Heat-induced-radiolabeling and click chemistry: A powerful combination for generating multifunctional nanomaterials

A key advantage of nanomaterials for biomedical applications is their ability to feature multiple small reporter groups (multimodality), or combinations of reporter groups and therapeutic agents (multifunctionality), while being targeted to cell surface receptors. Here a facile combination of techniques for the syntheses of multimodal, targeted nanoparticles (NPs) is presented, whereby heat-induced-radiolabeling (HIR) labels NPs with radiometals and so-called click chemistry is used to attach bioactive groups to the NP surface. Click-reactive alkyne or azide groups were first attached to the nonradioactive clinical Feraheme (FH) NPs. Resulting “Alkyne-FH” and “Azide-FH” intermediates, like the parent NP, tolerated 89Zr labeling by the HIR method previously described. Subsequently, biomolecules were quickly conjugated to the radioactive NPs by either copper-catalyzed or copper-free click reactions with high efficiency. Synthesis of the Alkyne-FH or Azide-FH intermediates, followed by HIR and then by click reactions for biomolecule attachment, provides a simple and potentially general path for the synthesis of multimodal, multifunctional, and targeted NPs for biomedical applications.


Syntheses of
Cell binding study of 89 Zr-Cy5.5-Protamine-FH ( 89 Zr-16) with MDA-MB-231 cells (Fig 5g and 5h) For the uptake of the radiolabeled Protamine-FH compound, two hundred thousand cells were seeded on 6-well Costar plates (Corning, NY) in 2 mL media, followed by overnight incubation at 37˚C, 5% CO2. They were then treated with 1X PBS-diluted compound, and placed in the incubator for 2 h. Subsequently, they were washed three times with 1X PBS, which was warmed at 37˚C, followed by 10-minute lysis in the presence of RIPA buffer. Prevention of endocytic uptake was performed by placing the cells for 1 h at 4˚C in the presence of the nanoparticles (final concentration = 10 nM). Cell-associated radioactivity was performed similar to the study investigating the uptake of radiolabeled-folate-FH.
To demonstrate that the association between Cy5.5 and NPs resulted from a click-based covalent reaction, and not adsorption of Cy5.5 to the NP surface two types of controls were run. First, the reaction between Azide-FH (4) and DBCO-Cy5.5 (6) was determined after incubation with DBCO-NH 2 which reduced n from 14.3 to 1.3 (Fig 2d). Second, DBCO-Cy5.5 (6) was reacted with FH (1) and gave no detectable reaction (Fig 2e). As summarized in Table 1, FH can be derivatized with click reactive azide or alkyne groups and the numbers of reactive groups per NP determined by reaction with an appropriate Cy5.5 fluorochrome.
Folate attachment by a copper mediated click reaction. We employed a copper mediated click reaction between the alkyne-functionalized FH's ( 89 Zr-Alkyne-FH, 89 Zr-5 or Alkyne-FH, 5) and Azide-Folate (10), to obtain folate functionalized NPs ( 89 Zr-Folate-FH, 89 Zr-11 or Folate-FH, 11) as shown in Fig 3. To demonstrate the presence of folates on the NP surface, NPs were reacted with an azide-bearing Cy5.5 (7) before and after their reaction with azide-folate. The number of folates per NP, p, was then determined by subtraction, p = m-q, where m is the number of reactive alkynes before folate attachment and q is the number of reactive alkynes remaining after folate attachment (Table 1). Using this technique there were 12.2 reactive alkynes on 89 Zr-Alkyne-FH ( 89 Zr-5) and 12.2 folates on 89 Zr-Cy5.5-Alkyne-FH ( 89 Zr-9). We term this as the"Before and After Fluorochrome Reaction and Subtraction Method" for determination of targeting groups on NP surfaces. RGD and protamine attachment by copperless click reactions. To demonstrate a surface modification of 89 Zr-Azide-FHs ( 89 Zr-4) or Azide-FH (4) using a copperless click reaction, the NP was reacted with a DBCO-bearing RGD peptide (Fig 4), and the number of surface RGDs determined with the "Before and After Fluorochrome Reaction and Subtraction Method" to be 11.7 RGD's per NP.

