Bioconjugation of calcium phosphosilicate composite nanoparticles for selective targeting of human breast and pancreatic cancers in vivo

Abstract

The early diagnosis of cancer is the critical element in successful treatment and long-term favorable patient prognoses. The high rate of mortality is mainly attributed to the tendency for late diagnoses as symptoms may not occur until the disease has metastasized, as well as the lack of effective systemic therapies. Late diagnosis is often associated with the lack of timely sensitive imaging modalities. The promise of nanotechnology is presently limited by the inability to simultaneously seek, treat, and image cancerous lesions. This study describes the design and synthesis of fluorescent calcium phosphosilicate nanocomposite particles (CPNPs) that can be systemically targeted to breast and pancreatic cancer lesions. The CPNPs are a approximately 20 nm diameter composite composed of an amorphous calcium phosphate matrix doped with silicate in which a near-infrared imaging agent, indocyanine green (ICG), is embedded. In the present studies, we describe and validate CPNP bioconjugation of human holotransferrin, anti-CD71 antibody, and short gastrin peptides via an avidin-biotin or a novel PEG-maleimide coupling strategy. The conjugation of biotinylated human holotransferrin (diferric transferrin) and biotinylated anti-CD71 antibody (anti-transferrin receptor antibody) to avidin-conjugated CPNPs (Avidin-CPNPs) permits targeting of transferrin receptors, which are highly expressed on breast cancer cells. Similarly, the conjugation of biotinylated pentagastrin to Avidin-CPNPs and decagastrin (gastrin-10) to PEG-CPNPs via PEG-maleimide coupling permits targeting of gastrin receptors, which are overexpressed in pancreatic cancer lesions. These bioconjugated CPNPs have the potential to perform as a theranostic modality, simultaneously enhancing drug delivery, targeting, and imaging of breast and pancreatic cancer tumors.

Figure 1

Zeta potential distributions for Citrate-CPNPs, Avidin-CPNPs, PEG-CPNPs, and Gastrin-10-PEG-CPNPs. The Citrate-CPNPs (blue line) displayed a mean zeta potential of −16 ± 1.3 mV, whereas PEGylation shifted the mean zeta potential to +3.0 ± 2.0 mV (red), gastrin 10 conjugation further shifted the mean zeta potential to +6 ± 3.2 mV (violet), and the Avidin-CPNPs (green line) had a mean zeta potential value of +29 ± 8.7 mV. All zeta potential distributions represent the average of three independent experiments.

Figure 2

Dynamic light scattering determinations for Citrate-CPNP, Anti-CD71-Avidin-CPNPs (Anti-CD71-Av-CPNP), Human Holotransferrin-Avidin-CPNPs (Tf-Av-CPNP), Pentagastrin-Avidin-CPNPs (PG-Av-CPNP), maleimidePEG-CPNPs (PEG-CPNP), and Gastrin 10-maleimidePEG-CPNPs (Gastrin-10-PEG-CPNP). All dynamic light scattering determinations are the mean of three independent experiments. Inset shows a typical TEM micrograph of Citrate-CPNPs.

Figure 3

The displacement of 2,6-ANS was utilized to evaluate the coupling of biotin to Avidin-CPNPs. (A) Fluorescence intensities for the first step of the 2,6-ANS assay. The addition of 2,6-ANS to the Avidin-CPNP complex results in a six fold increase in fluorescence as the fluorescent probe binds to the biotin binding site on avidin. The 2,6-ANS was added at increasing concentrations to Avidin-CPNPs and increased fluorescence, indicative of 2,6-ANS bound to avidin, was quantitatively determined. (B) Peak height of fluorescence shown on Figure 3A as a function of 2,6-ANS molarity. (C) Fluorescence intensities for the second step of the 2,6-ANS assay. The addition of biotin to the 2,6-ANS-Avidin-CPNP complex results in a decrease in fluorescence as biotin displaces the fluorescent probe from the biotin binding site on avidin. Biotin was added at increasing concentrations to the 2,6-ANS-Avidin-CPNP complex and a decrease in fluorescence, indicative of biotin displacing 2,6-ANS, was quantitatively determined. (D) Peak height of fluorescence shown on Figure 3C as a function of biotin molarity. All determinations are representative of three independent experiments.

Figure 4

Targeting transferrin receptors in an in vivo subcutaneous-tumor model of breast cancer. (A) Human MDA-MB-231 metastatic breast cancer cells were analyzed via flow cytometry for the presence of the transferrin receptor (CD71). (B) MDA-MB-231 cells were xenografted subcutaneously into athymic nude mice. One week following engraftment, ICG-loaded CPNPs were administered systemically via tail vein injection and near-infrared images were taken 96 hours post-injection. From left to right, mice receiving: (i) free ICG, (ii) ICG-loaded, PEG-CPNPs, (iii) ICG-loaded, Anti-CD71-Avidin-CPNPs, or (iv) ICG-loaded, Human Holotransferrin-Avidin-CPNPs. (C) Excised tumors (mice ii, iii, and iv from panel B), and spleen and stomach (mouse ii). All images are representative of four independent experiments.

Figure 5

ICG-loaded PEG-CPNP clearance via hepatobiliary secretion. 24 hours post-tail vein injection, the kidney, liver, spleen, and intestine were excised and imaged. Increased signal towards end of intestine as indicated by fecal pellets within intestine. All images are representative of three independent experiments.

Figure 6

Gastrin receptor-targeted CPNPs effectively targeted human BxPC-3 pancreatic cancer cells. BxPC-3 cells were exposed to fluorescein-loaded untargeted PEG-CPNPs, or Gastrin- 10-PEG-CPNPs, for 5 minutes followed by exchange to fresh media for 55 minutes, or exposure for 60 minutes. (A) Cells were fixed and visualized by microscopy. (B) Cells were fixed and analyzed by flow cytometry with graphs representing 10,000 collected events per sample.

Figure 7

Targeting gastrin receptors in an in vivo orthotopic-tumor model of pancreatic cancer. Human BxPC-3 pancreatic cancer cells were xenografted orthotopically into athymic nude mice. (A) One week following engraftment, ICG-loaded CPNPs were administered systemically via tail vein injection and near-infrared images were taken 24 hours post-injection. From left to right, mice receiving: (i) ICG-loaded, PEG-CPNPs, (ii) ICG-loaded, Gastrin- 10-PEG-CPNPs, or (iii) ICG-loaded, Pentagastrin-Avidin-CPNPs. (B) Excised, tumor-bearing, pancreases from each mouse, and excised brain (mouse ii). All images are representative of at least four independent experiment

Figure 8

Receptor, or surface feature, targeting strategies and pitfalls. CPNPs were designed to target transferrin receptors (CD71) or gastrin receptors via antibody or ligand coupling via non-covalent (avidin-biotin interactions) and covalent (PEG linker) coupling strategies. Receptor-targeted CPNPs utilizing coupled-antibodies may interact via epitopes separate from ligand-binding sites. Ligand-coupled CPNPs may interact with the receptor, however interference can occur in the form of steric hindrance (avidin or particle interferes with interaction), or ligand competition with the natural ligand.