Selective Capture and Recovery of Monoclonal Antibodies by Self-Assembling Supramolecular Polymers of High Affinity for Protein Binding.

The separation and purification of therapeutic proteins from their biological resources pose a great limitation for industrial manufacturing of biologics in an efficient and cost-effective manner. We report here a supramolecular polymeric system that can undergo multiple reversible processes for efficient capture, precipitation, and recovery of monoclonal antibodies (mAbs). These supramolecular polymers, namely immunofibers (IFs), are formed by coassembly of a mAb-binding peptide amphiphile with a rationally designed filler molecule of varying stoichiometric ratios. Under the optimized conditions, IFs can specifically capture mAbs with a precipitation yield greater than 99%, leading to an overall mAb recovery yield of 94%. We also demonstrated the feasibility of capturing and recovering two mAbs from clarified cell culture harvest. These results showcase the promising potential of peptide-based supramolecular polymers as reversible affinity precipitants for mAb purification.

Mechanisms of precipitate formation during the purification of an Fc-fusion protein.

Protein precipitates that arise during bioprocessing can cause manufacturing challenges, but they can also aid in clearance of host-cell protein (HCP) and DNA impurities. Such precipitates differ from many protein precipitates that have been studied previously in their heterogeneous composition, particularly in the presence of high concentrations of the product protein. Here, we characterize the precipitates that form after neutralization of protein A purified and viral-inactivated material of an Fc-fusion protein produced in Chinese hamster ovary cells. The physical growth of precipitate particles was observed by optical microscopy, transmission electron microscopy, dynamic light scattering, and small-angle and ultra-small-angle X-ray scattering to characterize the precipitate microstructure and growth mechanism. The precipitate microstructure is well-described as a mass fractal with fractal dimension approximately 2. The growth is governed by a diffusion-limited aggregation mechanism as indicated by a power-law dependence on time of the size of the principal precipitate particles. Optical microscopy shows that these primary particles can further aggregate into larger particles in a manner that appears to be promoted by mixing. Absorbance experiments at varying pH and salt concentrations reveal that the growth is largely driven by attractive electrostatic interactions, as growth is hindered by an increase in ionic strength. The solution conditions that resulted in the most significant particle growth are also correlated with the greatest removal of soluble impurities (DNA and HCPs). Proteomic analysis of the precipitates allows identification of O ( 100 ) unique HCP impurities, depending on the buffer species (acetate or citrate) used for the viral inactivation. Most of these proteins have pI values near the precipitation pH, supporting the likely importance of electrostatic interactions in driving precipitate formation.

Conformation Preservation of α-Helical Peptides within Supramolecular Filamentous Assemblies.

Hydrogen-bonded ß-sheets are the most commonly explored building motifs for creating peptide-based filamentous nanostructures; however, most bioactive epitopes must assume an α-helix conformation to exert their functions. Incorporating α-helical sequences into ß-sheet-forming peptides often involves the use of a flexible spacer to alleviate the steric impact of the intermolecular hydrogen bonding on the α-helical conformation. In this context, we report our findings on the alkylation-regulated conformation preservation of α-helical peptides within their filamentous assemblies. We found that the chemical conjugation of two short linear hydrocarbons (octanoic acids, C8) can retain the α-helical conformation of two protein A-derived peptide sequences while effectively driving their assembly into filamentous nanostructures. In contrast, the use of a single palmitoyl tail (C16) of similar hydrophobicity would lead to formation of ß-sheet assemblies. Our studies further demonstrated that the length of the conjugated hydrocarbon also plays an important role in partially preserving the native α-helical conformation, with longer ones promoting ß-sheet formation and short ones stabilizing α-helices to some extent. We believe that these findings offer important guiding principles for the alkylation of self-assembling peptides containing α-helical sequences.

Tuning Cellular Uptake of Molecular Probes by Rational Design of Their Assembly into Supramolecular Nanoprobes.

