Assembly of Bleomycin Saccharide-Decorated Spherical Nucleic Acids

Glyco-decorated spherical nucleic acids (SNAs) may be attractive delivery vehicles, emphasizing the sugar-specific effect on the outer sphere of the construct and at the same time hiding unfavorable distribution properties of the loaded oligonucleotides. As examples of such nanoparticles, tripodal sugar constituents of bleomycin were synthesized and conjugated with a fluorescence-labeled antisense oligonucleotide (AONARV7). Successive copper(I)-catalyzed azide-alkyne and strain-promoted alkyne-nitrone cycloadditions (SPANC) were utilized for the synthesis. Then, the glyco-AONARV7 conjugates were hybridized with complementary strands of a C60-based molecular spherical nucleic acid (i.e., a hybridization-mediated carrier). The formation and stability of these assembled glyco-decorated SNAs were evaluated by polyacrylamide gel electrophoresis (PAGE), UV melting profile analysis, and time-resolved fluorescence spectroscopy. Association constants were extracted from time-resolved fluorescence data. Preliminary cellular uptake experiments of the glyco-AONARV7 conjugates (120 nM solutions) and of the corresponding glyco-decorated SNAs (10 nM solutions) with human prostate cancer cells (PC3) showed an efficient uptake in each case. A marked variation in intracellular distribution was observed.</p

Pre-Targeting and Direct Immunotargeting of Liposomal Drug Carriers to Ovarian Carcinoma

Peer reviewe

Assembly of Bleomycin Saccharide-Decorated Spherical Nucleic Acids

Glyco-decorated spherical nucleic acids (SNAs) may be attractive delivery vehicles, emphasizing the sugar-specific effect on the outer sphere of the construct and at the same time hiding unfavorable distribution properties of the loaded oligonucleotides. As examples of such nanoparticles, tripodal sugar constituents of bleomycin were synthesized and conjugated with a fluorescence-labeled antisense oligonucleotide (AONARV7). Successive copper(I)-catalyzed azide-alkyne and strain-promoted alkyne-nitrone cycloadditions (SPANC) were utilized for the synthesis. Then, the glyco-AONARV7 conjugates were hybridized with complementary strands of a C60-based molecular spherical nucleic acid (i.e., a hybridization-mediated carrier). The formation and stability of these assembled glyco-decorated SNAs were evaluated by polyacrylamide gel electrophoresis (PAGE), UV melting profile analysis, and time-resolved fluorescence spectroscopy. Association constants were extracted from time-resolved fluorescence data. Preliminary cellular uptake experiments of the glyco-AONARV7 conjugates (120 nM solutions) and of the corresponding glycodecorated SNAs (10 nM solutions) with human prostate cancer cells (PC3) showed an efficient uptake in each case. A marked variation in intracellular distribution was observed.Peer reviewe

Flow cytometric analysis of cellular affinity.

<p>Directly targeted (black), pre-targeted (red) and non-targeted (blue) fluorescein-labeled liposomes were incubated with SKOV-3 (A) and SKOV3.ip1 (B–C) cells. In the pre-targeting group, the cells were incubated with neutravidin-cetuximab for 4 h, washed and incubated with biotin-liposomes for 2 h (A–B) or 4 h (C). In the other groups, the cells were incubated with the liposomes for 2 h (A–B) or 4 h (C). The green line is representing the background fluorescence of untreated cells.</p

Biodistribution of pre-targeted and non-targeted liposomes in mice bearing i.p. SKOV3.ip1 xenografts.

<p>Either neutravidin-cetuximab, at a dose of 20 µg of antibody, or PBS was injected i.p. to the mice. ^99mTc-labeled biotin-liposomes were injected 24 h later i.v. (A) or i.p. (B). Radioactivity of the indicated tissues was determined 24 h from liposome injections. The results are expressed as %ID/g tissue ± SD for pre-targeted liposomes and for non-targeted liposomes (<i>n</i> = 3–4).</p

Biodistribution of EGFR-targeted and non-targeted doxorubicin-liposomes in mice bearing i.p. SKOV-3 xenografts.

<p>Either targeted liposomal DXR or non-targeted liposomal DXR was injected i.v. at a dose of 2 mg/kg. DXR content was assayed in the indicated tissues at 24 h post-treatment (<i>n</i> = 3). Data are expressed as mean of % of injected dose (ID)/g tissue, ± SD.</p

SPECT-CT imaging.

<p>SPECT-CT images of pre-targeted and non-targeted ^99mTc-liposomes 4 h (A–B) and 24 h (C–D) after injection of liposomes, administrated either i.v. (A, C ) or i.p. (B, D). Neutravidin-cetuximab was injected to pre-targeted groups and PBS to non-targeted groups i.p. 24 h before liposome injections. Tumors are marked with white circles on the figures. Minimum and maximum values of intensity were adjusted to the same scale for 4 h images and for 24 h images, respectively.</p

Cytotoxicity of EGFR-targeted and non-targeted doxorubicin-liposomes and free doxorubicin.

<p>SKOV-3 (A) and CV-1 (B) cells were exposed to liposomal and free doxorubicin (DXR) (0.3–80 µM) for 2 h. After exposure to the drug, the cells were washed and incubated in growth medium for 5 days. Cell growth was assayed using Alamar Blue®. The data are presented as mean ± SD.</p

Cellular uptake of non-targeted and EGFR-targeted fluorescein-labeled liposomes.

<p>The SKOV-3 (A) or CV-1 (B) cells were either treated with or without competition with free cetuximab for 1 h at 4°C, washed and incubated with biotin-liposomes (0 µg cetuximab/µmol PL) or cetuximab-biotin-liposomes (3.75; 7.5 or 15 µg cetuximab/µmol PL) for 2 h at 37°C. After incubation, the cells were washed, detached and analyzed by flow cytometry for liposome uptake. The data indicates mean fluorescence ± SD. <i>p</i><0.005 (*), <i>p</i><0.001 (**).</p