Peptide–nanoparticle conjugates: a next generation of diagnostic and therapeutic platforms?

Peptide–nanoparticle conjugates (PNCs) have recently emerged as a versatile tool for biomedical applications. Synergism between the two promising classes of materials allows enhanced control over their biological behaviors, overcoming intrinsic limitations of the individual materials. Over the past decades, a myriad of PNCs has been developed for various applications, such as drug delivery, inhibition of pathogenic biomolecular interactions, molecular imaging, and liquid biopsy. This paper provides a comprehensive overview of existing technologies that have been recently developed in the broad field of PNCs, offering a guideline especially for investigators who are new to this field.

Nanomaterials (tens to a few hundreds of nanometers in size) possess novel physico-chemical properties distinct from those of conventional bulk materials. Their ultra-small size and high surface-area-to-volume ratio are advantageous in the development of engineered materials that can uniquely interact with a variety of nano- and micro-sized biomaterials [ 12 ]. The most straightforward approach to fabricate peptides-based nanostructures is self-assembly [ 13 , 14 ]. However, the spontaneity in the thermodynamic process does not allow the construction of nano-scale constructs having precisely regulated shape, size, and compositions. In contrast, peptide–NP conjugation offers enhanced control over the structural properties of nanostructures, allowing facile modification to overall shape, dimension, and size of the conjugates through engineering NP scaffolds tailored for intended applications. Another important aspect that the PNCs can provide is multivalency. Most interactions in biological systems are based on non-covalent interactions such as hydrogen bonds, ionic bonds, van der Waals forces, π–π stacking forces, and hydrophobic interactions. Although the individual bindings are relatively weak, their co-operative action enables strong binding kinetics (typically due to substantial decrease in dissociation kinetics through the multivalent binding effect) based on the principle that the collective binding strength depends exponentially on to the number of individual binding pairs (Fig. 2 a) [ 15 – 17 ]. In addition to the enhanced binding strength, multivalent interactions also provide improved selectivity by exploiting the density of interaction modules on a surface to recognize target polyvalent surfaces (Fig. 2 b) [ 18 ]. Fig. 2 a Comparison between monovalent- and multivalent interactions. b Selectivity in multivalent interactions. c Multidirectional ligand display and d statistical rebinding on a multivalent object The presence of multiple binding sites plays a role in allowing the strong multivalent bindings and in increasing statistical opportunities for multiple monovalent binding events to occur. As depicted in Fig. 2 c, the exposure of peptides in multiple directions results in greater opportunities to encounter binding partners [ 19 ]. During the dissociation process post binding, peptides on NP scaffolds express many re-binding sites, which can increase the retention time of target materials on the surface, known as the statistical re-binding mechanism (Fig. 2 d) [ 20 ]. Furthermore, co-conjugation with different types of peptides and/or other biological/non-biological materials offers additional functionalities for the hybrid materials, such as immune response evasion [ 21 ], theranostics [ 22 ], stimulus-responsive property [ 23 ], and multi-target directed treatment with a single material [ 24 ]. Consequently, displayed on a nanostructure surface, peptides can potentially compete with or outperform natural proteins despite their low individual affinity and selectivity [ 25 , 26 ]. The non-biological characteristics of NPs introduce novel properties and functions that are otherwise not obtained to their PNCs. For instance, NPs absorbing and emitting near infrared (NIR, 700–1100 nm) light have been actively utilized in in vivo imaging due to the advantages of deep imaging depth and high spatial resolution [ 27 ]. Some NPs produce reactive oxygen species (ROS) upon receiving the light energy, which can oxidize biomacromolecules and subsequently induce cell ablation (photodynamic therapy) [ 28 ]. In addition, the absorbed light energy can be converted to heat and sound energy using photothermal and photoacoustic effects of NPs, providing a non-invasive treatment option for diseases like cancer [ 29 , 30 ]. Magnetic nanoparticles (MNPs) are another promising class enabling the remote and active treatment of diseases. Responding to external magnetic stimuli, MNPs can be selectively accumulated at a target site in biological systems and release guest molecules in a dosage-controlled fashion [ 31 , 32 ]. Several in vitro studies have shown that MNPs, displaying multiple binding ligands, effectively discriminate target biomaterials from a mixture solution [ 33 ]. Furthermore, upon exposure to the magnetic field, the arrangement of MNPs on a surface can be controlled in various ways, resulting in the use of the MNPs for the development of novel cell culture scaffold [ 34 ].

