-azidopoly(ethylene glycol) cyanoacrylate] (P(HDCA-co-N3PEGCA)) copolymer, displaying azide functionalities at the extremity of the PEG chains. As a proof of concept, model alkynes were quantitatively coupled either to the P(HDCA-co-N3PEGCA) copolymers in homogeneous medium followed by self-assembly in aqueous solution or directly at the surface of the preformed P(HDCA-co-N3PEGCA) nanoparticles in aqueous dispersed medium, both yielding highly functionalized nanoparticles. This versatile approach, using alkyl cyanoacrylate derivatives, opened the door to ligand-functionalized and biodegradable nanoparticles with “stealth” properties for biomedical applications.


Nanoparticles developed from poly(alkyl cyanoacrylate)

(PACA) biodegradablepolymers have opened new and exciting perspectives in the field of drug delivery due to their nearly ideal characteristicsas drug carriersin connectionwith biomedical applications. Introduced more than 25 years ago in the field of pharmacology,1 PACA drug carriers have, indeed, demonstrated significant advantages for the treatment of numerous pathologies such as cancer2 and severe infections (viral, bacteriologic, parasite)3 as well as several metabolic and autoimmune diseases,4 which has been well-reviewed in the recent literature.5-7 Clinical trials have even shown that these nanodevices are safe and biocompatible when loaded with the anticancer drug doxorubicin.8

Throughout the last two decades, PACA nanoparticles with different features have been developed:9 nanospheres (matrixtype nanoparticles),1,10-15 nanocapsules (vesicular-type nanoparticles) either oil- or water-containing,16-25 as well as nanoparticles with controlled-surface properties;14,2,26-39 the later being considered as the second generation of drug delivery devices. Regarding this recent class of advanced PACA nanoparticles, the major breakthrough is undoubtedly the grafting of poly(ethylene glycol) (PEG), termed “PEGylation”. PEG is a hydrophilic and flexible polymer intensively employed in the pharmaceutical area, especially for drug delivery purposes such as polymer-protein/peptide bioconjugates40-45 or long-circulating nanoparticles.28,46-48 Indeed, PEG gives rise to several potential beneficial effects including increased bioavailability and plasma half-lives, biocompatibility/decreased immunogenicity, reduced proteolysis and enhanced solubility and stability, thus being consideredas a key material in this field.40 Considering nanoparticletechnology,non-“PEGylated”nanoparticlesare quickly eliminated from the bloodstream due to the adsorption of blood proteins (opsonins) onto their surface, which triggers the recognition by the macrophages of the mononuclear phagocyte system (MPS). As a consequence, these nanoparticles accumulate in the organs of the MPS such as the liver and the spleen, restricting the therapeutic activity of the entrapped compounds to hepatic diseases. In contrast, when covered by PEG chains, the obtained nanoparticles are able to efficiently escape this recognition system, resulting in long-circulating colloidal devices, also called “stealth” nanoparticles.46,48

Alkyl cyanoacrylatesmonomersare also well-knownfor their very high reactivity and the excellent adhesive properties of the resulting polymers. However, this unique feature tends to make the synthesis of well-defined and/or functionalizable poly(alkyl cyanoacrylate) architectures extremely difficult or even impossible. A significant step was accomplished to circumventthis important drawback via the synthesis of random poly[(hexadecyl cyanoacrylate)-co-methoxypoly(ethylene glycol) cyanoacrylate] (P(HDCA-co-MePEGCA)) comblike copolymers with amphiphilic properties.28 This original approach derived from tandem Knoevenagel condensation-Michael addition reaction to build the polymeric backbone, where the corresponding cyanoacetates were reacted with formaldehyde in the presence of dimethylamine as the catalyst (Scheme 1).

† Laboratoire de Physico-Chimie, Pharmacotechnie et Biopharmacie,

Univ. Paris-Sud, UMR CNRS 8612.

‡ Laboratoire Biocis, Univ. Paris-Sud, UMR CNRS 8076. § Center for Education and Research on Macromolecules (CERM), University of Liege.

Scheme 1. Synthesis of Random Poly[(hexadecyl cyanoacrylate)-co-methoxypoly(ethylene glycol) cyanoacrylate]

(P(HDCA-co-MePEGCA)) Copolymer via Knoevenagel Condensation-Michael Addition Reaction

10.1021/ma8013349 C: $40.75 2008 American Chemical Society Published on Web 10/24/2008

However, even though these “PEGylated” nanoparticleshave demonstrated a noticeable brain-targeting effect;6 they suffer from a crucial lack of specificity toward cells and/or tissues and can not be efficiently addressed. Thus, for the forthcoming years, the most exciting challenge in drug delivery, whatever the nature of the drug carriers(i.e., liposome,nanoparticles,etc.), will be undoubtedly the synthesis of efficient ligand-functionalized colloidal devices to achieve specific cells targeting based on a molecular recognition process. To the best of our knowledge concerning PACA technology, only one example of the so-called third generation PACA nanoparticles has been reported and involves poly[(hexadecyl cyanoacrylate)-coaminopoly(ethylene glycol) cyanoacrylate] (P(HDCA-co-

