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Synthesis of Patient-Specific Nanomaterials James Lazarovits, Yih Yang Chen, Fayi Song, Wayne Ngo, Anthony James Tavares, Yinan Zhang, Julie Audet, Bo Tang, Qiaochu Lin, Mayra Cruz Tleugabulova, Stefan Wilhelm, Jonathan Krieger, Thierry Mallevaey, and Warren C.W. Chan Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b03434 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 10, 2018
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Synthesis of Patient-Specific Nanomaterials
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James Lazarovits1, Yih Yang Chen1, Fayi Song1,2, Wayne Ngo1, Anthony J. Tavares1, Yi-Nan Zhang1, Julie Audet1,3,4, Bo Tang1,2, Qiaochu Lin5, Mayra Cruz Tleugabulova5, Stefan Wilhelm1, Jonathan Krieger6, Thierry Mallevaey5, and Warren C. W. Chan1,2,4,6,8* 1Institute
of Biomaterials and Biomedical Engineering, University of Toronto, Rosebrugh Building, Room 407, 164 College Street, Toronto, Ontario M5S 3G9, Canada.
2College
of Chemistry & Chemical Engineering, Chongqing University of Science & Technology, University Town, Shapingba District, Chongqing 401331, PR China.
3Terrence
Donnelly Center for Cellular and Biomolecular Research, University of Toronto, 160 College St, Room 230, Toronto, ON, M5S 3E1, Canada.
4Department
of Chemical Engineering, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada.
5Department
of Immunology, University of Toronto, Medical Sciences Building, Room 7308, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada.
6SPARC
BioCentre, The Hospital for Sick Children, The Peter Gilgan Centre for Research & Learning, 686 Bay Street, 21st Floor Toronto, ON M5G 0A4 Canada.
7Department
of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, M5S 3H6, Canada.
8Department
of Materials Science and Engineering, University of Toronto, Wallberg Building, 184 College Street, Suite 140, Toronto, Ontario M5S 3E4, Canada.
*Corresponding
author:
[email protected].
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ABSTRACT
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Nanoparticles are engineered from materials such as metals, polymers, and different carbon
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allotropes that do not exist within the body. Exposure to these exogenous compounds raises
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concerns surrounding toxicity, inflammation, and immune activation. These responses could
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potentially be mitigated by synthesizing nanoparticles directly from molecules derived from the
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host. However, efforts to assemble patient-derived macromolecules into structures with the same
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degree of size and shape tunability as their exogenous counterparts remains a significant challenge.
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Here we solve this problem by creating a new class of size- and shape-tunable personalized protein
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nanoparticles (PNP) made entirely from patient-derived proteins. PNPs are built into different sizes
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and shapes with the same degree of tunability as gold nanoparticles. They are biodegradable and
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do not activate innate or adaptive immunity following single and repeated administrations in vivo.
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PNPs can be further modified with specific protein cargos that remain catalytically active even
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after intracellular delivery in vivo. Finally, we demonstrate that PNPs created from different human
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patients have unique molecular fingerprints encoded directly into the structure of the nanoparticle.
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This new class of personalized nanomaterial has the potential to revolutionize how we treat
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patients and can become an integral component in the diagnostic and therapeutic toolbox.
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Keywords: nanotechnology, personalized medicine, patient-specific nanomaterials, protein corona, protein nanoparticles, mass spectrometry
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MANUSCRIPT
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Nanomaterials are integral components in the diagnostic and therapeutic toolbox and
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possess a unique set of optical, magnetic, and electrical properties that are derived from their
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material composition1. These parameters include the size, shape, and surface chemistry of the
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nanomaterial, all of which could dictate the biodistribution of the nanomaterial or its cellular
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interactions with the rest of the body2–4. Since these nanomaterials are similar in size to biological
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molecules, manipulating these physicochemical properties can theoretically control their transport
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to specific organs and cells. The core materials that have this high degree of synthetic tunability
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are built using substances that are exogenous to the body1. These include metals, polymers, and
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different carbon allotropes. When these foreign materials are assembled into nanoparticles greater
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than 6 nm, they cannot be renally excreted and are not eliminated from the body5. Therefore,
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exposure to these nanomaterials raises numerous concerns surrounding toxicity, inflammation, and
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activation of innate or adaptive immunity, especially following repeated administrations6–11. In
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order to mitigate these side effects, several groups have used endogenous materials found within
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the body, such as lipids, to produce biocompatible nanoparticles12,13. Since the body contains a
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reservoir of many different types of endogenous building block materials, we can use this supply
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to build libraries of non-toxic and non-immunogenic personalized nanostructures. However,
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current methods cannot produce personalized nanostructures with the same degree of size and
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shape tunability as their exogenous counterparts1. Here we demonstrate a solution to this problem
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with a simple three-step templating strategy to engineer personalized nanoparticles that achieves
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the same level of design freedom and complexity as exogenous nanomaterial classes while being
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non-toxic and non-immunogenic.
