Functionalization of Clinically Approved MRI Contrast Agents for the

Communication Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Functionalization of Clinically Approved MRI Contrast Agents for the Delivery of VEGF Michael Bietenbeck,†,⊥ Sabrina Engel,‡,⊥ Sebastian Lamping,‡ Uwe Hansen,§ Cornelius Faber,∥ Bart Jan Ravoo,*,‡ and Ali Yilmaz*,†

Bioconjugate Chem. Downloaded from pubs.acs.org by UNIV OF TEXAS AT DALLAS on 03/14/19. For personal use only.

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Division of Cardiovascular Imaging, Department of Cardiology I, University Hospital Münster, Albert-Schweitzer-Campus 1, 48149 Münster, Germany ‡ Organic Chemistry Institute and Center for Soft Nanoscience, Westfälische Wilhelms-Universität Münster, Corrensstrasse 40, 48149 Münster, Germany § Institute of Musculoskeletal Medicine, University Hospital Münster, Domagkstraße 3, 48149 Münster, Germany ∥ Translational Research Imaging Center, Department of Clinical Radiology, University Hospital Münster, Albert-Schweitzer-Campus 1, 48149 Münster, Germany S Supporting Information *

ABSTRACT: In combining the two clinically approved substances ferumoxytol and VEGF-165 via peptide coupling, we propose a straightforward approach to obtain a potentially ready-to-use theranostic contrast agent for specific cardiovascular diseases. Clinical and preclinical magnetic resonance imaging (MRI) studies have shown that intravenously applied superparamagnetic ferumoxytol nanoparticles accumulate in acute ischemic myocardial tissue. On the other hand, growth factors such as VEGF-165 (vascular endothelial growth factor) play a major role during angiogenesis and vasculogenesis. Promising clinical studies with systemic application of VEGF165 have been performed in the past. However, following untargeted systemic application, the biological half-life of VEGF-165 was too short to develop its full effect. Therefore, we hypothesized that ferumoxytol particles functionalized with VEGF-165 will accumulate in ischemic myocardial regions and can be detected by MRI, while the prolonged retention of VEGF-165 due to ferumoxytol-coupling will help to prevent adverse tissue remodeling. In addition, strategies such as magnetic targeting can be used to enhance targeted local accumulation. As a precondition for further preclinical research, we confirmed the successful coupling between ferumoxytol and VEGF-165 in detail (TEM, XPS, and IR spectroscopy), characterized the functionalized ferumoxytol particles (DLS, TEM, and MRI) and performed in vitro tests that showed their superior effect on cell growth and survival.

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applications.4 Today, most SPIONs possess a core−shell structure with a superparamagnetic core enclosed by an organic, nonmagnetic coating. This shell is required to prevent the core from further oxidation and precipitation.5 The SPIONs can then be postfunctionalized with (bio)molecules either to delay cellular uptake, to enable specific cell bonding6,7 or to carry therapeutics.8,9 Moreover, SPIONs can be detected and visualized in vivo by MRI.10,11 Besides the current use of SPIONs, e.g., in magnetic separation of biomolecules,12 extensive research in areas such as molecular imaging, (longitudinal) therapeutic monitoring and drug delivery was performed. 10,13−16 However, it remains challenging to functionalize SPIONs in such a way that they are subsequently well-tolerated in vivo and have a sufficiently long half-life in

ver the past decades, iron oxide nanoparticles have attracted broad attention across different fields of research due to their outstanding size-dependent magnetic behavior.1 Ferromagnetic materials below a certain size show superparamagnetic properties. These nanoparticles possess a very strong net magnetic moment only within an external magnetic field. With the external field switched off the net magnetic moment also dissipates.2 However, it is not only the particles’ size, but also their shape and chemical surface composition, that are crucial for magnetization, biocompatibility, and long-term stability. Accordingly, substantial effort was made regarding the development of reproducible synthesis procedures that ensure a controlled fabrication of magnetic nanoparticles with well-defined size and shape distributions.3 Different surface modifications and particle morphologies were explored to design superparamagnetic iron oxide nanoparticles (SPIONs) with characteristics adapted to the intended © XXXX American Chemical Society

