Targeting of Cancer Cells Using Quantum Dot-Polypeptide Hybrid Assemblies that Function as Molecular Imaging Agents and Carrier Systems

Abstract

We report a highly tunable quantum dot (QD)-polypeptide hybrid assembly system with potential uses for both molecular imaging and delivery of biomolecular cargo to cancer cells. In this work, we demonstrate the tunability of the assembly system, its application for imaging cancer cells, and its ability to carry a biomolecule. The assemblies are formed through the self-assembly of carboxyl-functionalized QDs and poly(diethylene glycol-L-lysine)-poly(L-lysine) (PEGLL-PLL) diblock copolypeptide molecules, and they are modified with peptide ligands containing a cyclic arginine-glycine-aspartate [c(RGD)] motif that has affinity for α_vβ₃ and α_vβ₅ integrins overexpressed on the tumor vasculature. To illustrate the tunability of the QD-polypeptide assembly system, we show that binding to U87MG glioblastoma cells can be modulated and optimized by changing either the conditions under which the assemblies are formed or the relative lengths of the PEGLL and PLL blocks in the PEGLL-PLL molecules. The optimized c(RGD)-modified assemblies bind integrin receptors on U87MG cells and are endocytosed, as demonstrated by flow cytometry and live-cell imaging. Binding specificity is confirmed by competition with an excess of free c(RGD) peptide. Finally, we show that the QD-polypeptide assemblies can be loaded with fluorescently labeled ovalbumin, as a proof-of-concept for their potential use in biomolecule delivery.

Keywords: hybrid materials; nanostructures; quantum dots; self-assembly; supramolecular materials.

Figure 1

Schematic of the application of quantum dot (QD)-polypeptide assemblies as dual imaging and targeted drug-delivery agents. The self-assembly between the QDs (red) and PEGLL-PLL diblock copolypeptide molecules (green), in which the PEGLL block adopts an α-helical conformation, forms an assembly that can take the shape of a shell-like structure. Receptor-mediated binding and endocytosis of assemblies modified with cancer-targeting ligands would allow molecular imaging and drug delivery to be performed simultaneously.

Figure 2

Fluorescence microscopy image of c(RGD)-modified assemblies, which appear as fluorescent “dots”, formed using c(RGDfC)-PEGLL₂₀-PLL₇₂ with a conjugation ratio of 4.6 and a charge ratio (R’) = 8.5 × 10⁻³ (see Experimental). The negative control (inset), which corresponds to a QD suspension having the same QD concentration as that in the QD- polypeptide assembly mixture, did not exhibit any fluorescence. Scale bar: 3 μm.

Figure 3

Flow cytometry data performed using U87MG cells to illustrate the tunability of the QD-polypeptide assembly system. (A) Comparison of the level of non-specific binding exhibited by unmodified assemblies formed using two different architectures—PEGLL₂₀-PLL₇₂ and PEGLL₁₁-PLL₁₂₉—with R’ = 5.8 × 10⁻³. Cells were incubated with varying concentrations of the unmodified assemblies; the concentration is reported as the concentration of the PEGLL-PLL molecules that constitute the assemblies. (B) Binding of QD-polypeptide assemblies as a function of the charge ratio (R’). c(RGD)-assemblies were formed using c(RGDfC)-PEGLL₂₀-PLL₇₂ conjugates having a conjugation ratio = 4.6; the Submitted to assembly concentration was ~ 0.7 pM. The level of integrin-mediated binding was estimated as the % difference between the mean fluorescence intensities associated with c(RGD)- modified and unmodified assemblies, and is depicted above the bars. See SI 4 for the calculation of the assembly concentration.

Figure 3

Flow cytometry data performed using U87MG cells to illustrate the tunability of the QD-polypeptide assembly system. (A) Comparison of the level of non-specific binding exhibited by unmodified assemblies formed using two different architectures—PEGLL₂₀-PLL₇₂ and PEGLL₁₁-PLL₁₂₉—with R’ = 5.8 × 10⁻³. Cells were incubated with varying concentrations of the unmodified assemblies; the concentration is reported as the concentration of the PEGLL-PLL molecules that constitute the assemblies. (B) Binding of QD-polypeptide assemblies as a function of the charge ratio (R’). c(RGD)-assemblies were formed using c(RGDfC)-PEGLL₂₀-PLL₇₂ conjugates having a conjugation ratio = 4.6; the Submitted to assembly concentration was ~ 0.7 pM. The level of integrin-mediated binding was estimated as the % difference between the mean fluorescence intensities associated with c(RGD)- modified and unmodified assemblies, and is depicted above the bars. See SI 4 for the calculation of the assembly concentration.

