Granulocyte Colony-Stimulating Factor Nanocarriers for Stimulation

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G-CSF nanocarriers for stimulation of the immune system (Part I): synthesis and biodistribution studies Gabriel De Crozals, David Kryza, Gloria Jiménez Sánchez, Stéphane Roux, Doriane Mathé, Jacqueline Taleb, Charles Dumontet, Marc Janier, and Carole Chaix Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00605 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

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Bioconjugate Chemistry

G-CSF nanocarriers for stimulation of the immune system (Part I): synthesis and biodistribution studies

Gabriel De Crozals† ҂, David Kryza ‡ ⱶ ҂, Gloria Jiménez Sánchez§, Stéphane Roux§, Doriane Mathé¥, Jacqueline Taleb ‡, Charles Dumontet¥*, Marc Janier ‡ ⱶ*, and Carole Chaix†*

†

Institut des Sciences Analytiques,

UMR CNRS 5280/Université Claude Bernard Lyon

1/ENS de Lyon, 5, rue de la Doua, 69100 Villeurbanne Cedex, France. ‡

UNIV Lyon - Université Claude Bernard Lyon 1, LAGEP UMR 5007 CNRS Villeurbanne,

France ⱶ

Hospices Civils de Lyon, plateforme Imthernat, Hôpital Edouard Herriot, 69437 Lyon, France

§

Institut UTINAM, UMR CNRS 6213-Université de Bourgogne Franche-Comté, 25030

Besançon Cedex, France. ¥

Cancer Research Center of Lyon, INSERM 1052/CNRS 5286/University Claude Bernard

Lyon 1, Lyon, France

* Co-corresponding authors : Charles Dumontet Email : [email protected]; +33 (0)4 78 77 72 36 Marc Janier Email : [email protected]; Phone : +33 (0)4 72 11 73 90 Carole Chaix Email : [email protected]; Phone : +33 (0)4 37 42 35 57

҂

: These authors contributed equally to this work.

1 ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT In the field of cancer immunotherapy, an original approach consists in using granulocyte colony-stimulating factor (G-CSF) to target and activate neutrophils, cells of the innate immune system. G-CSF is a leukocyte stimulating molecule which is commonly used in cancer patients to prevent or reduce neutropenia. We focused herein on developing a G-CSF nanocarrier which could increase the in vivo circulation time of this cytokine, keeping it active for targeting the spleen, an important reservoir of neutrophils. G-CSF-functionalized silica and gold nanoparticles were developed. Silica nanoparticles of 50 nm diameter were functionalized by a solid phase synthesis approach. The technology enabled us to incorporate multiple functionalities on the surface such as a PEG as hydrophilic polymer, DTPA as

111

In

chelating agent and G-CSF. The gold nanocarrier consisted of nanoparticles of 2-3 nm diameter elaborated with DTPA groups on the surface and functionalized with G-CSF. We studied the particle biodistribution in mice with special attention to organs involved in the immune system. The two nanocarriers with similar functionalization of surface showed different pathways in mice, probably due to their difference in size. Considering the biodistribution after G-CSF functionalization, we confirmed that the protein was capable to modify the pharmacokinetics by increasing the nanocarrier concentration in the spleen, a reservoir of G-CSF receptor expressing cells.


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INTRODUCTION Cancer immunotherapy is a promising treatment modality that is the subject of ongoing and extensive studies. In this field, a part of the current research focuses on the modulation of the immune response to tumor cells. One strategy to overcome the resistance of cancer cells is to stimulate the proliferation and the differentiation of neutrophils, innate immune cells with phagocytic capacities. G-CSF is a leukocyte stimulating molecule which is commonly used in cancer patients to prevent or reduce neutropenia, a potentially life-threatening complication of cytotoxic chemotherapy.1 It has been shown that the stimulation of granulocytes by G-CSF could be of interest to enhance the antitumor potential of therapeutic monoclonal antibodies.2 G-CSF recruits and activates neutrophils, thereby increasing their ability to perform Antibody Dependent Cellular Phagocytosis (ADCP), one of the mechanisms involved in the anticancer activity of therapeutic antibodies. The development of granulocytes and their precursors is induced by G-CSF binding to membrane G-CSF receptors. This receptor-mediated endocytosis mainly occurs in the spleen which is a reservoir of neutrophils. Matsuzaki et al. described that recombinant human G-CSF inoculation during 8 days resulted in a significant increase of spleen cell number.3 However this effect is reduced by the rapid enzymatic degradation of G-CSF after intravenous or subcutaneous injection. Treatments require frequent injections of high doses of the drug for several days to observe an efficient effect, or use of a pegylated form with a longer half-life. In this context, strategies to protect G-CSF against degradation and concentrate it in the granulocyte reservoirs are required to enhance the stimulation of the innate immune response. The current approaches described in the literature for G-CSF vectorization are mainly based on entrapment or encapsulation in liposomal or polymeric nanocarriers.4-6 These formulations act

on

the

cytokine

pharmacokinetics in

mice

without clearly improving the

pharmacodynamics compared to free G-CSF. In this field, the development of new G-CSF formulations that provide enhanced targeting of neutrophils is a great challenge for therapies based on stimulation of the innate immune system. Here we developed G-CSF-functionalized silica and gold nanoparticles with multimodal detection to study the in vivo biodistribution after intravenous injection in mice. The surface functionalization of the silica nanocarrier was carried out by a supported synthesis approach. While top-down approaches consist in coupling reactive molecules (or biomolecules) to nanoparticles in solution to form functionalized NP, bottom-up approaches involve direct 3 ACS Paragon Plus Environment