Received: February 20, 2019 Published: March 12, 2019 A

DOI: 10.1021/acs.bioconjchem.9b00142 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

concentration of VEGF at the target site being inflammatory myocardial lesions. To the best of our knowledge, this is the first study to demonstrate the successful conjugation of VEGF-165 to ferumoxytol. We consider this complex as a starting platform for further research in settings such as tissue remodeling after myocardial infarction, while we expect a comparatively short time for clinical translation since the individual substances ferumoxytol and VEGFare already legally and clinically approved. For an efficient functionalization of the carboxymethyldextran coating of ferumoxytol (ca. 750 kDa),41 peptide coupling between carboxylic acid moieties of the shell and free amine functionalities of the VEGF-165 signal protein (12 kDa) was performed. For this purpose, the SPIONs were activated by incubation with aqueous solutions of the coupling reagents 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) leading to the development of an active ester species. Finally, the nucleophilic attack of protein amines resulted in the formation of new peptide bonds. After functionalization, dynamic light scattering (DLS) measurements were performed (see Supporting Information, Figures S1−S2). In PBS buffer as well as in ultrapure water, no significant changes in the hydrodynamic diameter were detected after conjugation with VEGF-165, proving the formation of stable nanoparticles. Successful peptide coupling was proven by X-ray photoelectron spectroscopy (XPS), a quantitative method to characterize the elemental composition as well as the binding mode of these elements within a sample. For comparable results, measurements were performed of three different types of samples (for the XPS survey spectrum, see Supporting Information, Figure S3). First of all, pure, nonmodified ferumoxytol particles were analyzed. Regarding carbon (Figure 2a), there are three different energy signals for

blood to unfold their intended effect in the target organ or tissue. Even the slightest changes result in relevant variations of the characteristic properties of these nanoparticles, which is why it is necessary to analyze cytotoxicity, half-life, and so forth for each system. Aiming at accelerating these time-consuming steps, we developed a straightforward method by using clinically approved SPIONs and functionalized them with a clinically approved biomolecule (Figure 1). By implication, both

Figure 1. Schematic representation of the surface functionalization of ferumoxytol with VEGF-165 and sustained cell growth after incubation with the conjugated SPIONs.

compounds have been previously characterized in depth,17−25 and can be manufactured industrially in gram scale, and potential in vivo (side-) effects are rare and wellknown. We have decided to use ferumoxytol, an ultrasmall SPION with a carboxymethyl-dextran polymer shell, that was used off-label as a contrast agent in MRI until commercial distribution in Europe has been discontinued in 2015.26,27 While the iron-oxide core in ferumoxytol has a mean diameter of approximately 3.25 nm, the overall hydrodynamic diameter is around 30 nm.28 After intravenous injection, ferumoxytol distributes freely in blood until it is eventually internalized by circulating leukocytes and/or the mononuclear phagocyte system.29 Its intravascular half-life is approximately 14−15 h in humans, which is sufficient for MR-angiography, for example. In the case of an active inflammation ferumoxytol nanoparticles are increasingly taken up by macrophages and monocytes that are recruited to the site of inflammatory response. This in turn allows the use of ferumoxytol as an MRI tracer for inflammation, which was already demonstrated in multiparametric cardiovascular MRI of patients with acute myocardial infarction.30,31 Moreover, the particles’ in vivo distribution can be monitored by MRI thereby allowing us to assess therapeutic efficiency and/or locate specific cell populations.4,31 In addition, the magnetic properties of SPIONs can also be used for external magnetic targeting and thereby enable to enhance the accumulation of functionalized SPIONs locally.32 In principle, this allows higher doses of the active ligand at the desired target organ and lower concentrations elsewhere. On the other hand, the various isoforms of VEGF play a major role in the promotion of angiogenesis and vascular permeability,33−35 which makes VEGF relevant for many potential therapeutic applications.36−38 In a preclinical model of acute myocardial ischemia-reperfusion the sustained release of the predominant isoform VEGF-165 led to improved vasculogenesis.39,40 Considering the aforementioned properties and evaluated applications of SPIONs, a compound consisting of ferumoxytol and VEGF is a very attractive platform to establish a MR-visible complex that also increases the

Figure 2. XPS survey spectra of the unfunctionalized (black), activated (blue), and VEGF-functionalized (green) nanoparticles. The C 1s-signals (a−c) for each nanoparticle species are summarized in (d). The N 1s-signal (e) and the S 2p-signal (f) show significant changes after functionalization with VEGF.