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Proteins are an abundant class of endogenous materials that can be exploited as building
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blocks for nanoparticle synthesis. Protein adsorption to nanoparticle surfaces is seen as a major
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problem for nanomaterial design14, because it obfuscates a nanoparticle’s underlying synthetic
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properties. Previous work has shown that the types and abundances of proteins that adsorb to
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nanoparticles are predictable and reproducible, because it depends on the underlying nanoparticle
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size, shape and surface chemistry14–19. However, we can neither prevent protein adsorption, nor
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has the field found a way to exploit it for a useful application. In his study we discovered how to
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exploit protein adsorption on nanostructures to build personalized protein nanoparticles (PNP). To
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do this, we incubated nanoparticles with biological fluids that were isolated from animals and
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humans, crosslinked the surface-adsorbed proteins in place, and removed the internal nanoparticle
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core to leave a nanostructure composed entirely of endogenous and patient-derived proteins (Fig.
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1a). Since size, shape, and surface chemistry tunability depends on the nanoparticle core template,
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we used gold nanoparticles (AuNP) because they have the broadest size range of any nanomaterial
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(from 2 to 500 nm), can be constructed into many different shapes (rods, spheres etc.), and are
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readily surface-modified20–22. We combined the synthetic tunability offered by AuNPs with the
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phenomenon of protein adsorption to create PNP that have the same level of size and shape
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tunability as AuNP.
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Figure 1| Engineering different sizes, shapes and compositions of personalized nanoparticles from human serum. a. We expose gold nanoparticle (AuNP) templates to biological fluids, crosslink the surface-adsorbed proteins in place, and remove the metal core with etchants. Transmission electron microscopy images of b. 15 nm, e. 30 nm and h. 50 nm spheres and k. 30 x 10 nm rod templates show how to make PNPs in different sizes and shapes. c,f,i,l. Spherical PNPs increased by 15 nm and rods by 7 nm at each axis d,g,j,m. Furthermore, spheres and rods assume surface charge densities between -8 and -15 mV following serum incubation and between -5 and -13mV after etching respectively. Error bars denote ± the standard deviation. Scale bar, 100nm.
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We began with AuNPs20,21 that were 50 nm in diameter and coated them with pooled
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human serum. We separated the protein-coated AuNPs from unbound serum proteins using a
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rigorous centrifugation and washing protocol16. The surface-adsorbed proteins were crosslinked
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into a rigid ‘shell’ using a photochemically-activated, heterobifunctional, sulfo-LC-SDA
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crosslinker23. To make the PNP composed only of protein, we needed to remove the exogenous
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AuNP core. Cyanide-containing solutions have been used in templating strategies that incorporate
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nucleic acids24 and polymers25, but they are hazardous to work with and irreversibly denature
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proteins26,27. Consequently, we chose potassium-iodide and iodine, because they do not form
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irreversible side chain adducts27,28 (Fig.1b). After purification, we used inductively-coupled
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plasma mass spectrometry to measure the atomic gold content of our PNP, and confirmed that PNP
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were essentially metal-free with roughly 35 ppb gold content (Fig. S1). We also quantified the loss
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of gold and protein over the reaction and determined we achieved a 78% yield (Fig. S2).
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Transmission electron microscopy revealed that PNP were size and shape tunable because they
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remained spherical and consistently 15 nm larger than their AuNP templates. Their zeta potentials
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were always 7 mV less negative (Fig. 1c, d, Fig. S2, Table S1). Overall, these results show a
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patient-derived nanostructure that is free of exogenous materials and approximates the template
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core size and morphology.
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We next sought to expand our control over size and shape tunability. We therefore
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synthesized 15 nm and 30 nm spherical AuNPs, and 30 nm x 10 nm rod-shaped AuNPs29. We
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confirmed that PNPs were size and shape tunable, as spherical PNPs maintained the same 15 nm
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increase in diameter across all sizes (Fig. 1e, h, k, and Table S1). Based on the unique geometry
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of the nanorod templates, we found they increased by 7 nm along both the longitudinal and
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transverse dimensions (Fig. 1f, i, l and Table S1). All PNP structures were 5.0 to 13.5 mV less
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anionic than their respective AuNP templates (Fig. 1g, j, m). Thus, we used AuNPs to build size
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and shape tunable PNPs.