Drugging the ‘undruggable’ targets is one of the key challenges in pharmacological studies [ 50 ]. Approximately 80% of proteins that involved in human diseases lack binding sites for small molecule ligands [ 51 ]. One potential strategy to address this issue is to implement protein-based pharmaceuticals. However, low thermal stability and difficulty in preparation of such proteins have hindered their widespread application [ 52 ]. PNCs provide a new insight to tackle these formidable challenges. For instance, the Lim group demonstrated inorganic NPs that serve as a scaffold for stabilizing peptide folding structures, which can eventually enhance both target affinity and selectivity [ 53 ]. Figure 3 a illustrates α-helical structure stabilized by reduced conformational entropy cost achieved through the use of cyclic peptides and interaction with inorganic surface [ 54 ]. Based on this principle, bioactive α-helical p53 peptides stabilized on AuNP surfaces effectively recognized their target protein, MDM2, which is known to suppress the p53-mediated apoptotic pathway. The therapeutic potential of the cyclic peptide–nanomaterial conjugate system was also demonstrated by inhibiting the α-helix-mediated interaction between Rev protein and Rev response element (RRE) RNA, which regulates HIV-1 gene expression [ 24 , 55 ]. Fig. 3 a Molecular models depicting gold nanoparticle binding-induced stabilization of α-helical structure. b Interactions of free peptides and peptide–nanoparticle conjugates with HIV-1 spike proteins. c Peptide hybrid-functionalized gold nanoparticles inhibiting amyloid-β aggregation The multivalent property of PNCs is a powerful tool for controlling polyvalent macromolecular associations that frequently occur in nature. Chaiken et al. reported that AuNP–peptide triazole conjugates inactivates HIV-1 by disrupting the interactions between host receptor proteins and trimeric envelope glycoprotein (Env) spikes of the virus [ 56 , 57 ]. As AuNP diameter and peptide valency increase, the antiviral potency of the PNCs is greatly enhanced. This implies that a sufficient quantity of peptide triazoles over a large area is required for effective interaction with the multiple spikes on the viral surface (Fig. 3 b). Protein-misfolding diseases including Alzheimer’s disease (AD) are also difficult to target with conventional therapeutics [ 58 ]. Xiong et al. decorated AuNPs with peptides including two inhibitory peptide sequences for Aβ aggregation, VVIA and LPFFD, in order to develop a multivalent inhibitor for the aggregation of amyloid-β (Αβ) proteins [ 59 – 61 ]. The two peptide sequences were conjugated onto the AuNP surfaces and ordered/oriented in optimal conformation to effectively inhibit Aβ aggregation. Utilizing the two different peptides on a single NP was highly synergistic, preventing Aβ aggregation more strongly with less cytotoxicity, compared to the free peptides (Fig. 3 c). In some applications, PNC functionality can be significantly improved by precisely controlling the peptide valency. NPs that are covered with peptides at a higher density typically exhibit increased binding affinity [ 62 ]; however, precisely engineered binding modules that have a specific spacing or certain ligand density have been shown to further enhance the interaction with target molecules in a controlled manner [ 63 , 64 ]. One approach to controlling the ligand valency is to use dendrimers. Dendrimers are hyper-branched polymers that have precisely controlled size, surface property, composition, and density of functional groups through relatively simple chemical reactions [ 65 , 66 ]. In a recent study, Lauster et al. showed that polyglycerol dendrimers decorated with peptides targeting hemagglutinin (HA) can inhibit the infection of influenza A virus (IAV) [ 25 ], which uses multiple HAs for enhanced binding to the host cell surface [ 67 ]. Interestingly, despite the improved antiviral activity of the PNC utilizing the multivalent binding of the HA targeting peptides, the inhibitory capacity was not proportionally increased with an increase of the peptide density. Instead, higher valency reduced the inhibitory activity when it exceeded a certain threshold, indicating