MAPLE Deposition of Antibodies to EGFR

MAPLE (Fig. 3) is a deposition technique [28] derived from pulsed laser deposition (PLD) in order to avoid laser radiation-induced damage to biologic or organic molecules or compounds. Such a result is achieved carrying out laser deposition from a frozen target containing the guest material to be deposited in a volatile matrix [29–32]. As specific MAPLE characteristic, the presence of the matrix protects guest material from being damaged by laser radiation but deposition is essentially solvent-free since matrix molecules do not reach substrate. This allows multilayer depositions using the same solvent and depositions on soluble substrates. The steps for production of antibody-CNM-DOX nanoparticles were as follows:

A soluble layer was created on a MAPLE substrate (a microscope cover glass) by placing it at the bottom of a 5-mm deep container filled with sterile saline solution (9 mg/ml NaCl in water from Fresenius Kabi) and letting water to evaporate at 95 °C and atmospheric pressure for about 12 h.

A layer containing the required nanoparticles (OLC-DOX or UDD-DOX, see Table 1 for details) was deposited on the top of the soluble layer by taking advantage of the solvent-free behavior of MAPLE technique. The MAPLE target was obtained by dispersing nanoparticles in aqueous or saline solution. The target was frozen by placing it in thermal contact with liquid nitrogen. The vacuum chamber pressure was then lowered and laser turned on. Laser beam impinges at an angle of 45° on the target which, during the deposition, is moved by a computer controlled translation system in order to avoid overheating and drilling: an area of about 2 cm² is scanned, one or more times depending on the total number of pulses, during deposition time.

MAPLE deposition parameters

The soluble layer covered by a second layer containing OLC/UDD-DOX nanoparticles was used as a substrate for a second MAPLE deposition of anti-EGFR antibodies. The target was a saline solution of EGFR or EGR, the guest compound, frozen by thermal contact with liquid nitrogen. The MAPLE deposition (deposition parameters are detailed in Table 1) ensures again that previous layers were not damaged or removed.

Finally, the microscope cover glass with the resulting multilayer deposition over soluble substrate was immersed in a saline solution as solvent. The soluble substrate dissolved and the nanoparticle layer disintegrated into individual antibody-OLC/UDD-DOX units.

A scheme of matrix-assisted pulsed laser evaporation techniques shows principal of protein deposition on carbon surface. Antibodies to human epidermal growth factor receptor and epidermal growth factor were placed on target platen and evaporated on substrate. As substrates UDD-DOX (samples C46, C47) or OLC-DOX (samples C36, C37), films were used