Despite numerous advances in the field, the treatment of cancer remains a challenge. It is recognized that the use of biologic drugs can slow the growth of tumors while causing less systemic damage because they interfere with the signaling pathways in the tumor cells and cause programmed cell death (apoptosis). Effective means of delivering these protein-based therapies directly to a tumor, however, have not yet been developed. Such targeted delivery is expected to significantly enhance the efficacy of these drugs. Yi Tang, a professor of chemical and biomolecular engineering, and researchers at the Henry Samueli School of Engineering and Applied Science at University of California, Los Angeles (UCLA), along with collaborators at the University of Southern California, have developed a polymeric nanoencapsulation approach for the the intracellular delivery of proteins (1).
A goal of apoptosis
Currently, some of the most investigated cancer therapies are designed to induce the apoptotic cell death of tumors. Apoptin is one of the most widely studied (1). It exists as a high molecular weight complex and induces apoptotic cell death when selectively accumulated in the nucleus of tumor cells. This complex is comprised of 30 to 40 subunits that do not adopt a well-defined structure. Unfortunately, the researchers reported that current delivery systems suffer from safety issues or inefficient release of the protein in tumor cells.
The researchers reported that they elected to develop a delivery system for apoptin and specifically used recombinant maltose-binding-protein fused apoptin (MBP–APO) because it is expressed by Escherichia coli in a soluble form and behaves similarly to native apoptin (1). This form of apoptin also exists as a complex with a molecular weight of approximately 2.4 mDa and an average diameter of about 40 nm.
Encapsulation with preservation
The researchers reported that the challenge they faced was developing an encapsulation system that would result in particles of a suitable size for injection (approximately 100 nm) while preserving the “structure” of the MNP-APO during both the encapsulation and release processes in order to ensure effective interaction of the protein once it was delivered to the tumor. Therefore, both steps needed to take place under physiological conditions without the use of surfactants (1).
To address these issues, the researchers elected to use a slightly positively charged water-soluble polymeric nanocapsule (NC). The shell is designed so that no covalent bonds form with the protein and thus complete release is possible. The positive charge protects the capsule from degradation by various enzymes and enables cellular uptake of the NCs via endocytosis. In addition, the polymer network that forms the shell is linked together by compounds that contain disulfide bonds. These bonds degrade once the nanocapsule enters the reducing environment of the cytoplasm (1). To encapsulate the protein, the researchers dissolved the MNP-APO in a carbonate buffer, and then the monomers acrylamide and N-(3-aminopropyl)methacrylamide, as well as the crosslinker N,N′-bis(acryloyl)cystamine, were deposited electrostatically onto the protein (1). “These water-soluble compounds were chosen because they can be readily copolymerized to form a polymeric film. The acrylamide serves as the basic building block while N-(3-aminopropyl)methacrylamide is positively charged, and thus its incorporation makes the entire surface of the nanocapsule positive,” explains Tang.
The process was optimized by the researchers so that protein aggregation and precipitation were minimized and the stability of the NCs in the solution was maximized. Excess reagents were then removed using ultrafiltration. The morphology of the nanocapsules was characterized, and the release of the protein upon exposure to a reductive environment was also confirmed (1).
Successful uptake results
The researchers reported that the next step was evaluation of the performance of the nanocapsules with respect to cellular uptake. In order to track the NCs by fluorescent microscopy, for these experiments the MNP-APO was conjugated to the dye rhodamine prior to encapsulation (1). When the researchers added the encapsulated protein to appropriately grown MDA-MB-231 cells, the MNP-APO was detected in the cytoplasm within one hour, and NCs with lesser positive charges experienced lower cellular internalization. The fate of the NCs once taken up by HeLa cells was also tracked. The concentration of MNP-APO was highest in early endosomes at 30 minutes and then declined. After two hours, the protein was detected in the nuclei, indicating successful release of the apoptin (1). “This performance was in line with our expectations. We have used these systems on other types of cells, and a one-to-two hour period is typical for internalization and release,” notes Yang.
Effective tumor delivery
Finally, the researchers reported that they tested their delivery technology by injecting the nanocapsules into tumors (breast cancer cells) grafted onto mice (1). Tumors injected with a saline solution or nanocapsules synthesized without the disulfide bonds (and thus not degradable) continued to grow rapidly, while the growth of tumors treated with the active nanocapsules was significantly delayed. These tumors also exhibited the highest level of cell apoptosis, indicating that the inhibition of growth was likely due to apoptin released from the NCs, according to Tang (1).
“The results of this study have been very rewarding. We have confirmed that our redox-responsive polymeric nanocapsules can deliver a high molecular weight protein in vitro and in vivo. Importantly,” says Tang. “This approach does not present the risk of genetic mutation posed by gene therapies for cancer or the risk to healthy cells caused by chemotherapy, which does not effectively discriminate between healthy and cancerous cells.”
The scientists are currently working on improving the effectiveness of the nanocapsules. The research involves modifying the surface of the nanocapsules with ligands designed to target specific cancer cells, prolonging the circulation time of the capsules, and delivering other important anticancer protein-based drugs. If that can be achieved, Tang believes that this new nanocapsule technology can be utilized to develop anticancer treatments that can be delivered intravenously.
1. M. Zhao, et al., Nano Today 8 (1) 11-20 (2013).