that optimization of the surface engineering is required. Another advantage of PNCs is that they can utilize multiple therapeutic pathways by incorporating different types of molecules in a single nanoformulation system [ 68 ]. Recently, Blancafort et al. conjugated poly(glycidyl methacrylate) NPs with peptides targeting Engrailed 1 (EN1), an undruggable transcription factor associated with cell proliferation, metastasis, and chemoresistance of basal-like breast cancer [ 69 ]. An anticancer agent, docetaxel (DTX), was encapsulated in the internal void of this NP. Both in vitro and in vivo studies revealed that the combination of peptidic- and chemotherapeutic agents via PNC induced more apoptosis on cancer cells, compared with using either DTX or E

Liquid biopsy is of high potential significance as a novel tool for diagnosis and prognosis of human diseases [ 100 ]. It refers to any techniques that examine, detect, and analyze disease biomarkers in bodily fluids, most notably blood [ 101 ]. Given its less invasive nature unlike conventional solid biopsy, liquid biopsy would substantially decrease the chance to cause complications while increasing patients’ compliance, allowing more frequent screening, early detection capability, and more accurate monitoring of diseases [ 102 ]. As a result, liquid biopsy provides more comprehensive information from a disease across multiple time points, enabling rapid and effective treatment. Circulating tumor cells (CTCs) [ 101 ], exosomes [ 103 ], cell-free DNAs (cfDNAs) [ 104 ], and circulating microRNAs (miRNAs) [ 105 ] have emerged as potential biomarkers for monitoring human diseases. A number of studies have reported that the genomic or proteomic profiling of these biomarkers is associated with progression, proliferation, recurrence, chemo-sensitivity, and metastatic potential of the disease [ 106 , 107 ]. However, accurate analysis and sensitive detection still remain a challenge due to the low concentration of liquid biopsy biomarkers in human bodily fluids [ 108 ]. Moreover, molecular heterogeneity among the biomarkers, coupled with phenotypic changes that frequently occur during therapeutic treatment and disease progression, makes separation of the biomarker difficult, limiting further downstream analysis [ 109 ]. This section summarizes several new technologies that use PNCs to detect liquid biopsy biomarkers with high sensitivity and specificity (Table 3 ). Antibodies are one of the most extensively used capture agents for separation of disease-related biomarkers, due to their high selectivity and strong binding affinity to specific surface receptors [ 102 , 110 ]. Recent studies suggest that antibodies could be spliced into shorter peptides that still recognize specific surface receptors [ 111 , 112 ]. As molecules that are small, stable, and easy to synthesize, compared to antibodies, peptides provide an opportunity to potentially replace the whole antibodies by addressing the reproducibility and productivity issues that current antibody-based approaches typically have [ 74 ]. Despite these advantages, the low binding affinity to specific target tissues are the major drawbacks of peptides. However, these concerns could be potentially addressed through the PNC approaches. For example, the multivalent binding effect, as described above, could be easily incorporated to various PNCs, which would improve biomarker separation based on the peptide binding to target biomarkers [ 37 , 47 ]. Table 3 Peptide–nanoparticle conjugates for biomarker detection Biomarker Peptide (Pep) Nanoparticle (NP) In vitro studies Clinical application Refs. Target Affinity (method) Type Size In vitro model Capture/detection CTC EpCAM K D : 2.69 × 10 −10 M (SPR) Iron oxide magnetic NP 235 nm (NP) 305 nm (conjugate) MCF-7, SK-BR-3 (breast), PC3 (prostate), Hep G2 (Liver) 90% (capture) 93% (purity) > 90% (viability) N/A [ 111 ] HER2 Capacity: 70% Selectivity: 0.7, (FCM, compared to anti-HER2 Ab) Iron oxide magnetic NP 200 nm MCF-7, SK-BR3 (Breast), SKOV3 (ovarian) 75% (capture) N/A [ 112 ] EGFR K D : 4.59 × 10 −4 M (AFM) Magnetic nanovesicles 219 nm SMMC-7721 (hepatoma) 90% (capture) Tested with 25 lung cancer patients’ samples. [ 114 ] EGFR