Bioactivity of surface functionalized NPs
To demonstrate that the surface functionalized NPs synthesized possess bioactive functional groups three experiments were conducted (Fig 5): (i) the binding of RGD functionalized NPs (RGD-FH, 14, Fig 4) [38,39] and cell fluorescence examined by flow cytometry (Fig 5a and 5b). Fluorescence was higher for the RGD bearing RGD-FH (14) than the control NP at three NP concentrations, indicating NPsbearing RGD peptides bound to cellular integrins (Fig 5c). The RGD peptides on RGD-FH (14) bind integrins expressed on BT-20 cells.

Discussion
We have shown that both azides and alkynes can be attached to FH and withstand HIR, allowing a wide range of click chemistry surface functionalizations with radioactive or nonradioactive NPs. However, with iron oxide NPs like FH the reducing conditions used for some click reactions can reduce ferric to ferrous ions, a reaction evident by a change from a reddish to a black/brown color. Our conditions for copper mediated click reactions of the Alkyne-FH (5) with Azide-Cy5.5 (7) or Azide-Folate (10) were 0.2 mg Fe, 15 min, room temperature with molar ratios of 67 / 1 / 1 / 37 for Fe / Cu / (BimC 4 A) 3 / Na.Ascorbate in DPBS. In addition the copper catalyst should be freshly made and solutions should be oxygen free (see S1 File). As shown in Table 1, the measurements of nanoparticle size and relaxivity indicate a lack dissolution or aggregation due to surface functionalization. Copperless click reactions between Azide-FH (4) and click reactive, strained cyclic DBCO were used with RGD and protamine peptides; these are free from the issues than arise with the use of reductive conditions.
To determine the numbers of chemically reactive azide or chemically reactive alkyne functional groups on the NP surface we employed the "Before and After Fluorochrome Reaction and Subtraction Method." This was done because of the shortcomings of other analytical methods discussed below, with Cy5.5 selected as an optical reporter because of its high extinction coefficient, its lack of spectral overlap with iron oxides (see Fig 2b), and the commercial availability of click reactive Cy5.5s. Shortcomings of other analytical arise when engaging in defining nanoparticle chemistry for nanoparticles used in mouse imaging. This arises because typically small quantities of nanoparticles are synthesized and used, since one milligram is sufficient to inject four 25 g mice at a typical dose of 10 mg Fe/kg. Hence analytical methods for this setting must consume small amounts of NPs, as well as overcoming NP superparamagnetism (no NMR) and the intense UV and visible absorption of iron oxides. Here we show how reactions with a commercially available fluorochrome can be used to obtain the number of biomolecules per NP. By reacting commercially available, click reactive Cy5.5s with NPs, the number attached Cy5.5s can be determined spectrophotometrically, with the number of bioactive groups, being the difference between the number of Cy5.5s attached before and number attached after their reaction the biomolecule. (Fig 3). With the high extinction coefficient of Cy5.5, the reaction of Cy5.5 with NPs uses only about 0.02 mg Fe to interrogate the status of the NP surface. Conventional methods of analyzing NP surfaces, e.g. FTIR or SIMMS [43] require substantially greater NP amounts and specialized instrumentation. With this "Before and After Fluorochrome Reaction and Subtraction Method" the numbers of folates or RDGs were determined through the reaction of Cy5.5. The method is useful when the number of attached targeting groups per NP is high, and difference of the before after reaction (p = m-q, Fig 3) is large. However, to determine low numbers protamines attached per NP, (a low number being necessitated by protamine induced NP agglomeration, see below), we synthesized a DBCO-protamine-Cy5.5 (15,Fig 4, see S2 File), and determined the numbers of protamines per NP directly.
With regard to steric blockage with Cy5.5 dye attachment to the Feraheme nanoparticle surface, steric blockage is not seen because of the Feraheme nanoparticle's extremely large dimensions compared to a Cy5.5. The Feraheme nanoparticle has diameter of 25 nm, equivalent to a globular protein of 750 kDa [44]. The Cy5.5 dyes have molecular weights of about 700-800 daltons, so that attachment of, for example 20 Cy5.5s (MW = 16,000 Da), is readily accommodated on the Feraheme nanoparticle. Thus we have not observed steric blocking and the number of Cy5.5s can be used to represent the reactive sites under click chemistry surface modification employed in this study.