C 1s that are characteristic for C−C (284.9 eV), C−O (286.1 eV), and COOR (287.5 eV) bonds and result from the carboxymethyl-dextran coating of the particles. No signals were detected for the elements N and S (Figure 2e,f, black lines, and Figure S4a). In comparison, the C 1s signals of activated SPIONs after incubation with EDC and NHS (Figure 2b) do not show a significant shift of the C−C bond (284.9 eV), whereas a small shift is visible for the C−O bond (286.6 eV) and a stronger shift occurs for the COOR bond (288.2 eV). B


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Bioconjugate Chemistry These changes can be explained by the formation of an active ester species. Moreover, no S signal, but a weak signal for N (400.3 eV, Figure 2e,f and Supporting Information, Figure S4b) is present, caused by the active ester. In contrast, major differences are obtained for the VEGF-functionalized nanoparticles: there is no C 1s signal shift for the C−C bond (285.0 eV), whereas a significant change is present for the C−O bond (286.3 eV, Figure 2c). The energy signal for the COOR bond (288.1 eV) is almost equal to the value of the activated nanoparticles, but differs from the signal of the nonfunctionalized SPIONs, which leads to the conclusion that the carboxylic acid functionalities were converted into the active ester and afterward transformed into a peptide bond. To summarize, Figure 2d clarifies the different C 1s signal shifts. Furthermore, a strong energy signal can be monitored for N (400.1 eV, Figure 2e) due to the increased concentration of peptide bonds and free amines, which were brought along from the VEGF protein. Finally, a strong S 2p signal for S (Figure 2c) is visible for the functionalized SPIONs arising from the disulfide bridges and sulfur-containing amino acids of the VEGF-165. The XPS results were additionally confirmed by use of infrared (IR) spectroscopy. Accordingly, the range of wavelengths characteristic for vibrational modes of peptide bonds was analyzed in more detail (Figure 3). No significant peak is

Figure 4. Representative TEM images showing specifically VEGF-165 binding immunogold particles (darkest particles, 18 nm, black arrowheads) with (a) unfunctionalized ferumoxytol SPIONs as a negative control and (b) VEGF-functionalized ferumoxytol SPIONs. Unlabeled particles are marked with white arrows. Scale bar 100 nm.

bind, while selective binding between VEGF-functionalized nanoparticles and anti-VEGF immunogold particles occurs, which results in visible aggregates of iron oxide (low contrast) and gold (dark, Figure 4b). These specific interactions only occur when the conformation and functionality of VEGF remains unchanged on the surface of the nanoparticles. Further TEM images (see Supporting Information, Figure S7) without BSA blocking and incubation with gold do not show any aggregation of the ferumoxytol after conjugation with VEGF165, concluding that stable nanoparticles with functional growth factor on the surface were synthesized. The ferric concentration of the dissolved functionalized ferumoxytol particles was assessed with spectrophotometric methods. In line with the previously published report by Dadashzadeh et al.,43 UV−vis absorption in a serial dilution of 1−100 μg nonfunctionalized ferumoxytol was measured and the optimal wavelength of the calculated standard curve, relating absorbance and ferric content, was found to be 370 nm. Hence, a ferric concentration of 3.34 ± 0.14 mg/mL was determined in the suspension of functionalized ferumoxytol particles. Before testing for their proliferative capacities, MRI mapping experiments were performed on a clinical 1.5 T MRI to show that the bound ferumoxytol has not lost T2*-shortening effect. Mapping sequences consist of a rapid series of acquisitions that record the signal intensity evolution while the relaxation constant (here T2*) per voxel can be determined by appropriate regression analysis.44 Sequence acquisition parameters were set to Fast Field Echo, TR = 151 ms, TE = 1.54 ms, N(TE) = 60, voxel size 1 × 1 × 10 mm3. A series of diluted ferumoxytol suspensions and a sample of 150 μL VEGFfunctionalized ferumoxytol filled up to 20 mL with PBS were analyzed. By the use of exponential regression analyses a T2* map was calculated (Figure 5) and a ferric content of 3.42 ± 0.31 mg/mL was determined for the sample of functionalized