N/A AuNP 60 nm Tu212 (head and neck), H292, H460 (Lung), MDA-MB-231 (Breast) 1–720 CTCs/mL (sensitivity) > 10 4 :1 (specificity) * SERS detection Tested with 19 head and neck cancer patients’ samples [ 115 ] Exosome and extracellular Vesicle CD63 N/A Nickel Dynabeads N/A Human serum obtained from 10 healthy volunteers 54% (capture) Tested with 15 HCC and 18 pancreatic patients’ samples [ 118 ] Hsp70 N/A Streptavidin-coupled Dynabeads N/A Lysates from MCF-7 (breast) Similar with ultracentrifugation (capture) N/A [ 121 ] cfDNA and miRNA miR-21, miR-96, miR-125b N/A Nano metal—organic framework (NMOF, UiO-66) 125 nm Synthetic miRNA MCF-7, MDA-MB-231 (breast), MCF-10A (non-tumor, breast) < 10 pM (limit of detection) N/A [ 124 ] miR-21, miR-96, miR-125b N/A Nanosized graphene oxide 0.05–300 nm (lateral) 1.03 nm (height) Synthetic miRNA MCF-7, MDA-MB-231 (breast), MDA-MB-435 (melanoma), HeLa (Cervix) 1 pM (limit of detection) N/A [ 125 ] let-7b, let-7c, miR-21 N/A AuNP 10 nm Synthetic miRNA + human serum < 10 fM (limit of detection) one-base mismatch (selectivity) N/A [ 126 ] E542K, E545K, methylation of PIK3CA gene N/A AuNP 50 nm Synthetic ctDNA + human serum 50 fM (limit of detection) N/A [ 127 ] The Yang and Wang groups utilized peptides that recognize epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2) for CTC isolation [ 111 , 112 ]. These peptides were conjugated to iron oxide magnetic NPs for immunomagnetic separation. Although peptides themsel

Dr. Woo-jin Jeong is a postdoctoral researcher in the Prof. Seungpyo Hong’s laboratory in the School of Pharmacy at the University of Wisconsin-Madison. He received his Ph.D. degree in the Department of Materials Science and Engineering at Yonsei University. His research interests include the development of peptide-based nano- and micromaterials for cancer therapeutics and diagnostics. Dr. Jiyoon Bu is a Postdoctoral Researcher in the Hong lab in School of Pharmacy at the University of Wisconsin-Madison. He received his Ph.D. in Bio & Brain Engineering from the Korea Advanced Institute of Science and Technology (KAIST) in 2017. His research focus is on developing novel biomedical devices for cancer diagnostics and therapeutics, based on biomimetics, microfluidics, and nanoengineering. More specifically, he is involved in developing highly-sensitive liquid biopsy platforms and engineering target-specific immunomodulatory nanoparticles. Luke J. Kubiatowicz is an undergraduate in the Department of Engineering Physics at the University of Wisconsin-Madison. He works as a researcher in Dr. Seungpyo Hong’s laboratory within the Wisconsin Center for NanoBioSystems. His research area of interest is the utilization of nanotechnology for biomedical applications. Stephanie S. Chen is an undergraduate student in the College of Agricultural and Life Sciences at the University of Wisconsin-Madison. Her research interests include the development of peptide-dendrimer conjugates for drug transportation. Prof. YoungSoo Kim is an assistant professor in Integrated Science and Engineering Division and Department of Pharmacy at Yonsei University, Republic of Korea. He earned his bachelor degree in biochemistry at New York University in 2001 and his Ph.D. degree in chemistry at The Scripps Research Institute in 2006. Then, Kim joined the Brain Science Institute at Korea Institute of Science and Technology as a principal investigator and, in 2017, moved to Yonsei University. His work focuses on therapeutics and diagnostics of Alzheimer’s disease by utilizing chemical biology as a research tool. Prof. Seungpyo Hong is Professor in Pharmaceutical Sciences Division, School of Pharmacy at University of Wisconsin-Madison. He received his Ph.D. from the University of Michigan in 2006, followed by a postdoctoral training in the Langer lab at MIT. From 2008 to 2016, he was Assistant/Associate Professor in the College of Pharmacy at the University of Illinois at Chicago (UIC), and subsequently joined the UW-Madison faculty as full Professor in 2016. To date, Prof. Hong’s research has culminated in over 75 peer-reviewed articles that have a combined total citation number of over 11,000 times.

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