A consideration when performing NP surface chemistry on FH NP is avoiding NP agglomeration. FH (1) has a strong negative charge over a wide pH range, with a Zeta potential at pH 7.36 of -30.55 mV [9]. With our chemistry, FH (1) tolerated the attachment of negatively charged Cy5.5, the cRGD peptide or folate at levels of 10-20 targeting groups per NP with a maintenance of NP size of less than 50 nm, see Table 1. However, when highly positively charged protamine was employed, agglomeration was initially obtained. Protamine 1 consists of 32 amino acids of 22 of which are positive arginines and the rest are neutral amino acids [35]. (Protamine is a mixture of 4 similar peptides.) To attach the smaller numbers of protamines consistent with NP stability, and determine the number of protamines per NP, we synthesized a protamine with N-terminal DBCO and C-terminal Cy5.5 (DBCO-protamine-Cy5.5 (15), see S2 File), and determined the number of protamines per NP directly from the attachment of DBCO-Protamine-Cy5.5 (15), see Fig 4. Our surface modifications with Cy5.5 fluorochromes, folate, RGD peptide and protamine employed materials with molecular weights of less than 5000 daltons. Separation of radioactive nanoparticles from these low molecular weight, surface functionalizing compounds is readily accomplished with PD-10 and Sephadex G-50 columns. With our method, a wide range of nanoparticle surface functionalization strategies can be employed with similarly sized materials. However, use of larger biomolecules like antibodies will required a different separation method and may occur far more slowly under the conditions used here.
The combination of heat-induced NP radiolabeling and click chemistry NP surface functionalization outlined in Fig 1 provides a number  NPs as a starting NP, which is an approved drug. FH NPs have well-described physical properties, [9,44,45] a well-understood metabolism, [46][47][48] low toxicity, [49,50] low batch to batch variability, and the sterility required of a parenteral pharmaceutical. Due to the superparamagnetism of its iron oxide core, FH (1) has been widely used as an MR contrast agent, [51] which further aids in understanding diagnostic applications of this NP. Second, the carboxyl groups of FH's carboxymethyldextran (CMD) coating endow the NP with extraordinary heat stability [11,12,52] and can be reacted with primary amines. [13,42] CMD's carboxyl groups are not involved in the radiocation binding of the HIR reaction based on electron magnetic resonance indicating their binding to the iron oxide surface and the ability to perform HIR with NP's lacking carboxyl groups. [4] Thus, we have modified some of these carboxyl groups to obtain alkyne and azide functionalized NPs as shown in Fig 2. Third, FH's heat stability allows heatinduced radiolabeling, which permits the bonding of three cations used in nuclear imaging ( 111 In, 89 Zr, 64 Cu) without altering the NPs physical properties or pharmacokinetics. [4,5] Here we demonstrate a fourth advantage of FH NPs, that alkyne and azide functionalized FH NPs (Azide-FH, 4 & Alkyne-FH, 5) survive the HIR reaction (yielding 89 Zr-Azide-FH, 89 Zr-4 & 89 Zr-Alkyne-FH, 89 Zr-5). This allows syntheses of nonradioactive Azide-FH (4) and Alkyne-FH (5) at modest pace and desired scale, followed by HIR, followed by the rapid click chemistry attachment of biomolecules under mild reaction conditions (Fig 3). Here Azide-FH (4) or Alkyne-FH (5) were radiolabeled, surface modified, and purified in total time of less than 4 h, which is short compared to the radiochemical half-lives of 64 Cu, 111 In or 89 Zr.

Conclusions
As summarized in Table 1, the HIR/click combination is a powerful approach for generating multifunctional nanomaterials, which allows detection of the NPs made here by MRI (FH's superparamagnetism), by fluorescence (click attachment of fluorochromes), and by radioactivity (HIR). In the future, the general strategy outlined in Fig 1 may be used to obtain multimodal NPs for labeling circulating monocytes or resident macrophages, [5] for targeting NPs to cells expressing receptors (e.g. folate receptor, RGD binding integrins) or for the ex-vivo labeling cells through the membrane translocating activity of protamine. [35,37,41] The reactions demonstrated here, use of the widely available and well understood FH (1) for the synthesis of the nonradioactive Alkyne-FHs (5) and Azide-FH (4) intermediates, followed by HIR, then by click chemistry modification of the NP surface biomolecules, provides a path to the design of multimodal/multifunctional NPs suitable for a wide range of diagnostic and therapeutic applications.