Technology

Figure 2. Functionalized nanoparticles targeted to cell cytoplasm. Two different types of primary human fibroblast cells (A) and (B) were treated for 30 minutes with fluorescent nanoparticles functionalized to target cell cytoplasm, washed and stained with DAPI (blue fluorescence showing nuclei). The green fluorescence shows localization of the functionalized nanoparticles in the cell cytoplasm.

Figure 3. Functionalized nanoparticles targeted to the cytoplasm of human hematopoietic cells. Human hematopoietic cells were treated for 60 minutes with fluorescent nanoparticles functionalized to target cell cytoplasm, washed and stained with DAPI (blue fluorescence showing nuclei). The green fluorescence shows localization of the functionalized nanoparticles in the cell cytoplasm.

Stemgenics multi-specific nanoparticles are highly efficient in delivering bioactive molecules into the cytoplasm of adherent and non-adherent hematopoietic cells

Imaging of human cells treated with nanoparticles functionalized with cytoplasmic targeting elements and labeled with a green fluorescent dye demonstrates that functionalized nanoparticles exhibit nearly 100% efficiency in penetrating through plasma membrane and entering the cytoplasm of the cells. Representative high-resolution images of the cells with cytoplasmic localization of functionalized nanoparticles are depicted in Figure 2 (human fibroblasts) and Figure 3 (human hematopoietic cells).

Figure 4. Nuclear localization of nanoparticle multi-functionalized with proteins and cytoplasmic and nuclear targeting elements. After 26 hours of treatment of human fibroblasts with fluorescently (FITC) labeled multi-functionalized nanoparticles, live cells were washed and visualized by fluorescence microscopy. The high-resolution fluorescence imaging of live cells shows the multi-functionalized nanoparticles concentrated in the nuclei of treated cells.

Figure 5. Nuclear localization of FITC-labeled nanoparticle multi-functionalized with proteins and cytoplasmic and nuclear targeting elements in primary human fibroblasts. The cells treated with nanoparticles multi-functionalized with proteins, cytoplasmic and nuclear targeting elements were fixed, extensively washed, stained with DAPI and images acquired using high resolution fluorescence microscope.

Fluorescently (FITC) labeled nanoparticles multi-functionalized with cell reprogramming polypeptide molecules, cytoplasmic and nuclear targeting elements effectively enter the cells and concentrate in the nucleus of treated cells. Representative high-resolution images of fluorescently labeled nanoparticles in live cells (Figure 4) or fixed cells (Figure 5) demonstrate virtually 100% efficiency in intracellular delivery of proteins of interest into the cell nuclei using Stemgenics multi-functionalized nanoparticles.

Figure 6. Protein-functionalized nanoparticles trigger robust expression of downstream target genes in primary human fibroblasts.

To demonstrate that nanoparticles multi-functionalized with bioactive protein not only enter the nucleus but also modify expression of target genes, real-time RT-PCR analysis was used to analyze gene expression of actin and four target genes in control cells and cells treated with functionalized nanoparticles. The real-time RT-PCR results depicted in Figure 6 demonstrate that while actin expression was unchanged, the target genes were overexpressed 58, 8, 22, and 57 fold.

Figure 7. Reprogramming somatic cells into stem cells. The left panel shows fibroblast cells (skin cells) in culture: long narrow cells. The panel on the right shows reprogrammed stem cells: an early stage colony.

Figure 8. Reprogramming somatic cells into stem cells. The left panel shows fibroblast cells (skin cells) in culture: long narrow cells. The panel on the right shows reprogrammed stem cells: a late stage colony of 200 microns.

Culturing mature human cells with cell reprogramming nanoparticles functionalized with bioactive molecules produces early stage stem cell colonies of 100 microns as shown in Figure 7 and late stage stem cell colonies of 200 microns as shown in Figure 8.

Human primary fibroblasts (A) were reprogrammed into human pluripotent stem cells (niPSC) using nanoparticles multi-functionalized with Oct4, Sox2, Nanog and Lin28 transcription factors in complete absence of DNA and characterized by morphology, growth, and presence of characteristic pluripotency markers.

Figure 9. Multi-functionalized nanoparticles are highly efficient in cytoplasmic delivery of gene- specific siRNA into primary human cells. Fluorescence images of control and treated cells demonstrate 100% efficiency in intracellular delivery of gene-specific siRNA into the cell cytoplasm. Real-time quantitative RT-PCRs demonstrate that less than 60 picomoles of gene- specific siRNA is sufficient for 50% reduction in the level of the target gene expression levels.

Figure 10. siRNA-functionalized NIMFA-NP are highly efficient in knocking down target gene expression in primary human cells. Cells treated with control vehicle (1), NIMFA-NP functionalized with control scrambled siRNA (2) or NIMFA-NP functionalized with target gene-specific siRNA (3) were cultured for 24 hours followed by RNA isolation and RT-PCR using either gene-specific or GAPDH-specific primers.

Intracellular delivery of gene-specific siRNAs with Stemgenics NIMFA-NP

Fluorescence images of control and treated cells demonstrate 100% efficiency in intracellular delivery of gene-specific siRNA into the cell cytoplasm using Stemgenics multi-functionalized nanoparticles.

NIMFA-NP functionalized with gene-specific siRNA are highly efficient in knocking down target gene expression in human cells

Stemgenics NIMFA-NPs functionalized with 75 picomoles (pmol) of gene-specific siRNA virtually abolish expression of a target gene as determined by RT-PCR.

Figure 11. Fluorescence images of human cells treated with mRNA-functionalized nanoparticles demonstrate 100% efficiency in intracellular delivery of mCherry mRNA followed by its robust expression as evidenced by intracellular localization of mCherry red fluorescent protein.

Highly efficient intracellular delivery and robust expression of gene-specific mRNA using Stemgenics lipid-free multi-functionalized nanoparticles.

Gene-specific mRNA generated in-house from cDNA was further modified and functionalized onto Stemgenics nanoparticles. Primary human cells treated with mRNA-functionalized nanoparticles were cultured overnight, fixed, and fluorescent images acquired using high resolution fluorescence microscope.