Intracellular sensing of pathologically relevant biomolecules could provide essential information for accurate evaluation of disease staging and progression, yet the poor cellular uptake of water-soluble molecular probes limits their use as protease sensors. In other cases such as extracellular sensing, cellular uptake should be effectively inhibited. Self-assembly of molecular probes into supramolecular nanoprobes presents a potential strategy to alter their interaction mechanisms with cells to promote or reduce their cellular uptake. Here, we report on the design, synthesis, and assembly of peptide-based molecular beacons into supramolecular protease sensors of either spherical or filamentous shapes. We found that positively charged spherical nanobeacons demonstrate much higher cellular uptake efficiency than its monomeric form, thus making them most suitable for intracellular sensing of the lysosomal protease cathepsin B. Our results also suggest that assembly into filamentous nanobeacons significantly reduces their internalization by cancer cells, an important property that can be utilized for probing extracellular protease activities. These studies provide important guiding principles for rational design of supramolecular nanoprobes with tunable cellular uptake characteristics.

Enhanced cellular entry and efficacy of tat conjugates by rational design of the auxiliary segment.

Conjugation with a cell penetrating peptide such as Tat presents an effective approach to improve the intracellular accumulation of molecules with low membrane permeability. This strategy, however, leads to a reduced cellular entry of molecules that can cross cell membrane effectively. We report here that covalent linkage of an additional hydrophobic unit that mimics a hydrophobic domain near the Tat sequence can further improve the cellular uptake of the parental conjugate into cancer cells regardless of the membrane permeability of the unconjugated molecule. Both fluorescent imaging and flow cytometry measurements confirmed the effect of palmitoylation on the increased internalization of the Tat conjugates with either 5-carboxyfluorescein (5-FAM), a nonmembrane penetrating dye, or doxorubicin, an anticancer cancer drug that can readily diffuse across cell membranes. In the case of the Tat-doxorubicin conjugate, palmitoylation improves the conjugate's anticancer activity in both drug sensitive and resistant cervical cancer cell lines. We further demonstrate that modification of a Tat-5-FAM conjugate with a hydrophobic quencher could not only efficiently quench the fluorescence outside of cancer cell but also facilitate its entry into MCF-7 breast cancer cells. These results highlight the importance of rational molecular design of using peptide conjugation chemistry in cancer therapeutics and diagnostics.

Multimeric disintegrin protein polymer fusions that target tumor vasculature.

Recombinant protein therapeutics have increased in number and frequency since the introduction of human insulin, 25 years ago. Presently, proteins and peptides are commonly used in the clinic. However, the incorporation of peptides into clinically approved nanomedicines has been limited. Reasons for this include the challenges of decorating pharmaceutical-grade nanoparticles with proteins by a process that is robust, scalable, and cost-effective. As an alternative to covalent bioconjugation between a protein and nanoparticle, we report that biologically active proteins may themselves mediate the formation of small multimers through steric stabilization by large protein polymers. Unlike multistep purification and bioconjugation, this approach is completed during biosynthesis. As proof-of-principle, the disintegrin protein called vicrostatin (VCN) was fused to an elastin-like polypeptide (A192). A significant fraction of fusion proteins self-assembled into multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion proteins compete specifically for cell-surface integrins on human umbilical vein endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that, unlike linear RGD-containing protein polymers, the disintegrin fusion protein undergoes rapid cellular internalization. To explore their potential clinical applications, fusion proteins were characterized using small animal positron emission tomography (microPET). Passive tumor accumulation was observed for control protein polymers; however, the tumor accumulation of A192-VCN was saturable, which is consistent with integrin-mediated binding. The fusion of a protein polymer and disintegrin results in a higher intratumoral contrast compared to free VCN or A192 alone. Given the diversity of disintegrin proteins with specificity for various cell-surface integrins, disintegrin fusions are a new source of biomaterials with potential diagnostic and therapeutic applications.

Supramolecular nanostructures formed by anticancer drug assembly.

