Melanin-covered nanoparticles for protection of bone marrow during radiation therapy of cancer

Abstract

Purpose: Protection of bone marrow against radiotoxicity during radioimmunotherapy and in some cases external beam radiation therapy such as hemi-body irradiation would permit administration of significantly higher doses to tumors, resulting in increased efficacy and safety of treatment. Melanin, a naturally occurring pigment, possesses radioprotective properties. We hypothesized that melanin, which is insoluble, could be delivered to the bone marrow by intravenously administrated melanin-covered nanoparticles (MNs) because of the human body's "self-sieving" ability, protecting it against ionizing radiation.

Methods and materials: The synthesis of MNs was performed via enzymatic polymerization of 3,4-dihydroxyphenylalanine and/or 5-S-cysteinyl-3,4-dihydroxyphenylalanine on the surface of 20-nm plain silica nanoparticles. The biodistribution of radiolabeled MNs in mice was done at 3 and 24 h. Healthy CD-1 mice (Charles River Laboratories International, Inc., Wilmington, MA) or melanoma tumor-bearing nude mice were given MNs intravenously, 50 mg/kg of body weight, 3 h before either whole-body exposure to 125 cGy or treatment with 1 mCi of (188)Re-labeled 6D2 melanin-binding antibody.

Results: Polymerization of melanin precursors on the surface of silica nanoparticles resulted in formation of a 15-nm-thick melanin layer as confirmed by light scattering, transmission electron microscopy, and immunofluorescence. The biodistribution after intravenous administration showed than MN uptake in bone marrow was 0.3% and 0.2% of injected dose per gram at 3 and 24 h, respectively, whereas pre-injection with pluronic acid increased the uptake to 6% and 3% of injected dose per gram, respectively. Systemic MN administration reduced hematologic toxicity in mice treated with external radiation or radioimmunotherapy, whereas no tumor protection by MNs was observed.

Conclusions: MNs or similar structures provide a novel approach to protection of bone marrow from ionizing radiation based on prevention of free radical formation by melanin.

Fig. 1

Chemical structure of melanin: (a) structure of eumelanin oligomer and (b) structure of pheomelanin oligomer.

Fig. 2

Spectrophotometry evaluation of melanin formation from 3,4-dihydroxyphenylalanine (catalyzed by tyrosinase): (a) absorption of reaction mixtures in 250- to 750-nm range at 0 to 180 minutes after the start of the reaction and (b) kinetics of melanin formation over the period of 100 minutes was followed at 250, 300, and 700 nm.

Fig. 3

High-performance liquid chromatography of 3,4-dihydroxyphenylalanine/tyrosinase reaction mixture during various stages of melanin synthesis: (a) 0 minutes (before addition of tyrosinase), (b) 5 minutes after start of reaction, and (c) 20 minutes after start of reaction.

Fig. 4

¹H nuclear magnetic resonance of 3,4-dihydroxyphenylalanine (L-DOPA) solutions during various stages of melanin synthesis. The lowest spectrum represents L-DOPA solution before the addition of tyrosinase, and the spectra above are marked with the times passed after the addition of tyrosinase to L-DOPA. Arrows indicate melanin intermediate–associated peaks. Opaque gray regions represent background L-DOPA peaks and the residual water peak at 4.7 ppm.

Fig. 5

Characterization of melanin-covered nanoparticles (MNs). (a–c) Particle size determination by light scattering: (a) MNs made with 3,4-dihydroxyphenylalanine (L-DOPA), (b) MNs made with L-DOPA/5-S-cysteinyl-DOPA, and (c) plain silica nanoparticles. (d) Transmission electron microscopy image of MNs made with L-DOPA. The bar is 200 nm. (e) Immunofluorescence image of MNs made with L-DOPA obtained with 6D2 melanin-binding antibody.

Fig. 6

Biodistribution of intravenously injected melanin-covered nanoparticles (MNs) made with 3,4-dihydroxyphenylalanine (L-DOPA) in CD-1 mice: (a) particles only and (b) pre-injection with pluronic acid 12 h in advance. There was an approximately 30-fold increase in the uptake of MNs in the bone marrow after pre-injection of pluronic acid. The error bars represent standard error of the mean. ID/g = injected dose per gram.

Fig. 7

Protective effects of melanin-covered nanoparticles (MNs) on the bone marrow of mice during whole-body irradiation and radioimmunotherapy (RIT). (a, b) CD-1 mice were irradiated with 125 cGy of ¹³⁷Cs radiation: (a) white blood cell (WBC) counts and (b) platelet counts. controls = mice irradiated without any pre-treatment; non-mel = mice given non-melanized nanoparticles; mel = mice given MNs made with 3,4-dihydroxyphenylalanine. (c, d) Nude mice bearing A2058 melanoma tumors were given MNs 3 h before RIT with 1 mCi of ¹⁸⁸Re-labeled 6D2 antibody to melanin. Control mice were either given RIT without MNs or left untreated. (c) Change in tumor volume after RIT treatment. (d) White blood cell counts in RIT-treated mice. The error bars represent standard error of the mean.