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Multi-functionality Redefined with Colloidal Carotene Carbon Nanoparticles for Synchronized Chemical Imaging, Enriched Cellular Uptake and Therapy - PubMed

  • ️Fri Jan 01 2016

Multi-functionality Redefined with Colloidal Carotene Carbon Nanoparticles for Synchronized Chemical Imaging, Enriched Cellular Uptake and Therapy

Santosh K Misra et al. Sci Rep. 2016.

Abstract

Typically, multiplexing high nanoparticle uptake, imaging, and therapy requires careful integration of three different functions of a multiscale molecular-particle assembly. Here, we present a simpler approach to multiplexing by utilizing one component of the system for multiple functions. Specifically, we successfully synthesized and characterized colloidal carotene carbon nanoparticle (C(3)-NP), in which a single functional molecule served a threefold purpose. First, the presence of carotene moieties promoted the passage of the particle through the cell membrane and into the cells. Second, the ligand acted as a potent detrimental moiety for cancer cells and, finally, the ligands produced optical contrast for robust microscopic detection in complex cellular environments. In comparative tests, C(3)-NP were found to provide effective intracellular delivery that enables both robust detection at cellular and tissue level and presents significant therapeutic potential without altering the mechanism of intracellular action of β-carotene. Surface coating of C(3) with phospholipid was used to generate C(3)-Lipocoat nanoparticles with further improved function and biocompatibility, paving the path to eventual in vivo studies.

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Figures

Figure 1
Figure 1. Schematic representation of C3-NP usases.

(A) Graphical representation of a multifunctional nanoparticle system in anhydrous state presenting carotene functionalities on the surface for synchronous imaging, therapy and cellular transport. (B) Schematic portraying CNPs containing carotenoids driven into the cellular phospholipid bilayer. (C) Assembly of C3-NP from molecular to nanoscale, showing the final particle of use.

Figure 2
Figure 2. Physico-chemical characterization of C3-NPs and C3-Lipocoat nanoparticles.

(A) Hydrodynamic diameter; (B) Zeta potential; anhydrous state size of (C,D) C3-NPs and (E,F) C3 Lipocoat nanoparticles by TEM; (G,H) height profile of C3-NPs by AFM. The spectroscopic evaluation of integral photonic properties of C3-NPs and compared to bare-CNP and β-carotene itself for (I) fluorescence properties and (J) UV-vis absorption efficiency. Properties were well compared with bare-CNP and β-carotene formulations to show the co-existence in C3 particles.

Figure 3
Figure 3. Characteristic Infra-red spectral features.

Panel A shows the baseline corrected IR spectrum of a typical cell, C3 and C3 in cells. Spectra are offset for clarity. Panels B and C show the details of the IR spectra of the same in two different spectral regions. Characteristic spectral features of C3 are observed in cells as shown in Panel B at 1045 cm−1. Panel C displays the increase in the peak intensity ratio between ν2922 and ν2957 cm−1.

Figure 4
Figure 4. Infra-red cellular imaging.

IR images of the cell and C3 incubated in cells for 5 cases as mentioned in the rows. (A,D,G,J,M) Show the average, baseline corrected IR spectra with the variance, while two separate images corresponding to each of the conditions are displayed in the rest. The intensity of each pixel is calculated as the ratio between 2922 and 2958 cm−1 band.

Figure 5
Figure 5. Raman cellular imaging.

(A,C,E,I,K) Display the bright field images of breast cancer cells incubated with C3 at 5 different conditions. Raman images of the same regions are shown in (B,D,F,J,L) respectively. The C-H region intensity is plotted in the adjacent color bar while the C3 intensities are false-colored in red. Representative Raman spectra are also shown for cells only and cells incubated with C3 in M and N. Since the C-H region is not affected by C3, it is used to isolate cellular regions, while the Raman spectra of the CNPs are used to isolate the C3s.

Figure 6
Figure 6. In vitro cellular studies.

MTT assay for the evaluation of cytotoxic effects of β-carotene in free or passivated to CNPs with post coatings of amphiphilic phospholipidic assembly. Experiment was performed in (A,C) MDA-MB231 breast cancer and (B,D) C32 melanoma cells after incubation for (A,B) 48 and (C,D) 72 h at various concentrations (125, 62.5, 31.25, 15.6125, 7. 8 and 3.9 μM) of β-carotene in free or form of C3 nanoparticles and C3-Lipocoat nanoparticle formulations. (E) Comparative IC50 values of used formulations and (F) fold change across both the cell lines. Statistical analysis performed using Two-way ANOVA on IC50 values represented as *** for p < 0.001.

Figure 7
Figure 7. Mechanistic studies for induced apoptosis via reactive oxygen species (ROS) generation.

(A) DNA laddering assay performed on genomic DNA extracted from MDA-MB231 cells (lane 1) untreated; or treated with (lane 2) C3-Lipocoat; (lane 3) C3 nanoparticles; (lane 4) CNP and (lane 5) β-carotene at concentration of 100 μM. Here lane 6 represent DNA ladder of 1–10 kb. Scatter plots obtained from 4% paraformaldehyde fixed MDA-MB231 cells (B) untreated or treated with (C) β-carotene; (D) C3 and (E) C3-Lipocoat at concentration of 100 μM post propidium iodide (PI; 10 μg/mL) staining. Scattering events in R7 and R2 gates represent the cells from live cell and apoptotic cell population, respectively. (F) Scattering events revealed the increasing % of apoptotic cell population in order of β-carotene, C3 and C3-Lipocoat while % live population followed the reverse order with maximum in untreated cells. (G) Variation in ROS abundance with time of incubation and change in β-carotene formulation in MDA-MB231 with maximum fold increase by C3-Lipocoat compared to CNP itself (H). Statistical analysis performed using Two-way ANOVA on IC50 values represented as *** for p < 0.001.

Figure 8
Figure 8. Ex-vivo fluorescence imaging.

Panel A shows the 3D fluorescence experimental geometry. The objective generates a plane sheet of light that irradiates the sample. Fluorescence is acquired orthogonal to the light plane. Panel B shows a three dimensional rendering of background subtracted fluorescence images of pigskin treated with C3. Panel C shows the integrated fluorescence intensity with skin depth for both treated and untreated samples. Panel D shows the concentration profile of C3 within 2 layers of the skin upon diffusion.

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