We report here a supramolecular strategy to directly assemble the small molecular hydrophobic anticancer drug camptothecin (CPT) into discrete, stable, well-defined nanostructures with a high and quantitative drug loading. Depending on the number of CPTs in the molecular design, the resulting nanostructures can be either nanofibers or nanotubes, and have a fixed CPT loading content ranging from 23% to 38%. We found that formation of nanostructures provides protection for both the CPT drug and the biodegradable linker from the external environment and thus offers a mechanism for controlled release of CPT. Under tumor-relevant conditions, these drug nanostructures can release the bioactive form of CPT and show in vitro efficacy against a number of cancer cell lines. This strategy can be extended to construct nanostructures of other types of anticancer drugs and thus presents new opportunities for the development of self-delivering drugs for cancer therapeutics.

Cellular uptake and cytotoxicity of drug-peptide conjugates regulated by conjugation site.

Conjugation of anticancer drugs to hydrophilic peptides such as Tat is a widely adopted strategy to improve the drug's solubility, cellular uptake, and potency against cancerous cells. Here we report that attachment of an anticancer drug doxorubicin to the N- or C-terminal of the Tat peptide can have a significant impact on their cellular uptake and cytotoxicity against both drug-sensitive and drug-resistant cancer cells. We observed higher cellular uptake by both cell lines for C-terminal conjugate relative to the N-terminal analogue. Our results reveal that the C-terminal conjugate partially overcame the multidrug resistance of cervical cancer cells, while the N-terminal conjugate showed no significant improvement in cytotoxicity when compared with free doxorubicin. We also found that both N- and C-conjugates offer a mechanism to circumvent drug efflux associated with multidrug resistance.

Emerging biomaterials for downstream manufacturing of therapeutic proteins.

Downstream processing is considered one of the most challenging phases of industrial manufacturing of therapeutic proteins, accounting for a large portion of the total production costs. The growing demand for therapeutic proteins in the biopharmaceutical market in addition to a significant rise in upstream titers have placed an increasing burden on the downstream purification process, which is often limited by high cost and insufficient capacities. To achieve efficient production and reduced costs, a variety of biomaterials have been exploited to improve the current techniques and also to develop superior alternatives. In this work, we discuss the significance of utilizing traditional biomaterials in downstream processing and review the recent progress in the development of new biomaterials for use in protein separation and purification. Several representative methods will be highlighted and discussed in detail, including affinity chromatography, non-affinity chromatography, membrane separations, magnetic separations, and precipitation/phase separations. STATEMENT OF SIGNIFICANCE: Nowadays, downstream processing of therapeutic proteins is facing great challenges created by the rapid increase of the market size and upstream titers, starving for significant improvements or innovations in current downstream unit operations. Biomaterials have been widely used in downstream manufacturing of proteins and efforts have been continuously devoted to developing more advanced biomaterials for the implementation of more efficient and economical purification methods. This review covers recent advances in the development and application of biomaterials specifically exploited for various chromatographic and non-chromatographic techniques, highlighting several promising alternative strategies.

Bioinspired supramolecular engineering of self-assembling immunofibers for high affinity binding of immunoglobulin G.

Many one-dimensional (1D) nanostructures are constructed by self-assembly of peptides or peptide conjugates containing a short ß-sheet sequence as the core building motif essential for the intermolecular hydrogen bonding that promotes directional, anisotropic growth of the resultant assemblies. While this molecular engineering strategy has led to the successful production of a plethora of bioactive filamentous ß-sheet assemblies for interfacing with biomolecules and cells, concerns associated with effective presentation of α-helical epitopes and their function preservation have yet to be resolved. In this context, we report on the direct conjugation of the protein A mimicking peptide Z33, a motif containing two α-helices, to linear hydrocarbons to create self-assembling immuno-amphiphiles (IAs). Our results suggest that the resulting amphiphilic peptides can, despite lacking the essential ß-sheet segment, effectively associate under physiological conditions into supramolecular immunofibers (IFs) while preserving their native α-helical conformation. Isothermal titration calorimetry (ITC) measurements confirmed that these self-assembling immunofibers can bind to the human immunoglobulin G class 1 (IgG1) with high specificity at pH 7.4, but with significantly weakened binding at pH 2.8. We further demonstrated the accessibility of Z33 ligand in the immunofibers using transmission electron microscopy (TEM) and confocal imaging. We believe these results shed important light into the supramolecular engineering of α-helical peptides into filamentous assemblies that may possess an important potential for antibody isolation.