Figure 4

Binding and microscopy data to demonstrate the feasibility of the c(RGD)-assembly system as a targeted imaging and biomolecule carrier system. (A) Binding of c(RGD)- assemblies to U87MG cells as evaluated by flow cytometry. To demonstrate integrin-binding specificity, cells were incubated with a 2500-fold molar excess of free c(RGDfC) prior to addition of the c(RGD)-assemblies. The assembly concentration was ~ 0.7 pM. (B) Binding and uptake of c(RGD)-assemblies by U87MG cells as evaluated by confocal microscopy. U87MG cells were incubated with ~ 0.1 pM of c(RGD)-assemblies in the absence (left) and presence (right) of a 100-fold molar excess of c(RGDfC). Scale bar: 20 μm. In (A) and (B), assemblies were formed using c(RGDfC)-PEGLL₂₀-PLL₇₂ conjugates having a conjugation ratio of 4.6 with R’ = 8.5 × 10⁻³. (C) Confocal microscopy image showing the loading of Alexa Fluor 647 labeled ovalbumin into c(RGD)-assemblies. Co-localization of the ovalbumin fluorescence (red) with that of the QD-polypeptide assembly (blue) results in a pink color. A blow-up of the confocal microscopy image depicting the co-localization is shown (inset). Excess ovalbumin that was not loaded into the assemblies was not removed, and it formed aggregates (red) that were detected by confocal microscopy. Scale bar: 5 μm.

Figure 4

Binding and microscopy data to demonstrate the feasibility of the c(RGD)-assembly system as a targeted imaging and biomolecule carrier system. (A) Binding of c(RGD)- assemblies to U87MG cells as evaluated by flow cytometry. To demonstrate integrin-binding specificity, cells were incubated with a 2500-fold molar excess of free c(RGDfC) prior to addition of the c(RGD)-assemblies. The assembly concentration was ~ 0.7 pM. (B) Binding and uptake of c(RGD)-assemblies by U87MG cells as evaluated by confocal microscopy. U87MG cells were incubated with ~ 0.1 pM of c(RGD)-assemblies in the absence (left) and presence (right) of a 100-fold molar excess of c(RGDfC). Scale bar: 20 μm. In (A) and (B), assemblies were formed using c(RGDfC)-PEGLL₂₀-PLL₇₂ conjugates having a conjugation ratio of 4.6 with R’ = 8.5 × 10⁻³. (C) Confocal microscopy image showing the loading of Alexa Fluor 647 labeled ovalbumin into c(RGD)-assemblies. Co-localization of the ovalbumin fluorescence (red) with that of the QD-polypeptide assembly (blue) results in a pink color. A blow-up of the confocal microscopy image depicting the co-localization is shown (inset). Excess ovalbumin that was not loaded into the assemblies was not removed, and it formed aggregates (red) that were detected by confocal microscopy. Scale bar: 5 μm.

Figure 4

Binding and microscopy data to demonstrate the feasibility of the c(RGD)-assembly system as a targeted imaging and biomolecule carrier system. (A) Binding of c(RGD)- assemblies to U87MG cells as evaluated by flow cytometry. To demonstrate integrin-binding specificity, cells were incubated with a 2500-fold molar excess of free c(RGDfC) prior to addition of the c(RGD)-assemblies. The assembly concentration was ~ 0.7 pM. (B) Binding and uptake of c(RGD)-assemblies by U87MG cells as evaluated by confocal microscopy. U87MG cells were incubated with ~ 0.1 pM of c(RGD)-assemblies in the absence (left) and presence (right) of a 100-fold molar excess of c(RGDfC). Scale bar: 20 μm. In (A) and (B), assemblies were formed using c(RGDfC)-PEGLL₂₀-PLL₇₂ conjugates having a conjugation ratio of 4.6 with R’ = 8.5 × 10⁻³. (C) Confocal microscopy image showing the loading of Alexa Fluor 647 labeled ovalbumin into c(RGD)-assemblies. Co-localization of the ovalbumin fluorescence (red) with that of the QD-polypeptide assembly (blue) results in a pink color. A blow-up of the confocal microscopy image depicting the co-localization is shown (inset). Excess ovalbumin that was not loaded into the assemblies was not removed, and it formed aggregates (red) that were detected by confocal microscopy. Scale bar: 5 μm.

Figure 4

Binding and microscopy data to demonstrate the feasibility of the c(RGD)-assembly system as a targeted imaging and biomolecule carrier system. (A) Binding of c(RGD)- assemblies to U87MG cells as evaluated by flow cytometry. To demonstrate integrin-binding specificity, cells were incubated with a 2500-fold molar excess of free c(RGDfC) prior to addition of the c(RGD)-assemblies. The assembly concentration was ~ 0.7 pM. (B) Binding and uptake of c(RGD)-assemblies by U87MG cells as evaluated by confocal microscopy. U87MG cells were incubated with ~ 0.1 pM of c(RGD)-assemblies in the absence (left) and presence (right) of a 100-fold molar excess of c(RGDfC). Scale bar: 20 μm. In (A) and (B), assemblies were formed using c(RGDfC)-PEGLL₂₀-PLL₇₂ conjugates having a conjugation ratio of 4.6 with R’ = 8.5 × 10⁻³. (C) Confocal microscopy image showing the loading of Alexa Fluor 647 labeled ovalbumin into c(RGD)-assemblies. Co-localization of the ovalbumin fluorescence (red) with that of the QD-polypeptide assembly (blue) results in a pink color. A blow-up of the confocal microscopy image depicting the co-localization is shown (inset). Excess ovalbumin that was not loaded into the assemblies was not removed, and it formed aggregates (red) that were detected by confocal microscopy. Scale bar: 5 μm.