Ystem. La0.5Gd0.5(225Ac)PO4@GdPO4@Au NPs represent a novel system for targeted a radiotherapy. Adding a Au surface onto a LnPO4 core (Ln = La, 
Gd) allows for facile, reproducible surface functionalization. The addition of Gd into the particles creates a magnetic moment which is sufficient to separate the gold NPs containing Gd from any gold NPs produced in the gold coating step. This separation ensures that gold NPs without a radioactive core will not compete with the TAT conjugate for receptor sites. Compared with single a-emitting therapies, the use of in vivo a generators holds the potential to deliver a much larger biologically effective dose to target tissues. Effective design of in vivo TAT agents with isotopes 
like 225Ac requires two major components. First, the therapeutic agent must be able to deliver the generator radionuclide specifically to target tissue at a cytotoxic dose. The high, receptor-mediated uptake of particles in the lung endothelium demonstrates the ability of La0.5Gd0.5(225Ac)PO4@GdPO4@Au NPs to deliver 225Ac to a tissue target that is present in the vascular space. Second, the TAT must be able to retain the daughter products of the generator in the target tissue. Migration of daughter products to non-target tissue will severely limit the administered therapeutic dose. Retention of the decay daughters can be achieved in a number of ways. First, the radionuclide may be selected so that the daughter half-lives are sufficiently short that they will not have time to migrate throughout the body. Alternatively, the radionuclide can be chosen so that the daughter products exhibit similar in vivo behavior and remain in the target tissue. This is the GNF-7 principle behind the Gracillin recent successes using 223 RaCl2 for treatment of bone metastases [34]. The 223Ra daughter products either have short half-lives or have a high affinity for bone (211Pb, t1/2 = 36 m). While effective in this case, translation of this in vivo a generator to other tumor types would require a different mechanism of retaining the 211Pb and 211Bi daughters in the target tissue. A third solution to the daughter retention problem involves internalization 23727046 of the 1676428 parent radionuclide in the target cell itself [12]. This approach utilizes the internal milieu of the cell to contain the daughter decay products. Tumor targets for internalization occur largely in the extravascular space,Figure 5. MAb 201b antibody conjugation to multi-layered NPs. doi:10.1371/journal.pone.0054531.gGold Coated LnPO4 Nanoparticles for a RadiotherapyFigure 6. Biodistribution of NPs following tail vein injection in mice at 1 hour (n = 3). doi:10.1371/journal.pone.0054531.gwhich is difficult to access with larger constructs that promote endocytosis. Attempts to reduce 213Bi toxicity through targeted, metal-chelate based internalizing antibodies have shown only moderate success [35]. The NP construct described in this work improves 225Ac daughter retention relative to both chelate approaches and previous NP constructs. La0.5Gd0.5(225Ac)PO4@GdPO4@Au NPs contain 88 of the 221Fr daughter in vitro, compared with 50 retention observed with La(225Ac)PO4 NPs [28]. Additionally, the in vivo a-generator delivery agent has a negligible effect on the energies of the emitted a particles. A 6 MeV a-particle loses less than 0.2 of its energy in the layered NP whereas the range of the 100 keV recoiling daughters is ,20 nm in bulk LnPO4. Moreover, a portion of the kinetic energy of the daughter pa.Ystem. La0.5Gd0.5(225Ac)PO4@GdPO4@Au NPs represent a novel system for targeted a radiotherapy. Adding a Au surface onto a LnPO4 core (Ln = La, Gd) allows for facile, reproducible surface functionalization. The addition of Gd into the particles creates a magnetic moment which is sufficient to separate the gold NPs containing Gd from any gold NPs produced in the gold coating step. This separation ensures that gold NPs without a radioactive core will not compete with the TAT conjugate for receptor sites. Compared with single a-emitting therapies, the use of in vivo a generators holds the potential to deliver a much larger biologically effective dose to target tissues. Effective design of in vivo TAT agents with isotopes like 225Ac requires two major components. First, the therapeutic agent must be able to deliver the generator radionuclide specifically to target tissue at a cytotoxic dose. The high, receptor-mediated uptake of particles in the lung endothelium demonstrates the ability of La0.5Gd0.5(225Ac)PO4@GdPO4@Au NPs to deliver 225Ac to a tissue target that is present in the vascular space. Second, the TAT must be able to retain the daughter products of the generator in the target tissue. Migration of daughter products to non-target tissue will severely limit the administered therapeutic dose. Retention of the decay daughters can be achieved in a number of ways. First, the radionuclide may be selected so that the daughter half-lives are sufficiently short that they will not have time to migrate throughout the body. Alternatively, the radionuclide can be chosen so that the daughter products exhibit similar in vivo behavior and remain in the target tissue. This is the principle behind the recent successes using 223 RaCl2 for treatment of bone metastases [34]. The 223Ra daughter products either have short half-lives or have a high affinity for bone (211Pb, t1/2 = 36 m). While effective in this case, translation of this in vivo a generator to other tumor types would require a different mechanism of retaining the 211Pb and 211Bi daughters in the target tissue. A third solution to the daughter retention problem involves internalization 23727046 of the 1676428 parent radionuclide in the target cell itself [12]. This approach utilizes the internal milieu of the cell to contain the daughter decay products. Tumor targets for internalization occur largely in the extravascular space,Figure 5. MAb 201b antibody conjugation to multi-layered NPs. doi:10.1371/journal.pone.0054531.gGold Coated LnPO4 Nanoparticles for a RadiotherapyFigure 6. Biodistribution of NPs following tail vein injection in mice at 1 hour (n = 3). doi:10.1371/journal.pone.0054531.gwhich is difficult to access with larger constructs that promote endocytosis. Attempts to reduce 213Bi toxicity through targeted, metal-chelate based internalizing antibodies have shown only moderate success [35]. The NP construct described in this work improves 225Ac daughter retention relative to both chelate approaches and previous NP constructs. La0.5Gd0.5(225Ac)PO4@GdPO4@Au NPs contain 88 of the 221Fr daughter in vitro, compared with 50 retention observed with La(225Ac)PO4 NPs [28]. Additionally, the in vivo a-generator delivery agent has a negligible effect on the energies of the emitted a particles. A 6 MeV a-particle loses less than 0.2 of its energy in the layered NP whereas the range of the 100 keV recoiling daughters is ,20 nm in bulk LnPO4. Moreover, a portion of the kinetic energy of the daughter pa.