One-Component Supramolecular Filament Hydrogels as Theranostic Label-Free Magnetic Resonance Imaging Agents.

Gadolinium (Gd)-based compounds and materials are the most commonly used magnetic resonance imaging (MRI) contrast agents in the clinic; however, safety concerns associated with their toxicities in the free ionic form have promoted the development of new generations of metal-free contrast agents. Here we report a supramolecular strategy to convert an FDA-approved anticancer drug, Pemetrexed (Pem), to a molecular hydrogelator with inherent chemical exchange saturation transfer (CEST) MRI signals. The rationally designed drug-peptide conjugate can spontaneously associate into filamentous assemblies under physiological conditions and consequently form theranostic supramolecular hydrogels for injectable delivery. We demonstrated that the local delivery and distribution of Pem-peptide nanofiber hydrogels can be directly assessed using CEST MRI in a mouse glioma model. Our work lays out the foundation for the development of drug-constructed theranostic supramolecular materials with an inherent CEST MRI signal that enables noninvasive monitoring of their in vivo distribution and drug release.

Enzyme-Specific Doxorubicin Drug Beacon as Drug-Resistant Theranostic Molecular Probes.

We report here on the use of anticancer drug doxorubicin (Dox) to construct a Förster resonance energy transfer (FRET)-based theranostic molecular probe by covalently linking together through a lysine junction a fluorescent drug, a black hole quencher, and a cell-penetrating peptide. We show that upon cleavage by the target lysosomal protease cathepsin B (CatB) the designed drug beacon could release the fluorescent drug serving as an indicator for CatB. Our cell studies suggest that the drug-beacon design can help to circumvent the Dox drug resistance in NCI/ADR-Res ovarian cancer cells, showing significant improvement in cell cytotoxicity compared to the free drug. We believe our design opens up new opportunities to exploit the new functional and structural features of anticancer drugs in addition to their characteristic cytotoxicity.

Activatable nanoprobes for biomolecular detection.

Precise detection of pathologically relevant biomolecules could provide essential information on important intercellular, cellular, and subcellular events for accurate disease diagnosis and staging, thus leading to appropriate treatment recommendation. Activatable nanoprobes are nanoscale objects that can be turned on through specific reactions or interactions with biomolecules of interest, and afford some advantageous properties for improved detection of biomolecules both in vitro and in vivo. In this brief review, we highlight several recent examples in the development of activatable nanoprobes for biomolecule detection.

The Role of Micelle Size in Tumor Accumulation, Penetration, and Treatment.

The specific sizes that determine optimal nanoparticle tumor accumulation, penetration, and treatment remain inconclusive because many studies compared nanoparticles with multiple physicochemical variables (e.g., chemical structures, shapes, and other physical properties) in addition to the size. In this study, we synthesized amphiphilic block copolymers of 7-ethyl-10-hydroxylcamptothecin (SN38) prodrug and fabricated micelles with sizes ranging from 20 to 300 nm from a single copolymer. The as-prepared micelles had exactly the same chemical structures and similar physical properties except for size, which provided an ideal platform for a systematic investigation of the size effects in cancer drug delivery. We found that the micelle's blood circulation time and tumor accumulation increased with the increase in their diameters, with optimal diameter range of 100 to 160 nm. However, the much higher tumor accumulation of the large micelles (100 nm) did not result in significantly improved therapeutic efficacy, because the large micelles had poorer tumor penetration than the small ones (30 nm). An optimal size that balances drug accumulation and penetration in tumors is critical for improving the therapeutic efficacy of nanoparticulate drugs.

An amphipathic alpha-helical peptide from apolipoprotein A1 stabilizes protein polymer vesicles.

