No endosome required

Professor Vince Rotello takes us through nanoparticle-stabilised capsules – the ideal vehicle for protein delivery to the cell

The delivery of functional proteins to replace or augment protein levels within the cell is an emerging strategy for the treatment of disease states such as inflammation¹ or cancer².

There are two key challenges to this approach. First, the protein must be transported into the cell, something that doesn't happen for most proteins. Second, proteins need to have access to the cytosol, where they can perform their desired function. Most methods of transporting proteins inside cells goes through endocytosis, where the cell engulfs materials into vesicular components. Endosomes tend to trap and degrade proteins, making escaping endosomes or (even better) avoiding endosomal uptake critical to effective delivery³. Mechanical/physical approaches, such as microinjection and electroporation puncture the cell membrane and thus deliver the protein directly into the cytoplasm. However, these strategies have low in vivo efficiencies restricting their practical application⁴. Another method for the delivery of proteins is the covalent attachment of proteins to a carrier. These modified proteins are generally taken up via an endocytotic mechanism, resulting in limited release of the protein into the cytosol⁵.

[caption id="attachment_36206" align="alignright" width="300"] Figure 1: Structure and self-assembly strategy to generate NPSCs.[/caption]

Supramolecular drug and protein delivery vehicles are promising vehicles for delivery applications. The reversibility and modular nature of these structures make them versatile tools for interfacing with biological systems. Recently, a group at the University of Massachusetts Amherst led by Vincent M. Rotello has developed an adaptable delivery vehicle for hydrophobic therapeutics⁶ and in recent studies extended this work to include therapeutic proteins⁷. The system relies on self-assembly of nanoparticles and proteins at an oil/water interface to form nanoparticle-stabilised capsules (NPSCs, Figure 1). As shown in Figure 1, the multivalent interactions between the nanoparticles, fatty acid molecules, and protein provide a robust core shell structure with a biologically relevant size (~120 nm).

The recent study by the Rotello group focused on delivery of two proteins: green fluorescent protein (GFP) and Caspase-3 (CASP3), an enzyme that induces controlled cell death (apoptosis). Within 1 hour of treatment with NPSCs containing GFP, fluorescence was evenly distributed throughout HeLa cancer cells cell (Figure 2a). Comparison of the green fluorescence with red fluorescent protein (RFP) expressed by the cells indicated complete co-distribution of GFP and RFP, and hence complete access of the delivered GFP to the cytosol. A similar delivery strategy was used to deliver apoptosis-inducing protein CASP3. The HeLa cells treated with NPSCs containing CASP3 displayed a high level of apoptosis (Figure 2b) demonstrating that active enzymes can be delivered to the cell.

[caption id="attachment_36207" align="alignleft" width="200"] Figure 2. Delivery of proteins into HeLa cells by NPSCs: (a) GFP, showing cytosolar distribution (b) CASP3, demonstrating effective induction of apoptosis. Delivery of (c) GFP-PTS1, and (d) normal GFP into peroxisome-labeled HeLa cells. showing intracellular targeting of the tagged protein.[/caption]

A key strength of the supramolecular NPSC system is its modularity, allowing proteins to be swapped in and out without having to synthesise new materials.  To demonstrate this versatility, GFP fused with a peroxisomal targeting agent was incorporated into the NPSC. As expected, these fused GFP proteins were observed colocalised with peroxisomes after delivery (Figure 2c). In contrast, no colocalisation with peroxisomes occurred for normal GFP (Figure 2d).

In summary, these studies have demonstrated that NPSCs provide a rapid and effective means to deliver proteins in native form into the cytosol in vitro. This delivery opens up a range of applications in fundamental cell biology as well as in tissue engineering. If this method can be extended to in vivo systems, it could open up whole therapeutic areas to protein-mediated therapies.

References:   

1.  Jo, D.; Liu, D. Y.; Yao, S.; Collins, R. D.; Hawiger, J. “Intracellular Protein Therapy with Socs3 Inhibits Inflammation and Apoptosis” Nat. Med., 2005, 11, 892-898.
2. Leader, B.; Baca, Q. J.; Golan, D. E. “Protein Therapeutics: A Summary and Pharmacological Classification” Nat. Rev. Drug Discov., 2008, 7, 21-39.
3. Yan, M.; Du, J.; Gu, Z.; Liang, M.; Hu, Y.; Zhang, W.; Priceman, S.; Wu, L.; Zhou, Z. H.; Liu, Z.; Segura, T.; Tang, Y.; Lu, Y. “A Novel Intracellular Protein Delivery Platform Based on Single-Protein Nanocapsules” Nat. Nanotechnol., 2010, 5, 48-53.
4. Zhang, Y.; Yu, L. C. “Microinjection as a Tool of Mechanical Delivery” Curr. Opin. Biotechnol., 2008, 19, 506-510.
5. Kim, D.; Kim, C. H.; Moon, J. I.; Chung, Y. G.; Chang, M. Y.; Han, B. S.; Ko, S.; Yang, E.; Cha, K. Y.; Lanza, R.; Kim, K. S. “Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins” Cell Stem Cell, 2009, 4, 472-476.
6. Yang, X.C.; Samanta, B.; Agasti, S. S.; Jeong, Y.; Zhu, Z.; Rana, S.; Miranda, O. R.; Rotello, V. M. "Drug DeliveryUsing Nanoparticle-Stabilized Nanocapsules" Angew. Chem. Int. Ed., 2011, 50, 477-481.
7. Tang, R.; Kim, C. S.; Solfiell, D. J.; Rana, S.; Mout, R.; Velázquez-Delgado, E. M.; Chompoosor, A.; Jeong, Y.; Yan, B.; Zhu, Z.-J.; Kim, C.; Hardy, J. A.; Rotello, V. M. "Direct Delivery of Functional Proteins and Enzymes to the Cytosol Using Nanoparticle-Stabilized Nanocapsules" ACS Nano, 2013, 7, 6667-6673.