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Changing the composition of the adsorbed proteins influences biological fate14. It is also
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known that changing nanoparticle surface chemistry changes the enrichment pattern of proteins on
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the surface. Therefore, since different applications may require different compositions, we next
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explored the flexibility of our synthetic method by changing both the enrichment patterns of
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proteins, and the sources of the protein building blocks. We functionalized the surface of 50 nm
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AuNPs with neutral, anionic, or cationic ligands to modify the profile of adsorbed serum proteins
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species in order to yield compositionally distinct PNPs (Fig. 2a)14. We produced five unique
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classes of PNPs with different surface chemistries (Fig. 2b-d) (see Materials and Methods and
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Table S2), and the proteins were analyzed using high resolution liquid chromatography tandem
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mass spectrometry. Each PNP had compositionally distinct protein populations (Fig. S3). We
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confirmed each profile was distinct using both principal component (PCA) and canonical centroid
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analysis (CCA) (Fig. 2e, f and S4). Moving beyond serum, we next explored whether we could
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build PNPs from other biological fluids. We successfully engineered PNP from tears, saliva and
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breast milk (Fig. 2a). Each PNP contained a diverse array of protein building blocks that differed
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from one another in a statistically significant manner (Fig. 2g-l and S5). Interestingly, PNP size,
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shape and charge remained constant regardless of the incubated biological fluid (Table S2). Since
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these PNPs consist of crosslinked self-proteins, we next tested the immunogenicity and toxicity of
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PNPs in vitro and in vivo.
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Figure 2| Engineering protein nanoparticles by changing template surface chemistry and source biological fluid. a. Altering template surface chemistry to anionic, neutral, or cationic changes the distinct protein compositions found in PNPs. Different template surface chemistries such as b. Mercaptoundecanoic acid (MUA) c. mercaptoundecyltrimethylammonium bromide (MUB) d. 1kDa polyethylene glycol (PEG) e. 5kDa PEG produced reproducible PNP. f. Principal component analysis (PCA) derived from mass spectrometry data shows the variability and statistically unique compositions derived from the different template surface chemistries. Colored circles represent the 95% confidence region for each replicate (n =3). The greater the overlap between samples, the more related they are g. The statistically significant proteins that were discovered using the multivariable analysis of variance were classified according to their gene ontology function using the Universal Protein Database. Similarly, h. serum, i. breast milk, j. saliva and k. tears were isolated from patients and used to make PNP. Each PNP was significantly different from one another according to l. PCA and m. multivariable analysis of variance. Scale bar, 100nm.
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Since PNPs built from serum contain complement proteins within their structure (Fig. S4),
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we measured the dose-dependent relationship of serum-PNP on complement activation and
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hemolysis. We incubated 50 nm AuNP templates with isolated serum from inbred C57BL/6 mice
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to construct host-specific PNPs. We found that PNP did not activate complement pathways nor
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induce hemolysis (3a). However, PNPs showed some hemolytic activity at a very high dose of 250
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mg/kg (Fig. 3b). To show PNP do not chronically accumulate to these doses, we determined they
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were biodegradable by conjugating fluorescent dyes to their surface (Fig. S6), and, using
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fluorescence resonance energy transfer-based (FRET)30 (Fig. S7), we showed that PNPs degraded
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when exposed to Proteinase K (Fig. S8). We then exposed bone-marrow derived dendritic cells to
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PNPs and found that they did not exhibit any measurable activation (Fig. S9)31,32.
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Figure 3| In vitro and in vivo toxicity and immunogenicity study of PNP a. PNPs did not visibly activate the complement system compared to PBS at four test concentrations of 5x (250mg/kg), 1x (50 mg/kg), 0.2x (10mg/kg) and 0.04x (2 mg/kg). b. At the same test concentrations, PNPs were found to be non-hemolytic compared to PBS or human serum until 5x the injected dose (****,P>0.0001). c. Splenocytes count shows two times more cells in the LPS group compared to the PNP group (***P < 0.001), and no statistical difference among PBS, PNP and free serum protein. d. LPS groups was higher than PNP groups, and no differences were found between PBS, PNP and free serum for MHC II expression (****P < 0.001) and e. Total IgM and f. IgG antibody production (***P < 0.001). All in vitro studies had three technical and two biological replicates. In vivo studies were n =4 per condition. ± denotes standard deviation. Statistical significance was determined using one-way ANOVA with Tukey correction for multiple comparisons.
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After characterizing the safety and behaviour of PNP in vitro, we next performed both
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short-term and long-term studies in C57BL/6 mice in vivo. We first determined the short-term in
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vivo toxicity and immunogenicity by injecting C57BL/6 mice with 50mg/kg of C57BL/6-PNPs.
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PNP did not induce detectable amounts of chemokines and cytokines involved in early
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inflammation and immune responses (Fig. S10) nor did they induce any liver toxicity (Fig. S11).
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We next evaluated whether PNP induced adaptive immunity following repeated administrations
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over a 28-day period. Mice were injected with 50 mg/kg of vehicle, PBS, free serum protein, or a
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positive control of lipopolysaccharide (LPS) at 2mg/kg on days 0 and 14. Their weights were
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recorded every 3 days. Mice treated with LPS lost 20% of their body weight 3 days after both the
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first and second injections (Fig. S12), whereas PBS, protein and PNP did not. Histopathology
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analysis indicated severe inflammation and neutrophil infiltration following LPS treatment, but no
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inflammation in the PBS, protein and PNP groups (Fig. S13). Splenocytes were counted to
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determine the degree of immune cell recruitment, and we found no difference between PBS,
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protein, and PNP groups, but a two-fold increased cell number was found in the LPS group (Fig.
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3C, ***P