synthesis of molecules (or biomolecules) from the surface of NP.7,

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8

Solid phase synthesis

(SPS) was recently extended as bottom-up approach for efficient and modular functionalization of silica nanoparticles.9 This method provides many advantages including a high surface coverage and easy multifunctionalization, the control of molecule orientation and limitation of non-specific adsorption. A wide range of molecules is available as phosphoramidite derivatives and thus can be directly incorporated on NP by SPS. We focused herein on elaborating the amino-PEG linker by this bottom-up technique to further graft the DTPA chelating molecule. Then, SiO2NP-DTPA were used to elaborate NP-protein conjugates. By our strategy of support-assisted functionalization, we obtained a multifunctional silica nanocarrier that combines polyethylene glycol moieties for stealthness, chelating molecules for radiolabeling, and carboxylic groups for protein coating through covalent amide bonds. The SiO2NP-DTPA/G-CSF conjugate was synthesized and characterized by different physical and analytical methods. In parallel, ultra-small AuNPDTPA (~3 nm diameter) was synthesized on the basis of the monophasic protocol of Brust.10 AuNP-DTPA/G-CSF conjugate was obtained by coupling the protein with the DTPA according to the same grafting protocol as for silica-NP. We studied the particle biodistribution in mice with special attention to organs involved in the immune system. The relationship between G-CSF loading and spleen uptake was evaluated. The SiO2NP-DTPA/G-CSF biodistribution was compared to that of the AuNP-DTPA/G-CSF conjugate. It is now recognized that the size (or rather the hydrodynamic diameter Dh) plays an important role in the biodistribution of nanoparticles because it determines their mode of elimination. The large particles (Dh > 10 nm) are inevitably captured by the liver whereas nanoparticles with a hydrodynamic diameter of less than 5–6 nm lead to renal elimination whatever their surface charge.11 It has been reported in a previous work that AuNP-DTPA NPs with a Dh of 6 nm were mainly removed from the body by renal clearance. After intravenous injection, these gold nanoparticles freely circulate in blood stream with only a low accumulation in liver and spleen.12, 13 We compared herein the biodistribution of SiO2NPDTPA and AuNP-DTPA after their functionalization by the G-CSF in order to evaluate the impact of the NP size and nature on the distribution pathways induced by the protein.

RESULTS AND DISCUSSION Silica nanoparticle solid-phase functionalization 4 ACS Paragon Plus Environment

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With the aim to obtain a maximal enhancement of the immunostimulating effect induced by G-CSF, we envisaged a high protein loading on the nanoparticles. We thus focused on an original bottom-up strategy involving solid phase synthesis (SPS) to functionalize silica NP in a highly controlled manner. To adapt SPS to nanosized material, we elaborated a solid support composed of nanoparticles covalently grafted on the surface of porous micrometric glass beads. Controlled pore glass (CPG), a solid support widely used in DNA synthesis, was chosen for this purpose. The CPG with 300 nm pore diameter was preferred to ensure the grafting of 50 nm NP inside the pores without decreasing SPS efficiency. This innovative support solved a major issue which is to maintain the nanoparticles held in the column during synthesis. Aggregation problems commonly associated with NP functionalization were also avoided. As the washing steps were directly performed by the automated synthesizer, centrifugation/redispersion steps, which often lead to aggregation, were minimized. Concerning the design of the nanoparticles, the fluorescent rhodamine core facilitated the handling and quantification of the nanoparticles, while the silica material was chosen for its biocompatibility. In addition, the NP size of 50 nm was expected to be small enough to allow circulation in blood and large enough to be detected by the immune system.14 The general strategy, depicted in Figure 1, involved three important steps: i) nanoparticle functionalization with diethylene triaminepentaacetic acid ligands (DTPA) by SPS, ii) covalent coupling of G-CSF proteins on activated DTPA in solution, and iii) particle radiolabeling through the chelation of indium-111 by residual DTPA ligands.

Figure 1. Synthesis of radiolabeled nanoparticle-protein conjugates (SiO2NP-DTPA/G-CSF) via solid phase synthesis (SPS). i) Functionalization of silica nanoparticles grafted on controlled pore glass (CPG) solid support: sequential incorporations of polyethyleneglycol phosphoramidite (x2), aminohexyl phosphoramidite, and diethylenetriaminepentaacetic acid (DTPA) dianhydride, followed by the release of NPs from the CPG support; ii) G-CSF protein