Figure 3. IR spectra of the unfunctionalized (black) and VEGFfunctionalized (green) ferumoxytol nanoparticles and zoom into the region for the characteristic signals for the peptide bonds.

visible in the range of 1750−1375 cm−1 examining the nonfunctionalized ferumoxytol particles (black lines), whereas two strong absorption bands (green lines) are present for the VEGF-functionalized SPIONs. The amide band ranging from 1600 to 1700 cm−1 is the most intense absorption band in proteins and derives mainly from the stretching vibrations of the CO and CN groups, whereas the amide band between 1580 and 1510 cm−1 is governed by the in-plane NH bending as well as from the CN and CC stretching vibrations. The occurrence of these two characteristic bands further proves the presence of the VEGF proteins on the surface of the ferumoxytol nanoparticles.42 To demonstrate that the VEGF-165 signal protein is immobilized on the surface of the ferumoxytol SPIONs without losing its original structure and functionality, transmission electron microscopy (TEM) was performed. Samples of unfunctionalized and VEGF-functionalized nanoparticles were absorbed on TEM grids separately, washed with PBS, and blocked with BSA to reduce unspecific binding. Afterward, the grids were incubated with anti-VEGF antibodies, washed and finally incubated with 18-nm-sized gold conjugated immunoglobulins. Subsequently obtained TEM images (see Figure 4a) show that pure ferumoxytol and immunogold particles do not

Figure 5. T2*-color map of various dilutions of nonfunctionalized (1)−(7) and a sample of VEGF-functionalized ferumoxytol X. C


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lowest in the presence of pure ferumoxytol particles (2.7× the starting cell number). While the control wells showed a comparable increase in cell number to those, whose media was supplemented with 10 ng VEGF-165 (4.2× vs 3.7×), HUVECs treated with an extra 50 ng VEGF-165 proliferated as well as those cells treated with functionalized ferumoxytol (5.7× vs 6.2×). These different proliferation densities match with the visual impression of the microscopic images (Figure 7). The first row shows the equal seeding density of all HUVEC cultures, whereas the second row markedly depicts the different proliferation rates after 72 h. Importantly, only in those wells that were supplemented with functionalized ferumoxytol or 50 ng VEGF-165 living HUVECs properly attached and growing at the bottom were seen after 72h (Figure 7, second row of column b and e). In clinical phase II/III studies that examined the effect of intracoronary and intravenous VEGF infusions on the neoangiogenesis in ischemic myocardial tissue, high doses of exogenous protein were used. For example, 50 ng/kg BW/min over 20 min of intracoronary infusion followed by three intravenous infusions of VEGF, each lasting 4 h.45 In a 70 kg BW patient, this corresponds to a total dose of 70 μg VEGF during the first intracoronary infusion. In order to apply the same amount of VEGF, approximately 21 mL of our ferumoxytol-VEGF complex will be necessary. This in turn corresponds to a total iron dose of 3.22 g. For the treatment of iron deficiency anemia a total dose of 1020 mg is suggested by the manufacturer, but its application is divided into two infusions, which should be 3−8 days apart. In order to comply with this maximum dose, increasing the loading capacity of ferumoxytol with VEGF will be the next goal of our synthesis. Following our current protocol, it is fair to assume a very high coupling efficiency, since no protein at all was determined in an ELISA-assay of the supernatant after coupling (data not shown). In a best-case scenario, our synthesis yields approximately 1 mL of complex with a ferumoxytol (ca. 750 kDa) loading of 3.4 mg that binds 5 μg VEGF-165 (12 kDa). Thus, only 1 in 10 ferumoxytol particles carries VEGF-165 in our setting. In this context, it might be sufficient to increase the amount of VEGF-165 during the synthesis to increase the loading of VEGF per ferumoxytol particle to the desired levels. In summary, we have synthesized a potentially ready-to-use, theranostic contrast agent for MRI that consists of ferumoxytol