L4F, an alpha helical peptide inspired by the lipid-binding domain of the ApoA1 protein, has potential applications in the reduction of inflammation involved with cardiovascular disease as well as an antioxidant effect that inhibits liver fibrosis. In addition to its biological activity, amphipathic peptides such as L4F are likely candidates to direct the molecular assembly of peptide nanostructures. Here we describe the stabilization of the amphipathic L4F peptide through fusion to a high molecular weight protein polymer. Comprised of multiple pentameric repeats, elastin-like polypeptides (ELPs) are biodegradable protein polymers inspired from the human gene for tropoelastin. Dynamic light scattering confirmed that the fusion peptide forms nanoparticles with a hydrodynamic radius of approximately 50nm, which is unexpectedly above that observed for the free ELP (~5.1nm). To further investigate their morphology, conventional and cryogenic transmission electron microscopy were used to reveal that they are unilamellar vesicles. On average, these vesicles are 49nm in radius with lamellae 8nm in thickness. To evaluate their therapeutic potential, the L4F nanoparticles were incubated with hepatic stellate cells. Stellate cell activation leads to hepatic fibrosis; furthermore, their activation is suppressed by anti-oxidant activity of ApoA1 mimetic peptides. Consistent with this observation, L4F nanoparticles were found to suppress hepatic stellate cell activation in vitro. To evaluate the in vivo potential for these nanostructures, their plasma pharmacokinetics were evaluated in rats. Despite the assembly of nanostructures, both free L4F and L4F nanoparticles exhibited similar half-lives of approximately 1h in plasma. This is the first study reporting the stabilization of peptide-based vesicles using ApoA1 mimetic peptides fused to a protein polymer; furthermore, this platform for peptide-vesicle assembly may have utility in the design of biodegradable nanostructures.

Design and construction of supramolecular nanobeacons for enzyme detection.

Molecular beacons are typically water-soluble molecules that can convert specific chemical reactions or binding events into measurable optical signals, providing a noninvasive means to help understand cellular and subcellular activities at the molecular level. However, the soluble form of the current molecular beacon design often leads to their poor stability and facile degradation by nonspecific enzymes, and as a result, this undesired activation could give rise to false signals and thus poses a limitation for accurate detection of enzymatic activities. Here we report a proof-of-concept design and synthesis of a new type of supramolecular nanobeacon that is resistant to nonspecific enzymatic degradation in the self-assembled state but can be effectively cleaved by the target enzyme in the monomeric form. Our results show that the nanobeacon with a GFLG peptide linker could serve as an indicator for the presence of a lysosomal enzyme, cathepsin B.

Self-assembly of natural and synthetic drug amphiphiles into discrete supramolecular nanostructures.

Molecular assembly provides an effective approach to construct discrete supramolecular nanostructures of various sizes and shapes in a simple manner. One important technological application of the resulting nanostructures is their potential use as anticancer drug carriers to facilitate targeted delivery to tumour sites and consequently to improve clinical outcomes. In this carrier-assisted delivery strategy, anticancer drugs have been almost exclusively considered as the cargo to be carried and delivered, and their potential as molecular building blocks has been largely ignored. In this discussion, we report the use of anticancer drugs as molecular building units to create discrete supramolecular nanostructures that contain a high and quantitative drug loading and also have the potential for self-delivery. We first show the direct assembly of two amphiphilic drug molecules (methotrexate and folic acid) into discrete nanostructures. Our results reveal that folic acid exhibits rich self-assembly behaviour via Hoogsteen hydrogen bonding under various solvent conditions, whereas methotrexate is unable to assemble into any well-defined nanostructures under the same conditions, despite its similar chemical structure. Considering the low water solubility of most anticancer drugs, hydrophilic segments must be conjugated to the drug in order to bestow the necessary amphiphilicity. We have demonstrated this for camptothecin through the attachment of beta-sheet-forming peptides with overall hydrophilicity. We found that the intermolecular interactions among camptothecin segments and those among beta-sheet peptides act together to define the formation of stable one-dimensional nanostructures in dilute solutions, giving rise to nanotubes or nanofibers depending upon the processing conditions used. These results lead us to believe that self-assembly of drugs into discrete nanostructures not only offers an innovative way to craft self-delivering anticancer drugs, but also extends the paradigm of using molecular assembly as a toolbox to achieve functional nanostructures, to a new area which is specifically focused on the direct assembly of functional molecules (e.g. drugs, or imaging agents) into nanostructures of their own.