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coupling with EDC/sulfo-NHS activated NP-DTPA; iii) Radiolabeling with Indium-111 through DTPA chelation. The NP-CPG support was prepared according to a previous paper with some modifications.9 CPG beads were first derivatized with amine groups to enable the incorporation of a hydroquinone linker (Q-linker). Q-linker was introduced by Pon et al. for the elaboration of ultramild DNA synthesis supports.15 This linker showed faster cleavage than conventional succinyl linker in mild basic conditions. By analogy with DNA synthesis, we observed a fast release of the nanoparticles from the CPG support, and more importantly, the release step was performed in less drastic conditions (100 mM DBU in H2O/acetonitrile instead of 1 % aqueous NH4OH). Indeed, no degradation of the silica nanoparticles was observed in 100 mM DBU. Once the Q-linker incorporated, the CPG support was capped with trimethylsilyl groups to avoid undesired reactions on the residual amine functions of CPG. The nanoparticles were then grafted onto the CPG support via ester bonds formed with Q-linker. The grafting step was monitored by measuring absorbance of the NP solution. We clearly observed the progressive discoloration of the supernatant along with the coloration of the solid support (Figure S1). Albeit complete grafting was obtained in 2 hours, the suspension was stirred overnight allowing the stabilization of SiO2NP-CPG bonds. The homogeneity of the NP grafting was evaluated by SEM observations. The SiO2NP-CPG support exhibits a homogeneous monolayer of nanoparticles located both inside and outside the pores (Figure S2 A-B). The particles are well separated from each other. As described in a previous paper, the proportion of the NP surface that is accessible for further functionalization was estimated to be 60 % of the whole NP surface.9 After SPS functionalization with two PEG and one amino phosphoramidite derivatives, we observed by SEM the disappearance of the nanoparticles grafted outside the pores (Figure S2 C-D). While the outermost surface of the CPG beads only represents a small proportion of the total accessible surface, we assume that most of the particles were protected and stayed grafted inside the pores during SPS. Moreover, it should be noted that only the most exposed surface to mechanical friction was concerned by this abrasive phenomenon. In a typical sample, both abraded zones and preserved zones were found (Figure S2 F). The SPS was alternatively performed on 35-40 mg or 150-200 mg of NP-CPG using the standard 1 µmol or 10 µmol coupling programs in a DNA automated synthesizer respectively. The silica nanoparticles were functionalized with successive incorporations of two polyethylene glycol phosphoramidites and one 5’-aminohexyl phosphoramidite. The total 6 ACS Paragon Plus Environment

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duration of the synthesis was approximately 30 minutes. Incorporation yields were monitored by measuring absorbance of dimethoxytrityl cations (DMT) released at the end of each incorporation cycle. Coupling yields per cycle comparable to standard SPS were found for PEG (95.1 ± 6.5 %) and amine (88.5 ± 3.5 %) incorporations. The final loading of the support was 6.50 ± 0.18 µmol g-1 (calculated for the aminohexyl phosphoramidite incorporation). SPS led to nanoparticles coated with hydrophilic PEG linkers bearing terminal amine functions and intermediate phosphodiester linkages that generate negatively charged particles. The DTPA groups were then incorporated by reaction of DTPA dianhydride on the amine functions (Figure S3). Due to the sensitivity of G-CSF protein to basic conditions, the release of the nanoparticles in solution was performed prior to protein coupling.16 The cleavage of the particles from the CPG support was achieved in mild basic conditions over a few hours. The progressive release of the particles from the support was monitored visually then controlled by absorbance measurements at 557 nm (Figure S4). Preparation of SiO2NP-DTPA/G-CSF G-CSF proteins were coupled to SiO2NP-DTPA via a simple and well-described EDC/sulfoNHS chemistry.17 A ratio of 3 equivalents of protein per NP was used for the coupling reaction. As discussed previously, the SPS approach provided NPs highly functionalized with DTPA groups. With this material, we studied the protein coupling reaction with a protein concentration ranging from 1 to 64 equivalents per NP. Although we demonstrated that it is possible to immobilize up to 34 protein molecules per SiO2NP-DTPA (confirming the efficiency of the loading strategy), the SiO2NP-DTPA/G-CSF-3 conjugate (coupling reaction with 1 eq. of NP and 3 eq. of protein) was preferred for pharmacokinetic studies in order to compare with AuNP-DTPA functionalized with the same ratio of G-CSF per NP (3/1). After the coupling reaction, the SiO2NP-DTPA/G-CSF conjugate was characterized by HPLC. The conjugate peak was observed at 12.7 min on the chromatogram A (Figure 2). In the same experimental conditions, the G-CSF retention time by HPLC was 13.7 min (Chromatogram B on Figure 2). Considering that the residual presence of free G-CSF was negligible on chromatogram A, it can be concluded that the G-CSF coupling on SiO2NPDTPA was total after reaction. Furthermore, the conjugate was formed only when DTPA functions were pre-activated by EDC/sulfo-NHS reagents since no conjugate peak was observed on the control HPLC without activation (Chromatogram C on Figure 2). That is to 7 ACS Paragon Plus Environment


say that after purification, no residual protein was adsorbed on the SiO2NP-DTPA and the immobilization resulted only from a covalent grafting on the silica surface.

Absorbance (A.U.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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A B C 0

5

10 15 Time (min)

20

Figure 2. HPLC characterization after 18 hours of the coupling reaction of SiO2NP-DTPA/GCSF with (A) or without (C) EDC/sulfo-NHS activation of the DTPA groups prior to reaction; G-CSF control (B). The absorbance was measured at 560 nm; A.U.: arbitrary unit. The covalent coupling was further confirmed by dynamic light scattering (Figure 3). We compared the apparent hydrodynamic size of SiO2NP-NH2, SiO2NP-DTPA, SiO2NP-DTPA with non-activated acids in the presence of G-CSF, and SiO2NP-DTPA/G-CSF. While a slight increase was observed with the grafting of DTPA on NP-NH2, the presence of the protein induced a more pronounced effect. Even under non-coupling conditions, the adsorption of GCSF onto the nanoparticles induced a substantial increase of size. However, the gain was higher with the SiO2NP-DTPA/G-CSF conjugates. Electrophoretic mobility studies were performed and zeta potential measurements were compared. All the NP samples gave negative values at pH 7 due to the presence of a majority of phosphate functions on the surface. Nevertheless, slight differences may be considered. Zeta values of SiO2NP-DTPA was the most negative due to the presence of DTPA on the surface and SiO2NP-DTPA/G-CSF gave the lowest negative zeta value due to the presence of G-CSF. With an isoelectric point of 6.1, the protein is nearly neutral at pH 7, and partially masks the negative charge of the particles. Zeta measurements are in good agreement with the apparent hydrodynamic size of samples and confirmed the efficiency of the protein grafting on NP-DTPA. TEM images confirmed that SiO2NP-DTPA/G-CSF were intact and non-aggregated (Figure 4).