ferumoxytol, which is in good agreement with the value calculated from the spectrophotometric measurement. For the HUVEC (human umbilical vein endothelial cells) proliferation assay, 4000 cell/cm2 were plated per (6-)well prepared with 0.2% gelatin in EBM-2 basal medium supplemented with growth factor containing EGM-SingleQuot-Kit. All experiments were carried out between passages 2 and 5. Cultures were allowed to grow for 24 h at 37 °C in a humidified atmosphere of 95% air and 5% CO2, before serum starvation in basal medium containing only 0.5% FCS. After 6 h the cell medium was exchanged again, now supplemented with a reduced concentration of growth factors (1/3 R3-IGF, 1/3 hEGF, 1/8 hFGF-B, no FCS or VEGF) and the respective test substance (N = 3): 300 μg/mL un- or VEGF-coated ferumoxytol and 10 or 50 ng/mL VEGF. While cell growth was documented every 24 h, living cells were collected and counted in a hemocytometer after 72 h. If we compare the numeric results (Figure 6) with the individual microscopic photographs (Figure 7), it becomes

Figure 6. Proliferation rates of HUVEC cultures in growth factor reduced media plus different supplementsexpressed as multiples of the initial seeding cell number after 72 h.

evident that the VEGF peptide has preserved both its structure and its function: After 72 h of cell culture, HUVEC growth was

Figure 7. Representative microscopic images either taken after HUVEC seeding (upper row) or at the end of the proliferation assay, after 72 h (lower row). The enlargement is 4-fold except in the small sections in the lower row: here, it is 10-fold. (a) Control culture with growth factor reduced medium only, (b) +300 μg/mL functionalized ferumoxytol, (c) +300 μg/mL ferumoxytol, (d) +10 ng/mL VEGF, (e) +50 ng/mL VEGF. D