H2NPEGCA)) nanospheres displaying folic acid groups,49,50 to target the folate receptor which is overexpressed at the surface of many tumor cells. However, this approach is restricted to amine-reactivecompounds and led to only ∼15% folate content at the surface of the nanospheres.49,50

In order to extend this concept of functionalizable poly(alkyl cyanoacrylate) “PEGylated” nanoparticles, we have chosen to take advantage of Huisgen 1,3-dipolar cyclo-addition (termed click chemistry)51,52 between alkyne and azide derivatives due to its high efficiency and its mild experimental conditions.53-56 Indeed, click chemistry has recently received intense interest as a well-established synthetic route to obtain tailor-made complex materials and has been exploited in many areas such as dendrimers,57-59 bioconjugates,60-62 therapeutics63-65 and functionalized polymers.6-68

Herein, we propose a general methodology to obtain highly functionalized PACA biodegradable nanoparticles from a novel poly[(hexadecyl cyanoacrylate)-co-azidopoly(ethylene glycol) cyanoacrylate](P(HDCA-co-N3PEGCA))copolymer,as a clickable polymeric scaffold (Figure 1). This versatile approach allowed either: (i) the early coupling reaction to proceed in homogeneous medium with clickable P(HDCA-co-N3PEGCA) copolymers followed by the formation of functionalized nano-

particles by self-assembly in aqueous solution (Figure 1a) or (i) an effective coupling reaction directly at the surface of clickable P(HDCA-co-N3PEGCA) nanoparticles (Figure 1b). Depending on the characteristics of the desired alkyne moiety

(nature, solubility, size, etc.), one of the pathways would be more appropriate than the other one; for instance, if large molecules such as peptide sequences or proteins are required, the click reaction at the surface of the azido-functionalized PACA nanoparticles would be more suitable than early on the copolymerin homogeneousmedium, which would undoubtedly alter the following nanoprecipitation process (due to modified hydrophilic-lipophilic balance (HLB)).

In the literature, click chemistry has been employed with nanoparticles based on well-defined poly(acrylic acid)-bpolystyrene (PAA-b-PS) block copolymers.69,70 O’Reilly et al. used nitroxide-mediated polymerization (NMP) or reversible addition-fragmentation transfer (RAFT) to prepare shell crosslinked PAA-b-PS nanoparticles bearing azide functionalities at their surface, on which an alkyne-fluorescein dye has been successfullyclicked.69 More recently, Opsteen et al. synthesized an azido-terminated PAA-b-PS copolymer by atom-transfer radical polymerization (ATRP) to form water-containing nanocapsules covered by azide groups, followed by click reaction with a wide variety of alkyne-ligands based on dansyl dye, biotin, or enhanced green fluorescent protein (EGFP).70 Even though these two studies clearly demonstrated the feasibility of the click reaction at the surface of these model nanoparticles, a similar approach employing well-established biodegradable polymers such as PACA is highly desirable regarding biomedical applications, where biocompatible and/or biodegradable, ligand-functionalized, colloidal drug carriers are in great demand.

Experimental Section

Materials. Poly(ethylene glycol) monobenzyl ether (BnPEG70,

Mw/Mn ) 1.06) was purchased from Polymer Source and used as received.Ethyleneoxide (EO, >9%) was purchasedfrom Chemo- gas. Poly(ethylene glycol) monomethyl ether (PEG43, Mn,NMR ) carbodiimide (DCC, >9%, Fluka), methanesulfonyl chloride

(MsCl, 9.7%, Aldrich), sodium azide (NaN3, 9.5%, Aldrich), 4-dimethylaminopyridine (DMAP, 9%, Aldrich), formaldehyde

(37% in water, Aldrich), pyrrolidine (9%, Aldrich), anhydrous magnesium sulfate (MgSO4, >9%, Aldrich), sodium ascorbate

Figure 1. General approach to prepare functionalized poly(alkyl cyanoacrylate) nanoparticles: click reaction on the poly[(hexadecyl cyanoacrylate)- co-methoxypoly(ethylene glycol) cyanoacrylate] (P(HDCA-co-N3PEGCA)) copolymer followed by self-assembly in aqueous solution (a) or click reaction at the surface of preformed P(HDCA-co-N3PEGCA) nanoparticles (b).

Macromolecules, Vol. 41, No. 2, 2008 Poly(alkyl cyanoacrylate) Nanoparticles 8419