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20

NP-NH2 NP-DTPA

15 Intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NP-DTPA + G-CSF NP-DTPA/G-CSF

10

5

0 10

100 Hydrodynamic size (nm)

Dh (nm)

PdI

1000

ζ (mV)

SiO2NP-NH2

77.2 ± 3.8

0.107

-46.2 ± 7.3

SiO2NP-DTPA

80.0 ± 4.0

0.098

-50.8 ± 3.3

SiO2NP-DTPA + G-CSF*

98.8 ± 8.9

0.196

-48.3 ± 9.5

123.2 ± 11.1

0.157

-44.0± 2.3

SiO2NP-DTPA/G-CSF

* Control reaction without activation of carboxylic acid functions

Figure 3. Hydrodynamic diameter (Dh), polydispersity index (PdI), and zeta potentials (ζ) of SiO2 nanoparticles at different functionalization stages.

Figure 4. Transmission Electronic Microscopy (TEM) image of Si02NP-DTPA/G-CSF.



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Radiolabeling and in vivo biodistribution SiO2NP-DTPA/G-CSF SiO2NP-DTPA/G-CSF were labeled with indium-111 for in vivo biodistribution studies. Radiolabeling was obtained with a radiochemical purity > 96 % after purification on Sephadex G25 column. After 6 hours of incubation at 37°C in phosphate buffer saline pH 7.4, the radiochemical purity (RCP) remained greater than 95%, indicating a suitable kinetics stability to perform in vivo experiments. A ratio of 3 G-CSF per SiO2NP was preferred for this study in order to compare the in vivo distribution with those obtained with the AuNPDTPA/G-CSF. Quantitative tissue distribution analysis was performed at different times following intravenous (i.v.) injection of NPs. Tissue distribution was expressed as the percentage of injected dose per gram of organ (Figure 5). After i.v. administration, high levels of radioactivity were observed in the liver at 30 min, 2 and 4-6 hours post injection, as frequently observed for large NPs.18 The liver uptake was found to be important for both functionalized and non-functionalized NP (~50% of ID/g of tissue for SiO2NP-DTPA and ~60% of ID/g of tissue for SiO2NP-DTPA/G-CSF). Nevertheless, the slight difference observed with SiO2NP-DTPA/G-CSF probably resulted from a higher hydrodynamic diameter of the protein-functionalized NP which further enhances liver uptake. No significant lung accumulation was observed for either nanovectors at any time point, indicating a good colloidal stability. Furthermore, the brain uptake was very low at all time points which suggests that radiolabeled NPs could not cross the blood-brain barrier.


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SiO2NP-DTPA % injected dose / g

80.00 70.00

30 min

60.00

2h

50.00

6h

40.00 30.00 20.00 10.00 0.00

SiO2NP-DTPA/G-CSF 80.00

% injected dose / g

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


70.00

30 min

60.00

2h

50.00

4h

40.00 30.00 20.00 10.00 0.00

Figure 5. Biodistribution study of SiO2NP and SiO2NP/G-CSF after indium-111 labeling; results are expressed as % of injected dose per gram of tissue (mean value of four experiments). The splenic distribution was clearly improved by the G-CSF. At 2 hours post injection, spleen uptake was significantly higher for radiolabeled SiO2NP-DTPA/G-CSF with values of 18.9±0.6 %ID/g compared to the SiO2NP-DTPA (10.0±1.7 %ID/g). It is also worth noting that the main excretion route of both SiO2NP-DTPA and SiO2-DTPA/G-CSF was not achieved by direct renal clearance as we mainly observed accumulation in liver and spleen. But silica is a biodegradable material and elimination is likely to occur through a slow hydrolysis process which was not observed during the 4-6 h of the study. A control was achieved with a radiolabeled SiO2-DTPA/caseinβ conjugate. The caseinβ has a MM of ~24 kDa, close to G-CSF MM (19.6 kDa). The biodistribution study of this control conjugate indicated a value of 1.9 %ID/g in spleen, 2 hours after injection. This experiment shows a 11 ACS Paragon Plus Environment


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different biodistribution of SiO2-DTPA/caseinβ compared to SiO2-DTPA/G-CSF (18.9±0.6 %ID/g in spleen at 2 hours), confirming our hypothesis of G-CSF receptor targeting in splenocytes.

AuNP-DTPA/G-CSF To confirm that the protein induced an increased concentration of the nanovectors in the spleen, the same functionalization was achieved on gold NPs. Ultrasmall AuNPs (~3 nm diameter) were selected for this study as we know that their renal clearance is very rapid and NPs do not accumulate in organs, except those involved in the renal clearance (kidneys and bladder).12, 13 Gold nanoparticles functionalized with DTDTPA ligands (AuNP-DTPA) were synthesized on the basis of the monophasic protocol of Brust.10 The reduction of the gold salt by a strong reducing agent (NaBH4) in presence of DTDTPA ligands provides ultrasmall gold nanoparticles. AuNP-DTPA are composed of a gold core (2-3 nm) and of a highly hydrophilic multilayered DTDTPA shell. The TEM characterization of the nanoparticles is shown in the supporting

data

(Figure

S5).

