Surface Modification of Polyanhydride Microspheres

Polyanhydrides are emerging as a new class of biodegradable polymers for drug delivery.1-3 The degradation of polyanhydride delivery systems is largely through surface erosion, potentially leading to zero-order release of encapsulated drugs.4,5 Recently, the Food and Drug Administration has approved the use of the polyanhydride poly[sebacic acid-co-1,3-bis(p-carboxyphenoxy)propane] to deliver drugs for treatment of brain cancer.6 This is one of the few examples where an implantable synthetic degradable polymer has been approved for human use. The use of polyanhydride polymers in oral delivery of insulin and genes further broadens the scope of their applications in drug delivery.7 Despite numerous studies on polyanhydrides, the compositions thus far developed do not have the capability to target specific organs or cell types. To achieve this goal, it would be important to have an approach that could enable the chemical functionalization of microspheres to allow the attachment of targeting ligands (e.g. antibodies, etc). The recognition of ligands on the surface of delivery systems by organ-specific or cellspecific receptors may result in organ-targeted drug delivery8,9 or in improved drug uptake through ligand-induced endocytosis.10,11 Here we describe a one-step procedure to covalently modify the surface of poly(sebacic acid) (pSA)12 microspheres with ligands containing amino groups. Amino groups react with the anhydride groups on the surface of microspheres, which results in covalent attachment through an amide bond (Scheme 1). Experimental SectionsMaterialsspSA was synthesized from sebacic acid following a published procedure.12 L-Argininamide dihydrochloride and guanidine hydrochloride were purchased from Sigma. N-(5-Aminopentyl)biotinamide, 6-((biotinoyl)amino)hexanoic acid, and fluorescein-conjugated avidin were purchased from Molecular Probes (Eugene, Oregon). The pSA (Mw ) 34 kDa) microspheres (mean diameter ) 24 ( 1 μm) were formed by solvent evaporation.13 Surface Modification of Microspheres by ArgininamidesThe pSA microspheres (30 mg for each concentration of argininamide) were suspended in solutions containing different concentrations of argininamide (0, 0.40, 1.2, 4.0, 12, and 40 mM, 2 mL each) in borate buffer (0.20 M, pH 9.0) at room temperature. After 15 min, the surface modification was stopped by acidifying the solution to ∼pH 6 by addition of acetate buffer (0.20 M, pH 5.0, 5 mL).14 The microspheres were collected after centrifugation (10 000 rpm for 10 min) at 4 °C, washed with 2 M aqueous NaCl, distilled water (twice), and ethanol, and dried in vacuo. The surface density of argininamide was analyzed by X-ray photoelectron spectroscopy (XPS), and its bulk concentration was analyzed by 1H NMR (400 MHz) of microspheres dissolved in CDCl3. The size of the microspheres was measured by a Coulter Multisizer (Coulter Electronic Limited, Luton, U.K.). Guanidine Hydrochloride ExperimentsThe pSA microspheres (20 mg) were suspended in 30 mM guanidine hydrochloride solution in borate buffer (0.20 M, pH 9.0, 2 mL) at rt. After 15 min, acetate buffer (0.20 M, pH 5.0, 5 mL) was added. The microspheres were collected, washed with 2 M NaCl, distilled water (twice), and ethanol, and dried in vacuo. Self-Assembled Monolayer (SAM) ExperimentssThe gold substrates were prepared by e-beam evaporation of 5 nm of titanium and 200 nm of gold to a single-crystal silicon wafer in a deposition chamber. The gold-coated wafers were cut into ca. 1 cm × 2 cm pieces and self-assembled monolayers (SAMs) were formed by immersing the gold substrates in an ethanolic solution of 16-mercaptohexadecanoic acid (2 mM, 10 mL) at room-temperature overnight.15 One sample was used as an unmodified control. Two other samples were soaked in 10 mM argininamide solution in borate buffer (0.20 M, pH 9.0) at rt. After 15 min, one sample was taken out and blown dry by Dust-Off (Falcon Safety Products, Inc., NJ). The other sample was washed by 2 M aqueous NaCl and distilled water (twice) and finally blown dry. XPS analyses showed nitrogen signals in the unwashed SAM sample but not in the washed SAM sample nor the control sample (data not shown). The carboxylic acid groups on the SAMs were further transformed to interchain anhydrides using trifluoroacetic anhydride following a literature procedure.15 The resulting SAM sample was soaked in 10 mM argininamide solution in borate buffer (0.20 M, pH 9.0) at rt for 15 min. The sample was washed by 2 M aqueous NaCl and distilled water (twice) and blown dry. XPS analyses showed the appearance of nitrogen signals. Avidin Binding to the Surface of Biotin-Attached pSA MicrospheressThe pSA microspheres (10 mg) were suspended in a solution ofN-(5-aminopentyl)biotinamide (10.0 * To whom correspondence should be addressed. Telephone: (617) 253-3107. Fax: (617) 258-8827. Email: rlanger@mit.edu. † Massachusetts Institute of Technology. ‡ Massachusetts General Hospital. § Harvard University. Scheme 1sSurface modification of pSA microspheres with ligands containing amino groups via amide formation.