The targeted intracellular delivery of cytochrome C protein to tumors using lipid-apolipoprotein nanoparticles

Abstract

Intracellular-acting therapeutic proteins offer a promising clinical alternative to extracellular-acting agents, but are limited in efficacy by their low permeability into the cell cytoplasm. We have developed a nanoparticle (NP) composed of lipid (DOTAP/DOPE) and apolipoprotein (APOA-I) to mediate the targeted delivery of intracellular-acting protein drugs to non-small cell lung tumors. NPs were produced with either GFP, a fluorescent model protein, or cytochrome C (cytC), an inducer of apoptosis in cancer cells. GFP and cytC were separately conjugated with a membrane permeable sequence (MPS) peptide and were admixed with DOPE/DOTAP nanoparticle formulations to enable successful protein loading. Protein-loaded NPs were modified with DSPE-PEG-Anisamide to enable specific NP targeting to the tumor site in a xenograft model. The resulting particle was 20-30 nm in size and exhibited a 64-75% loading efficiency. H460 cells treated with the PEGylated MPS-cytC-NPs exhibited massive apoptosis. When MPS-GFP-NPs or MPS-cytC-NPs were intravenously administered in H460 tumor bearing mice, a specific tumor targeting effect with low NP accumulation in the liver was observed. In addition, MPS-cytC-NP treatment provoked a tumor growth retardation effect in H460 xenograft mice. We conclude that our NP enables targeted, efficacious therapeutic protein delivery for the treatment of lung cancer.

Figure 1

Schematic picture depicting our NP manufacturing platform for protein delivery.

Figure 2

TEM images of PEGylated NPs admixed with MPS-GFP (a) and MPS-cytC (b).

Figure 3

Uptake and intracellular accumulation in H460 cells after 6 h incubation with Alexa 488-labeled MPS-cytC-NPs (green) measured using confocal microscopy. Cell nuclei were stained with DAPI (blue) and cell mitochondria were stained with MitoRed (red).

Figure 4

Apoptotic induction as detected by Annexin V (x axis) and PI (y axis) staining of H460 cells after 12 h treatment with different formulations. The percentage of cells in the Q2 and Q4 quadrants is shown in Table 1 and is expressed as mean ± SEM (N=3)

Figure 5

Distribution of intravenously administered free GFP, free MPS-GFP, GFP + NP, and MPS-GFP-NP in major organs (heart, liver, spleen, kidney, lung, tumor) of H460 xenograft mice imaged using a Xenogen IVIS imaging system. GFP-MPS-NPs were prepared with or without DSPE-PEG/DSPE-PEG-AA as indicated. A control mouse group was treated with empty NP-PEG-AA. Organs were imaged using a kinetic IVIS optical imaging program v 3.1 (a). Confocal microscopy images were taken of tumor tissue from mice treated with MPS-GFP-NP-PEG-AA (b). Blue coloring indicates DAPI nuclear staining while green coloring represents GFP expression.

Figure 6

Distribution of intraveneously administered Alexa-488 labeled cytC, cytC + NP, or MPS-cytC-NPs in major organs (heart, liver, kidney, lung, spleen, tumor) of H460 xenograft mice imaged using a Xenogen IVIS imaging system. NP formulations were prepared with or without DSPE-PEG/DSPE-PEG-AA as indicated. Empty NP-PEG-AA, free cytC and free MPS-cytC served as control treatments. Organs were imaged (a) and quantified (b) using a kinetic IVIS optical imaging program v 3.1. Error bars indicate the Standard Deviation of the data.

Figure 7

Localization of activated Caspase-3 by immunohistochemistry in the tumor sections of mice treated with PBS (a), empty NP (b), cytC (c), cytC + NP-PEG-AA (d), MPS-cytC (e), and MPS-cytC-NP-PEG-AA (f). All tissues were stained with Caspase-3 antibody (brown staining).

Figure 8

Tumor growth retardation effect of different formulation treatments in an H460 xenograft mouse model. CytC (40 µg/kg) or MPS-cytC (160 µg/kg) were admixed with NPs and the resulting particles were further PEGylated with a 1:1 mixture of DSPE-PEG₂₀₀₀ and DSPE-PEG₂₀₀₀-AA. Each formulation was administered to mice once every two days via i.v administration (a). The dosage effect of MPS-cytC-NP-PEG-AA in H460 tumor model was evaluated after intravenous injection every other day. Doses for this study included 80, 160 and 320 µg/kg of MPS-cytC-NP-PEG-AA (P value: * < 0.05, ** < 0.001), Mean ± SEM (n=5).(b)