DTDTPA

is

a

dithiolated

derivative

of

DTPA

(diethylenetriaminepentaacetic acid) which is well known for its ability to form stable complexes with gadolinium ions for magnetic resonance imaging (MRI) and with indium-111 ions for single photon emission computed tomography (SPECT). These nanoparticles have been successfully developed for MRI guided radiotherapy.19, 20 Although bis-amide derivative of DTPA (such as DTDTPA) form less stable complexes with Gd than DTPA, we demonstrated that the stability constant of DTDTPA-Gd complexes onto the gold core is very close to the one of DTPA-Gd in contrast to the case of DTDTPA-Gd in solution. Moreover, the inertness of DTDTPA-Gd is higher when the gadolinium chelates are immobilized onto the gold cores. In other words, AuNP-DTPA are well suited for a safe immobilization of ions of interest for medical imaging.21 Furthermore, the surface functionalization is very similar to the one of SiO2NP-DTPA. The same protocol was applied for coupling G-CSF on the AuNPs.13,

22

A ratio of 3 G-CSF per AuNP was used for the grafting reaction in order to

optimize the loading. The excess of proteins was further removed by centrifugation as described in the experimental section. The indium-111 labeling was achieved with 99% radiochemical purity for in vivo biodistribution studies (Figure 6). The comparison between the biodistribution of SiO2NP-DTPA and AuNP-DTPA reveals that the in vivo behavior of these nanoparticles is as expected different. Whatever the delay between the intravenous 12 ACS Paragon Plus Environment

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injection and the sacrifice, the gold nanoparticles are mainly present in kidneys whereas SiO2NP-DTPA are present in a large amount in the liver. This difference in the biodistribution is assigned to the difference in size since the surface chemical composition is similar for both types of nanoparticles. In agreement with results reported in the literature,11 the largest nanoparticles are quickly uptaken by phagocyte-rich organs (liver, spleen) while the smallest ones are removed from body by renal clearance. However, the renal clearance of AuNPDTPA seems to be slower than the hepatic uptake of SiO2NP-DTPA since the amount in blood is higher for AuNP-DTPA. The higher circulation time of AuNP-DTPA is also reflected by the temporal evolution of radioactivity in the organs after intravenous injection. Even if this level is very low in comparison to the value measured in kidneys, the radioactivity is detected in almost all organs 30 minutes after intravenous injection of gold nanoparticles (~5% in liver and spleen) in contrast to the case of SiO2NP-DTPA. However, the radioactivity quickly decreases. This can be explained by the circulation of blood in these organs and not by the accumulation since the evolution of radioactivity is the same in the blood. After G-CSF functionalization, the main excretion route of the AuNP-DTPA was still through the renal elimination pathway. Only a small delay in the excretion was observed in presence of the protein. This result indicates that the hydrodynamic diameter of AuNP-DTPA/G-CSF was probably less than 8-10 nm, the maximum threshold allowing efficient filtration in the kidneys.11 This hypothesis was corroborated by the Dh values of AuNPs recorded by Vinluan et al., before and after insulin grafting.23 However, this has not been validated experimentally as we failed to measure the hydrodynamic diameter of AuNP-DTPA/G-CSF with good accuracy due to the precision limit of the DLS instrument. We concluded that G-CSF functionalization does not alter the renal clearance pathway of the AuNP-DTPA, which is an important point to avoid long-term toxicity of the nanocarrier. As for SiO2NP-DTPA/G-CSF, the in vivo biodistribution clearly revealed that the splenic and hepatic uptake of the AuNPDTPA was increased by the presence of G-CSF proteins on AuNP-DTPA. A 2 hours post injection, the liver and spleen uptake of AuNP-DTPA/G-CSF were significantly higher with values of 7.5 %ID/g and 4.0 %ID/g respectively compared to liver and spleen uptake of the AuNP-DTPA with values of 2.3 %ID/g and 1.5 %ID/g respectively. As for the SiO2NPDTPA/G-CSF, the increase of the liver uptake observed with the AuNP-DTPA/G-CSF probably resulted from a size increase of the functionalized nano-object. This is corroborated by a higher level of radioactivity in blood (30 minutes after intravenous injection of protein13 ACS Paragon Plus Environment


conjugated gold nanoparticles (AuNP-DTPA/G-CSF) and by the evolution of the radioactivity in kidneys. Considering the biodistribution study of the two nanocarriers elaborated with either silica or gold material and functionalized with G-CSF, we assume that the protein is capable to modify their distribution. In the study, the diameter of the two nano-objects was also different in order to compare the uptake (50nm and 3 nm for SiO2NP-DTPA and AuNPDTPA measured by TEM respectively). We confirmed that the splenic uptake was increased by the protein on the NP surface, regardless of the nature and size of the nanocarrier and probably due to the expression of G-CSF receptor in splenocytes.

AuNP-DTPA 50.0

% injected dose / g

30 min

40.0

2h 4h

30.0 20.0 10.0 0.0

AuNP-DTPA/G-CSF 50.0 30 min

% injected dose / g

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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40.0 30.0

2h 4h

20.0 10.0 0.0

Figure 6. Biodistribution study of AuNP-DTPA and AuNP-DTPA/G-CSF after indium-111 labeling; results are expressed as % of injected dose per gram of tissue (mean value of four experiments).