(Aldrich), copper sulfate pentahydrated (CuSO4.5 H2O, >9%, Aldrich),4-pentyn-1-ol(95%, Acros),triethylamine (TEA, Aldrich) and N-[2-(dimethylamino)ethyl]-N′,N′,N′-trimethyl-1,2-ethanediamine (PMDETA, 9%, Aldrich) were used as received. Copper bromide(CuBr,97%, Aldrich)was purifiedaccordingto the method of Keller and Wycoff.71 2-Propanol (9.5%) and N,N-dimethylformamide (DMF) were purchased from Fluka. All other solvents (tetrahydrofuran,(THF),methanol(MeOH),dichloromethane(DCM),

diethyl ether (Et2O), chloroform (CHCl3), ethanol (EtOH), ethyl acetate (EtOAc) and hexane) were purchased at the highest grade from Carlo Erba. Propynyl-dansyl (alkyne-dansyl) was synthesized from dansylchloride (Acros, 98%) and propargyl amine (Acros, 9%) as described elsewhere.70 Analytical Techniques. 1H and 13C NMR spectra were per- formed in deuteratedchloroform(CDCl3) or deuteratedwater (D2O) at ambient temperature on a Bruker Avance (300 MHz unless otherwise specified and 75 MHz, respectively). IR spectra were obtained on a Fourier Transform Bruker Vector 2 spectrometer. Size exclusionchromatography(SEC) was performedat 30 °C with two columns from Polymer Laboratories (PL-gel MIXED-D; 300 × 7.5 m; bead diameter: 5 µm; linear part: 400 to 4 × 105 g·mol-1) and a differentialrefractive index detector (Spectrasystem

RI-150 from Thermo Electron Corp.). The eluent was CHCl3 at a flow rate of 1 mL·min-1 and toluene was used as a flow-rate

marker. The calibration curve was based on poly(ethylene glycol) standards (peak molar masses, Mp ) 200 to 23 600 g·mol-1)o r poly(methyl methacrylate)(PMMA) standards (peak molar masses,

Mp ) 625 to 625 500 g·mol-1) from Polymer Laboratories. Unless otherwise indicated, the calibration based on PEG standards was used. This technique allowed Mn (the number-averagemolar mass), Mw (the weight-averagemolar mass) and Mw/Mn (the polydispersity index, PDI) to be determined. Nanoparticles diameter (Dz) was measured by dynamic light scattering (DLS) with a Nano ZS from

Malvern (173° scattering angle) at a temperature of 25 °C. The particle size distribution is generally considered as narrow when below 0.10.

Synthesis of Hexadecyl Cyanoacetate.Hexadecyl cyanoacetate was synthesized as follows. In a 250 mL round-bottom flask containing hexadecane-1-ol (10.65 g, 4 mmol), cyanoacetic acid (7.48 g, 8 mmol), EtOAc (5 mL) and DCM (50 mL) was introduced dropwise by a syringe over ca. 20 min, a solution of DCC (9.98 g, 48.4 mmol) and DMAP (120 mg, 0.82 mmol) in DCM (50 mL). The reaction medium was stirred during 24 h at ambienttemperatureunder argon atmosphere.The solid was filtered off and the solvents were removed under reduced pressure. The solid was then purified by flash chromatography (SiO2, hexane/ EtOAc; 5:1; v:v) to give a fine, white powder: 12.9 g (95%). 1H

Synthesis of Poly(ethylene glycol) Monobenzyl Ether

(BnPEG47). BnPEG47 was synthesized by ring-opening polymerization of EO as follows. Benzyl alcohol (1.70 g, 16 mmol) and naphthalene potassium (2.68 g, 16 mmol) were added to 250 mL of freshly distilled THF under nitrogen. After 10 min of stirring, the solution was transferred to a 1 L stainless steel reactor and EO (35 g, 0.79 mol) was added to the solution. The polymerization of EO proceeded for 4 h at 50 °C. An excess of diluted hydrochloride aqueous solution was then added to stop the reaction and the polymer was quantitavely recovered by precipitation into a large

Synthesis of N3PEG70CA Following Path A. Synthesis of

Benzylpoly(ethylene glycol) Acetate (BnPEG70OAc, 2). Ina5 0m L round-bottom flask, a solution of BnPEG70 (1.0 g, 0.32 mmol), DMAP (30 mg, 0.24 mmol), TEA (124 µL, 0.8 mmol), and acetic anhydride (90 mg, 0.8 mmol) in DCM (15 mL) was allowed to stir at room temperature during 2 h under argon atmosphere. The mixture was then washed three times with 1 M aqueous HCl solution and once with brine. The organic phase was dried over

MgSO4, filtered and concentratedunder reduced pressure. The solid was dissolved in a minimal amount of DCM and precipitated by dropwise addition in a large volume of cold Et2O. The product was collected by filtration as a fine, white powder: 993 mg (98%).

Synthesis of Poly(ethylene glycol) Acetate (PEG70OAc, 3). A suspension of 2 (500 mg, 0.156 mmol), EtOH (4 mL), acetic acid

(600 mg, 10.0 mmol), and Pd(OH)2/C (80 mg, 0.057 mmol) was hydrogenated at 6 bar in a steel autoclave during 15 h at ambient temperature under vigorous stirring. After the reaction, the catalyst was filtered off and the resulting solution was concentrated under reduced pressure and dried under vacuum to give a slightly yellow µL, 0.64 mmol) in DCM (7.6 mL) was cooled to 0 °C. MsCl (40 µL, 0.51 mmol) was then introduced dropwise by a syringe over ca. 15 min. The mixture was then stirred under argon atmosphere at 0 °C during 2 h and overnight at room temperature. The mixture was then washed three times with 1 M aqueous HCl solution and once with brine. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The solid was dissolved in a minimalamount of DCM and precipitatedby dropwiseaddition

(Parte 1 de 4)