Concentration-Independent Multivalent Targeting of Cancer Cells by Genetically Encoded Core-Crosslinked Elastin/Resilin-like Polypeptide Micelles.

Valency is a fundamental principle to control macromolecular interactions and is used to target specific cell types by multivalent ligand-receptor interactions using self-assembled nanoparticle carriers. At the concentrations encountered in solid tumors upon systemic administration, these nanoparticles are, however, likely to show critical micelle concentration (CMC)-dependent disassembly and thus loss of function. To overcome this limitation, core-crosslinkable micelles of genetically encoded resilin-/elastin-like diblock polypeptides were recombinantly synthesized. The amphiphilic constructs were covalently photo-crosslinked through the genetically encoded unnatural amino acid para-azidophenylalanine in their hydrophobic block and they carried different anticancer ligands on their hydrophilic block: the wild-type tenth human fibronectin type III domain, a GRGDSPAS peptide-both targeting αvß3 integrin-and an engineered variant of the third fibronectin type III domain of tenascin C that is a death receptor 5 agonist. Although uncrosslinked micelles lost most of their targeting ability below their CMC, the crosslinked analogues remained active at concentrations up to 1000-fold lower than the CMC, with binding affinities that are comparable to antibodies.

Quantitative Study of the Interaction of Multivalent Ligand-Modified Nanoparticles with Breast Cancer Cells with Tunable Receptor Density.

Multivalent nanoparticles that target a cell surface receptor that is overexpressed by cancer cells are a promising delivery system for cancer therapy. However, the impact of the receptor density and nanoparticle ligand valency on the cell uptake has not been studied in a system where both variables can be systematically tuned over a wide range. To address this lacuna, we report cell-uptake studies on a genetically engineered breast cancer cell line with tunable ErbB2 expression by a polypeptide micelle with tunable ligand valency. We examined the uptake of ErbB2-targeting micelles at 5 ligand densities and 11 receptor densities. We identified a matching pattern between receptors and ligands in which a receptor-to-ligand density ratio of 0.7-4.5 and a minimum of ∼1.6 bonds are required to initiate receptor-mediated endocytosis. Lower and upper limits of receptor density in the cell-uptake profile suggested a standard by which to categorize breast cancer patients as ErbB2-low, ErbB2-medium, and ErbB2-high, with each group expected to respond differently to multivalent therapeutic nanoparticles. At ErbB2-medium and ErbB2-high levels, increasing the ligand valency to 40-valent ErbB2-targeting peptides for a 20 nm radius nanoparticle accelerated the cell uptake, suggesting that the use of nanoparticles with high ligand valency for drug delivery will greatly benefit patients in these two groups. This study advances our understanding of how to rationally optimize nanotechnology for targeted drug delivery.

A Recombinant Secondary Antibody Mimic as a Target-specific Signal Amplifier and an Antibody Immobilizer in Immunoassays.

We construct a novel recombinant secondary antibody mimic, GST-ABD, which can bind to the Fc regions of target-bound primary antibodies and acquire multiple HRPs simultaneously. We produce it in tenth of mg quantities with a bacterial overexpression system and simple purification procedures, significantly reducing the manufacturing cost and time without the use of animals. GST-ABD is effectively conjugated with 3 HRPs per molecule on an average and selectively bind to the Fc region of primary antibodies derived from three different species (mouse, rabbit, and rat). HRP-conjugated GST-ABD (HRP-GST-ABD) is successfully used as an alternative to secondary antibodies to amplify target-specific signals in both ELISA and immunohistochemistry regardless of the target molecules and origin of primary antibodies used. GST-ABD also successfully serves as an anchoring adaptor on the surface of GSH-coated plates for immobilizing antigen-capturing antibodies in an orientation-controlled manner for sandwich-type indirect ELISA through simple molecular recognition without any complicated chemical modification.

An enhanced ascorbate peroxidase 2/antibody-binding domain fusion protein (APEX2-ABD) as a recombinant target-specific signal amplifier.

A recombinant target-specific signal amplifier was constructed by genetically fusing the enhanced ascorbate peroxidase 2 (APEX2) and an antibody-binding domain (ABD). The fusion protein APEX2-ABD possessed the peroxidase activity and the antibody-binding capability simultaneously and replaced the conventional HRP-conjugated secondary antibodies in a TSA assay for amplifying fluorescence signals.

Developing genetically engineered encapsulin protein cage nanoparticles as a targeted delivery nanoplatform.