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CONCLUSION In the present work, we developed a G-CSF-nanocarriers with the potential to target and activate phagocytic cells of the innate immune system, in particular neutrophils. Silica nanoparticles of 50 nm diameter were functionalized on surface with a PEG as hydrophilic polymer, DTPA as indium-111 chelating agent and the granulocyte colony stimulating factor (G-CSF). G-CSF is a leukocyte stimulating protein used in the clinic to treat neutropenia by increasing the number of neutrophils in blood, more particularly after chemotherapy or irradiation. In our approach, the vectorization intends to protect G-CSF against degradation in biological fluids and reinforce its action by a concentration effect in vivo. A solid-phase synthesis (SPS) approach was developed to functionalize the NPs. SPS gave the benefit of automated process, which provides control and reproducibility of the functionalization. It allowed us to incorporate multiple functionalities on surface. The in vivo pharmacokinetic study obtained with the SiO2NP-DTPA of 50 nm diameter was compared to the one of AuNPDTPA of 2-3 nm diameter with similar functions on the surface. Protein functionalization modified the biodistribution of the two nanocarriers. A clear enhancement of the splenic uptake was observed in both cases. As silica NPs rapidly concentrated in liver and spleen after injection, gold NPs circulated more efficiently and then concentrated in kidneys. This interesting comparison proved that two nanocarriers with similar surface functionalization showed different elimination routes in mice, probably due to their difference of size. Considering the biodistribution after G-CSF functionalization, we confirmed that the protein was able to modify the pharmacokinetics by increasing the nanocarrier concentration in the spleen, a reservoir of G-CSF receptor-expressing cells.

EXPERIMENTAL Materials Controlled pore glass (200-400 Mesh, CPG-3000 Å), 3-aminopropyltriethoxysilane (APTES), dimethylaminopyridine (DMAP),

N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium

hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIEA), trimethylsilyl chloride (TMS-Cl), hexamethyldisilazane (HMDS), diethylenetriaminepentaacetic dianhydride (DTPA dianhydride),

1,8-diazabicyclo[5.4.0]undec-7-ene

(DBU),

piperidine,

trimethylamine,



succinic

anhydride,

Page 16 of 24

N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

hydrochloride

(EDC.HCl), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), N,N-dimethylformamide (DMF), and other solvents were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). 68 % nitric acid was purchased from VWR (Strasbourg, France). HydroquinoneO,O’-diacetic acid (HQDA) was purchased from Alfa Aesar (Karlsruhe, Germany). The dithiolated

bisamide

derivative

of

diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic

(DTDTPA) was elaborated as described in a previous paper.21

9-O-dimethoxytrityl-

triethyleneglycol-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite phosphoramidite),

acid

(PEG

6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-

diisopropyl)phosphoramidite (aminohexyl phosphoramidite), and other reagents for DNA synthesis were purchased from Glen Research (Sterling, Virginia, USA). Rhodamine-labeled silica nanoparticles were purchased from Nano-H (Lyon, France). G-CSF (Lenograstim) was purchased from Chugai Pharma as lyophilisate. Each lyophilisate contained 263 µg of G-CSF (MM = 19.6 kDa) and excipients (arginine, phenylalanine, methionine, mannitol, polysorbate 20, and hydrochloric acid). They were dissolved in 1 mL of sterile water before use. Preparation of the SiO2NP-CPG solid support Controlled pore glass beads (CPG) with 300 nm pore size were functionalized with carboxylic acid ligands as described previously with some modifications.9 Briefly, non-functionalized CPG were treated with concentrated nitric acid under reflux for 1 h to activate silanol functions. The particles were extensively washed with deionized water to remove any traces of acid. The particles were then modified with amino groups by incubation in a 10 % (v/v) aqueous solution of APTES at pH 2 for 2.5 h at 70 °C, followed by stabilization of the silane grafting in an oven at 120 °C overnight. The resulting CPG-NH2 were further functionalized with a baso-clivable hydroquinone linker arm (Q-linker) using a coupling method adapted from Pon et al.24 The dry CPG-NH2 (1.0 g) were incubated with HQDA (0.4 mmol, 90 mg), DMAP (0.38 mmol, 46 mg), HBTU (0.38 mmol, 144 mg), DIEA (0.8 mmol, 140 µL), and dry DMF (10 mL) in a glass screw capped vial. DMF was chosen in replacement of acetonitrile for better solubilization of the reagents. The mixture was shaken at 25 °C for 2 h. The CPG-COOH support was then washed with DMF, acetonitrile, and dichloromethane for rapid air drying.


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Residual silanol groups and residual amine functions were capped with trimethylsilyl groups to avoid undesired growing chains during automated solid-phase synthesis. The solid support was thus treated with an equimolar mixture of TMS-Cl (4.5 mL, 35.6 mmol) and HMDS (7.5 mL, 35.6 mmol) at room temperature for 16 h. The support was washed with acetonitrile, water, then a second time with acetonitrile, dichloromethane in a fritted-glass filter, and airdried. In