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(6) Liu, H., Zhang, J., Chen, X., Du, X. S., Zhang, J. L., Liu, G., and Zhang, W. G. (2016) Application of iron oxide nanoparticles in glioma imaging and therapy: From bench to bedside. Nanoscale 8, 7808−7826. (7) Abakumov, M. A., Nukolova, N. V., Sokolsky-Papkov, M., Shein, S. A., Sandalova, T. O., Vishwasrao, H. M., Grinenko, N. F., Gubsky, I. L., Abakumov, A. M., Kabanov, A. V., et al. (2015) VEGF-targeted magnetic nanoparticles for MRI visualization of brain tumor. Nanomedicine 11, 825−833. (8) Polyak, B., and Friedman, G. (2009) Magnetic targeting for sitespecific drug delivery: applications and clinical potential. Expert Opin. Drug Delivery 6, 53−70. (9) Meikle, S. T., Piñeiro, Y., Bañobre López, M., Rivas, J., and Santin, M. (2016) Surface functionalization superparamagnetic nanoparticles conjugated with thermoresponsive poly(epsilon-lysine) dendrons tethered with carboxybetaine for the mild hyperthermiacontrolled delivery of VEGF. Acta Biomater. 40, 235−242. (10) Weissleder, R., Nahrendorf, M., and Pittet, M. J. (2014) Imaging macrophages with nanoparticles. Nat. Mater. 13, 125−138. (11) Petros, R. A., and DeSimone, J. M. (2010) Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug Discovery 9, 615−627. (12) Samanta, A., and Ravoo, B. J. (2014) Magnetic Separation of Proteins by a Self-Assembled Supramolecular Ternary Complex. Angew. Chem., Int. Ed. 53, 12946−12950. (13) Ittrich, H., Peldschus, K., Raabe, N., Kaul, M., and Adam, G. (2013) Superparamagnetic iron oxide nanoparticles in biomedicine: applications and developments in diagnostics and therapy. RoFo 185, 1149−1166. (14) Tietze, R., Lyer, S., Dürr, S., and Alexiou, C. (2012) Nanoparticles for cancer therapy using magnetic forces. Nanomedicine 7, 447−457. (15) Kelkar, S. S., and Reineke, T. M. (2011) Theranostics: Combining Imaging and Therapy. Bioconjugate Chem. 22, 1879−1903. (16) Masthoff, M., Gran, S., Zhang, X., Wachsmuth, L., Bietenbeck, M., Helfen, A., Heindel, W., Sorokin, L., Roth, J., Eisenblätter, M., et al. (2018) Temporal window for detection of inflammatory disease using dynamic cell tracking with time-lapse MRI. Sci. Rep. 8, 9563. (17) Quinlan, E., López-Noriega, A., Thompson, E. M., Hibbitts, A., Cryan, S. A., and O’Brien, F. J. (2017) Controlled release of vascular endothelial growth factor from spray-dried alginate microparticles in collagen-hydroxyapatite scaffolds for promoting vascularization and bone repair. J. Tissue Eng. Regener. Med. 11, 1097−1109. (18) Jay, S. M., and Saltzman, W. M. (2009) Controlled delivery of VEGF via modulation of alginate microparticle ionic crosslinking. J. Controlled Release 134, 26−34. (19) Aday, S., Zoldan, J., Besnier, M., Carreto, L., Saif, J., Fernandes, R., Santos, T., Bernardino, L., Langer, R., Emanueli, C., et al. (2017) Synthetic microparticles conjugated with VEGF165 improve the survival of endothelial progenitor cells via microRNA-17 inhibition. Nat. Commun. 8, 747. (20) Simón-Yarza, T., Formiga, F. R., Tamayo, E., Pelacho, B., Prosper, F., and Blanco-Prieto, M. J. (2013) PEGylated-PLGA microparticles containing VEGF for long term drug delivery. Int. J. Pharm. 440, 13−18. (21) Golub, J. S., Kim, Y., Duvall, C. L., Bellamkonda, R. V., Gupta, D., Lin, A. S., Weiss, D., Robert Taylor, W., and Guldberg, R. E. (2010) Sustained VEGF delivery via PLGA nanoparticles promotes vascular growth. Am. J. Physiol. Circ. Physiol. 298, H1959. (22) Shi, Y., Zhou, M., Zhang, J., and Lu, W. (2015) Preparation and cellular targeting study of VEGF-conjugated PLGA nanoparticles. J. Microencapsulation 32, 699−704. (23) Maia, J., Vazão, H., Pedroso, D. C. S., Jesus, C. S. H., Brito, R. M. M., Grãos, M., Gil, M. H., and Ferreira, L. (2012) VEGFfunctionalized dextran has longer intracellular bioactivity than VEGF in endothelial cells. Biomacromolecules 13, 2906−2916. (24) Oduk, Y., Zhu, W., Kannappan, R., Zhao, M., Borovjagin, A. V., Oparil, S., and Zhang, J. (2018) VEGF nanoparticles repair the heart after myocardial infarction. Am. J. Physiol. Circ. Physiol. 314, 278−284.

nanoparticles and growth factor VEGF-165. Since both constituents are already clinically approved, we expect a relatively quick translation into the clinic, providing that our in vitro results are also confirmed in subsequent in vivo preclinical studies. With this study, we could demonstrate the successful peptide coupling using XPS and IR, while TEM measurements proved that the signal proteins preserve their functionality after binding to the surface of ferumoxytol. In line with this, HUVEC cell proliferation assays demonstrated both visually and in counted cell numbers that the functionalization of ferumoxytol with VEGF-165 resulted in a markedly positive impact on both cell viability as well as proliferation rates. On the other hand, our newly synthesized compound is still MRIactive as seen in T2*-weighted MRI acquisitions. Since the preparation of this novel contrast agent is operationally simple, we consider our findings as a starting point for further preclinical research (e.g., in animal models of myocardial infarction or myocarditis).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.9b00142.

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Experimental details including nanoparticle synthesis and further analysis data (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Bart Jan Ravoo: 0000-0003-2202-7485 Author Contributions ⊥

M.B. and S.E. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.E. would like to thank the Fonds der Chemischen Industrie for a doctoral fellowship. We thank Karin Schlattmann for excellent technical assistance.

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REFERENCES

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