Protein cage nanoparticles are excellent candidates for use as multifunctional delivery nanoplatforms because they are built from biomaterials and have a well-defined structure. A novel protein cage nanoparticle, encapsulin, isolated from thermophilic bacteria Thermotoga maritima, is prepared and developed as a versatile template for targeted delivery nanoplatforms through both chemical and genetic engineering. It is pivotal for multifunctional delivery nanoplatforms to have functional plasticity and versatility to acquire targeting ligands, diagnostic probes, and drugs simultaneously. Encapsulin is genetically engineered to have unusual heat stability and to acquire multiple functionalities in a precisely controlled manner. Hepatocellular carcinoma (HCC) cell binding peptide (SP94-peptide, SFSIIHTPILPL) is chosen as a targeting ligand and displayed on the surface of engineered encapsulin (Encap_loophis42C123) through either chemical conjugation or genetic insertion. The effective and selective targeted delivery of SP94-peptide displaying encapsulin (SP94-Encap_loophis42C123) to HepG2 cells is confirmed by fluorescent microscopy imaging. Aldoxorubicin (AlDox), an anticancer prodrug, is chemically loaded to SP94-Encap_loophis42C123 via thiol-maleimide Michael-type addition, and the efficacy of the delivered drugs is evaluated with a cell viability assay. SP94-Encap_loophis42C123-AlDox shows comparable killing efficacy with that of free drugs without the platform's own cytotoxicity. Functional plasticity and versatility of the engineered encapsulin allow us to introduce targeting ligands, diagnostic probes, and therapeutic reagents simultaneously, providing opportunities to develop multifunctional delivery nanoplatforms.

Development of P22 viral capsid nanocomposites as anti-cancer drug, bortezomib (BTZ), delivery nanoplatforms.

Genetic and chemical engineering approaches are used to employ P22 viral capsids as nanoplatforms for developing an efficient delivery vehicle. Catechol ligands are chemically attached to the interior surface of P22 viral capsid for subsequent encapsulation of an anticancer drug, bortezomib (BTZ), through boronic acid-diol complexation. For targeted delivery, hepatocellular carcinoma (HCC)-targeting peptide (SP94, SFSIIHTPILPL) is synthesized and chemically conjugated to the exterior surface of the P22 viral capsid nanocomposites. Effective targeted delivery of synthesized P22 viral capsid nanocomposites is demonstrated by fluorescent cell imaging and the efficacy of delivered P22 viral capsid nanocomposites is evaluated using a cell viability assay.

Genetically engineering encapsulin protein cage nanoparticle as a SCC-7 cell targeting optical nanoprobe.

BACKGROUND: Protein cage nanoparticles are promising nanoplatform candidates for efficient delivery systems of diagnostics and/or therapeutics because of their uniform size and structure as well as high biocompatibility and biodegradability. Encapsulin protein cage nanoparticle is used to develop a cell-specific targeting optical nanoprobe. RESULTS: FcBPs are genetically inserted and successfully displayed on the surface of encapsulin to form FcBP-encapsulin. Selectively binding of FcBP-encapsulin to SCC-7 is visualized with fluorescent microscopy. CONCLUSIONS: Encapsulin protein cage nanoparticle is robust enough to maintain their structure at high temperature and easily acquires multifunctions on demand through the combination of genetic and chemical modifications.

Implementation of P22 viral capsids as intravascular magnetic resonance T1 contrast conjugates via site-selective attachment of Gd(III)-chelating agents.

P22 viral capsids and ferritin protein cages are utilized as templating macromolecules to conjugate Gd(III)-chelating agent complexes, and we systematically investigates the effects of the macromolecules' size and the conjugation positions of Gd(III)-chelating agents on the magnetic resonance (MR) relaxivities and the resulting image contrasts. The relaxivity values of the Gd(III)-chelating agent-conjugated P22 viral capsids (outer diameter: 64 nm) are dramatically increased as compared to both free Gd(III)-chelating agents and Gd(III)-chelating agent-conjugated ferritins (outer diameter: 12 nm), suggesting that the large sized P22 viral capsids exhibit a much slower tumbling rate, which results in a faster T1 relaxation rate. Gd(III)-chelating agents are attached to either the interior or exterior surface of P22 viral capsids and the conjugation positions of Gd(III)-chelating agents, however, do not have a significant effect on the relaxivity values of the macromolecular conjugates. The contrast enhancement of Gd(III)-chelating agent-conjugated P22 viral capsids is confirmed by in vitro phantom imaging at a short repetition times (TR) and the potential usage of Gd(III)-chelating agent-conjugated P22 viral capsids for in vivo MR imaging is validated by visualizing a mouse's intravascular system, including the carotid, mammary arteries, the jugular vein, and the superficial vessels of the head at an isotropic resolution of 250 µm.