parallel,

the

fluorescent

nanoparticles

were

silanized

with

3-

(triethoxysilyl)propylhydroxyhexyl urea (TESPHU) as described in our previous reports.9, 25 To ensure the covalent grafting between the CPG-COOH solid support and the nanoparticles, carboxylic acids were activated with HBTU (0.38 mmol, 144 mg), DMAP (0.38 mmol, 46 mg), DIEA (0.8 mmol, 140 µL) in anhydrous acetonitrile (10 mL) at room temperature for 3 h. After a rapid washing of the support with dry DMF, the solution of nanoparticles (10 mL, 10 mg/mL in DMF) was added dropwise to the CPG beads under agitation. The suspension was stirred at room temperature overnight. The solid support turned pink as the color of the supernatant faded. The supernatant was removed and the NP-CPG solid support was rinsed with DMF. The absorbance of the supernatant was measured at 557 nm by a UV-visible spectrophotometer for the quantification of the grafted nanoparticles. Finally, residual activated acids were capped with 1 % (v/v) piperidine in DMF for 1 h at room temperature. After extensive washings with DMF, acetonitrile, and dichloromethane, the NP-CPG were dried in air and kept in the dark at room temperature in a dry place. NP-CPG supports were analyzed by SEM for controlling the homogeneity of the NP grafting. Nanoparticle functionalization by automated solid-phase synthesis Once immobilized on a porous CPG support, the nanoparticles were easily functionalized by phosphoramidite chemistry using an Applied Biosystems 394 RNA/DNA synthesizer. Two sizes of columns were alternatively used with comparable phosphoramidite coupling efficiencies. The 1 µmol scale columns were filled with about 35-40 mg of NP-CPG, while about 150-200 mg of NP-CPG were introduced in the 10 µmol scale columns. Phosphoramidite incorporations were performed using standard 1 µmol or 10 µmol coupling programs. A PEG-PEG-C6NH2 sequence was synthesized as the linker between the nanoparticles and the DTPA. Two PEG phosphoramidites and one aminohexyl phosphoramidite (C6NH2) were sequentially coupled to the nanoparticles. The coupling yields



Page 18 of 24

were monitored by trityl quantification at 498 nm (or at 477 nm for MMT groups of aminohexyl phosphoramidite) for each incorporation. Preparation of the chelating nanoparticles DTPA molecules were coupled onto the amine functions for both radiolabeling and attaching G-CSF proteins. After the solid-phase synthesis, the NP-CPG were transferred into a glass screw capped vial and suspended in a freshly prepared DTPA dianhydride solution (10 mM in dry DMF). Then, a small amount of triethylamine (16 µL, i.e. 0.4 % (v/v) of the total volume) was introduced using a syringe. The suspension was stirred at 25 °C for 24 h. The NP-CPG support was then extensively washed with DMF, acetonitrile, and dichloromethane, and dried in air. Release of the nanoparticles from the solid support NP-DTPA were released in solution by treatment under mild basic conditions. The solid supports were incubated in 100 mM DBU solution in a mix of acetonitrile/water 1/1 (v/v). The suspension was stirred at 600 rpm in a thermomixer at 30 °C. The color of the support gradually faded until the support became blank (typically in few hours). The NP release was monitored by absorbance measurements of the supernatant at 557 nm. The nanoparticles were purified by centrifugal filtration with 30K Amicon Ultra filters (5000 g, 10 min) to yield NPDTPA final solutions after at least three washing cycles with deionized water (final concentration of about 1.0-5.0x1013 NP/mL). Preparation of NP/G-CSF Before protein coupling, carboxylic acid groups of NP-DTPA were activated by a standard EDC/sulfo-NHS protocol. Nanoparticles were centrifuged in Eppendorf tubes at 10 000 g for 10 min. Once the supernatant removed, the nanoparticles were redispersed in 10 mM EDC.HCl and 10 mM sulfo-NHS in a mix of DMF/water 1/1 (v/v). The solution was stirred at 20 °C for 2 h. In parallel, G-CSF protein (Lenograstim, 34 million UI mL-1, 263 µg) was purified by dialysis in a 20K dialysis cassette for 5 h. By this treatment, excipients that could interfere with protein coupling such as arginine, phenylalanine and methionine, were removed. The activated NP-DTPA were centrifuged at 10 000 g for 15 min, resuspended and washed twice with deionized water to remove the DMF and the residual EDC/sulfo-NHS. NPDTPA/G-CSF-3 were prepared by mixing activated NP-DTPA (1.3 mL, 4.6x1013 NP) with purified G-CSF (17 µL, 4.5 µg, 1.36x1014 proteins) at 20 °C for 18 h. The excess of proteins 18 ACS Paragon Plus Environment

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was further removed by centrifugation. The conjugates were characterized by HPLC after 18 h reaction. Characterization of NP/G-CSF HPLC: The solutions were analyzed with an Agilent 1200 HPLC on a Widepore C5 4 x 3 mm precolumn. Retention times were determined by absorbance monitoring at 280 and 560 nm. Gradient elution was achieved using the mobile phase consisting of (A) water/acetonitrile (50:50, v/v) with 0.05 mol L-1 triethylammonium acetate and (B) acetonitrile with 0.05 mol L1

triethylammonium acetate at a flow rate of 0.3 mL min-1. The gradient program was a linear

increase of mobile phase (B) from 25 % to 75 % for 10 min, then a linear decrease from 75 % to 25 % (B) for 5 min, followed by having the system equilibrated with 25 % (B) for 5 min. DLS/Zeta potential: Dynamic Light Scattering (DLS) measurements were carried out with a Zetasizer Nano ZS (Malvern Instruments) to determine hydrodynamic diameter and zeta potential of NPs. The percentage was recorded in intensity-distribution size value. SEM/TEM: Scanning electron microscopy (SEM) pictures were obtained using a FEI Quanta FEG 250 with an accelerating voltage of 25 kV (Centre Technologique des Microstructures – Université Claude Bernard Lyon 1, Villeurbanne, France). SEM samples were metallized in sputtering mode with a 4 nm layer of Cu. Transmission electron microscopy (TEM) pictures were obtained using a Philips CM120 with an accelerating voltage of 120 kV. The nanoparticles were examined after deposition of 5 µL of diluted solutions on a carbon formvar-coated copper grid (Electron Microscopy Sciences) and evaporation to dryness. AuNP-DTPA synthesis and characterization For a typical preparation of gold particles, 200 mg (51 × 10–5 mol) of HAuCl4•3H2O, dissolved in 60 mL of methanol, were placed in a 250 mL round-bottom flask. 256 mg (50 × 10–5 mol) of DTDTPA in 40 mL of water and 2 mL of acetic acid were added to the gold salt solution under stirring. The mixture turned from yellow to orange. After 5 min, 185 mg (489 × 10–5 mol) of NaBH4 dissolved in 14 mL of water were added to the orange mixture under vigorous stirring at room temperature. At the beginning of the NaBH4 addition, the solution became first dark brown then a black flocculate appeared. The vigorous stirring was maintained for 1 h before adding 5 mL of 1 M aqueous hydrochloric acid solution. After the partial removal of the solvent under reduced pressure and at a maximum of 40°C, the precipitate was filtered on a polymer membrane and washed thoroughly and successively with 19 ACS Paragon Plus Environment


Page 20 of 24

0.01 N HCl, water, and diethylether. The resulting black powder (AuNP-DTPA) was dried and stocked in the solid state or dispersed in 10 mL of 0.01 M NaOH solution (up to 200 mg of dry powder). The size of the gold core of AuNP-DTPA nanoparticles were characterized by transmission electron microscopy (TEM) using a JEOL 2010 microscope operating at 200 kV. Radiolabeling and in vivo biodistribution For quantitative biodistribution studies, NP-DTPA/G-CSF were radiolabeled with Indium-111 radionuclide (half-life 2.8 days, stability constant DTPA-In3+ close to 1029). Briefly, 30-70 MBq of high-purity

111

In chloride (specific activity >185 GBq/µg indium-111) in diluted

hydrochloric acid (Covidien, Petten, The Netherlands) was added to 0.5 mL of a solution of the nanoparticles in the presence of 300µL of citrate buffer (50mM, pH5). Indium-111 links to the DTPA hydrophilic layer of the nanoparticles via coordination bonds. The mixture was incubated for 30 min at 40°C. NP-DTPA111In/G-CSF-X were separated from 111In chloride by PD-10 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) as follows. The PD-10 column was first washed with 15 mL of 50 mM citrate buffer (pH = 5). The radiolabeling solution was then loaded on the PD-10 column, and fractions of 0.5 mL were eluted with 50 mM citrate buffer (pH = 5). NP-DTPA111In/G-CSF were first eluted. The radioactivity of each fraction was counted using an ionization chamber (Capintec Radioisotope Calibrator CRC-15, Capintec Inc.). Finally, the four fractions with the highest radioactivity were pooled and the pH of the labeled mixture was then adjusted to 7 using NaOH. Radiochemical purity (RCP) was determined by instant thin layer chromatography (ITLC). ITLC of the purified NPDTPA111In/G-CSF was performed using silica gel plates (Gelman Science Inc., Ann Arbor, MI, USA) in 50 mM citrate buffer (pH = 5) as the solvent, and a TLC scanner (MiniGita, Raytest, Isotopenmessgeräte, GmbH, Straubenhardt, Germany) in order to determine the percentage of free residual

111

111

In chloride. NP-DTPA111In/G-CSF remained at the origin, whereas

In chloride migrated with an Rf of 0.8-0.9. For stability testing, an aliquot of the

purified NP-DTPA111In/G-CSF were incubated at 37 °C in 2 mL phosphate buffer saline (pH 7.4) and radiochemical purity was evaluated as described above. All animal experiments were approved by the local animal ethics committee of University Claude Bernard Lyon 1, according to French legislation, and carried out in line with current guidelines. Adult Bl57 mice (20-25 g) were obtained from Janvier Laboratory (Saint Berthevin, France). All animals were housed under standard environmental conditions (free access to food and water and a 12/12 h light/dark cycle), and acclimated for at least 48 h 20 ACS Paragon Plus Environment

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before experimentation. For all experiments, a gaseous anesthesia protocol was applied using 1-2% isoflurane (Laboratoire Bellamont, Boulogne Billancourt, France) in a mixture of O2/N2 (25/75%) at 0.8 L/min after an induction process. Biodistribution of NP-DTPA111In/G-CSF was studied in order to evaluate the potential of the multimodal nanohybrid agent. Radiolabeled nanoparticles were injected intravenously through a caudal vein in mice after anesthesia had been performed. Mice were sacrificed at 0.5, 2, 4 or 6h post-injection, and organs of interest, such as the heart, lungs, spleen, liver, brain, kidneys, bones, muscles, skin, digestive tract, and cadaver were collected. Urine and feces were also collected each day and weighed and counted for 5 min in an automatic gamma scintillation counter (Wizard gamma counter, Perkin-Elmer, USA). Tissue distribution was expressed as the percentage injected dose per gram tissue (% ID/g). The injected dose was calculated by adding activity of all organs, cadaver, and excretions (feces and urine).

ACKNOWLEDGMENTS The authors would like to thank Christian Chapelle (Pulsalys/SATT de Lyon) for his constant support during the project. Financial support was provided by Pulsalys/SATT de Lyon, the Ministry of Research and Education and the Lyric Grant INCa-DGOS-4664.

SUPPORTING INFORMATION Visual control of the NP grafting on CPG; SEM of SiO2NP-CPG supports; schematic representation of the DTPA linker; conditions of NP release in solution; TEM images of SiO2NP-DTPA and AuNP-DTPA.

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Silica and gold nanoparticles of 50 and 3 nm diameter respectively were functionalized with granulocyte colony-stimulating factor (G-CSF). The cytokine was able to modify the pharmacokinetics by increasing the nanocarrier concentration in the spleen. 133x79mm (96 x 96 DPI)

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Granulocyte Colony-Stimulating Factor Nanocarriers for Stimulation

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