Granulocyte-Colony Stimulating Factor Nanocarriers for Stimulation of

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G-CSF nanocarriers for stimulation of the immune system (Part II): Dose-dependent biodistribution and in vivo anti-tumor efficacy in combination with Rituximab David Kryza, Gabriel De Crozals, Doriane Mathe, Jacqueline Taleb, Marc Janier, Carole Chaix, and Charles Dumontet Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00606 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 31, 2017

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Bioconjugate Chemistry

G-CSF nanocarriers for stimulation of the immune system (Part II): Dose-dependent biodistribution and in vivo anti-tumor efficacy in combination with Rituximab David Kryza†,‡,*,¥ , Gabriel De Crozals§,¥, Doriane Matheⱶ, Jacqueline Taleb Sidi-Boumedine†, Marc Janier†,‡,#, Carole Chaix§, #, Charles Dumontet‡,ⱶ, #. †

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

France ‡

Hospices Civils de Lyon, 69437 Lyon, France

§

Institut des Sciences Analytiques, UMR 5280 CNRS/Université Claude Bernard

Lyon 1/ENS de Lyon, 69100 Villeurbanne Cedex, France. ⱶ Cancer Research Center of Lyon, INSERM 1052/CNRS 5286/University Claude Bernard Lyon 1, Lyon, France

*Corresponding author:

David KRYZA, Hospices Civils de Lyon, plateforme Imthernat, Hôpital Edouard Herriot, 5 place d'Arsonval, F-69437 Lyon cedex 03

E-mail: [email protected] Tel: +33 469 85 60 06; Fax: +33 472 11 69 57 ¥ #

:These authors contributed equally to this work. : These authors should be considered as co-director

ACS Paragon Plus Environment

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ABSTRACT The purpose of immuno-modulation is to increase or restore the action of immunocompetent cells against tumors with or without the use of monoclonal antibodies. The innate immune system is a key player in various pathological situations but cells of this system appear to be inhibited or insufficiently active in malignancy or severe infectious diseases. The present study was designed to investigate therapeutic value of nanoparticles (NPs) coupled with bioactive hematopoietic growth factors acting on the innate immune system. The use of nanoparticles (NPs) allowing multimodal detection and multifunctional grafting are currently of great interest for theranostic purposes. In the present work, we have evaluated the impact of the number of G-CSF grafted on the surface on the NPs on the biodistribution in mice thanks to indium 111 radiolabeling. Furthermore, we have investigated whether grafted GCSF NPs could stimulate the immune innate system and enhance the therapeutic efficacy of the monoclonal antibody rituximab in mice bearing human lymphoma xenografts. Following intraveinous (i.v) administration of NP-DTPA and NP-DTPA/G-CSF-X high levels of radioactivity were observed in liver. Furthermore, spleen uptake was correlated with the number of G-CSF molecules grafted on the surface of the NPs. Combining NP-DTPA/GCSF-34 with rituximab strongly reduced RL tumor growth compared to rituximab alone or in combination with conventional G-CSF + rituximab. The use of highly loaded G-CSF NPs as immune adjuvants could enhance the antitumor activity of therapeutic monoclonal antibodies by amplifying tumor cell destruction by innate immune cells.


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INTRODUCTION There has been continuous interest in therapeutic strategies based on stimulation of the immune

system

with

granulocyte-monocyte-colony

stimulating

factor

(GM-CSF),

granulocyte-colony stimulating factor (G-CSF), interferon-alpha or interleukin-2. Stimulation of the innate or the adaptative immune systems involves increasing or restoring the action of immunocompetent cells with respect to tumors or infectious agents. The innate immune system is a key yet neglected player in a variety of pathological situations. Cells of the innate immune system (granulocytes, monocytes and natural killer cells (NK)) play a major role but are often inhibited or insufficiently active in tumor progression or during severe infection. These cells play an essential adjuvant in the treatment of malignancies with therapeutic monoclonal antibodies (mAbs), through various mechanisms involving effector cell mediated destruction of tumor cells. ADCC (antibody dependent cellular cytotoxicity), and ADCP (antibody dependent cellular phagocytosis) have been found to be major mechanisms of antitumor activity in patients. Furthermore, NK cells have been found to be the major effector cells for ADCC and positive results of Mabs therapy have been associated with these cell populations.1 While many efforts have gone towards stimulating NK cells,2 little has been done to recruit/activate granulocytes or monocytes in this defense mechanism, although they are very abundant effector cells. Preliminary data in the literature with GM‐CSF in patients receiving anti‐GD2 Mab for the treatment of neuroblastoma also support the interest of this type of stimulation.3 We have recently shown that pegylated-G-CSF could enhance the therapeutic activity of Mabs in preclinical models.4

Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein recombinant growth factor that is mainly used to reduce the severity or duration of chemotherapy-induced neutropenia in cancer patients,5 for hematopoietic stem cell harvesting6 and also associated to anti-infectious therapy.7 G-CSF has been proven not only to increase the number of granulocytes but although to increase and enhance the antitumor effects of various chemotherapeutic agents in preclinical models. The recombinant form of G-CSF has a very short blood half-life even if high doses are administrated and is mainly inactivated by enzymatic degradation leading to administer G-CSF by frequent injections, or to use a pegylated form with a longer half-life. In such context, various efforts have been attempted to increase the efficacy of G-CSF by preventing it rapid degradation.8-10



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The use of nanoparticles (NPs) with a size below 100 nm and offering multiple properties (multimodal detection and/or multifunctional NP) is currently of great interest for imaging applications, targeting and therapy.11 It has been shown that these nanoparticles circulate with a longer half‐life than conventional biomolecules, and that they can be highly loaded with active agents (labeling, targeting or therapeutic molecules).12-13 Thanks to their high surface to volume ratio, they can carry a large amount of active agents to the area targeted in the organism. In the present study, we synthesized three G-CSF-functionalized silica nanoparticles (SiO2NP-DTPA/G-CSF) with various amounts of proteins, bearing X=3, 11 or 34 G-CSF proteins. Their in vivo biodistribution thanks to indium 111 radiolabeling was assessed and compared to non-functionalized NP-DTPA in nude mice. Furthermore, the therapeutic potential of SiO2NP-DTPA/G-CSF as adjuvants in combination with mAbs therapy (rituximab) was evaluated in vivo in a xenograft mouse model of human follicular lymphoma.

RESULTS Preparation of SiO2NP-DTPA

Silica NP functionalization with DTPA was carried out by an original strategy of solid phase synthesis (SPS).14 The approach consisted in immobilizing the silica nanoparticles on the surface of a porous micrometric glass material named CPG (controlled pore Glass) in order to obtain assemblies which can be used in an automatic instrument of DNA synthesis. The NP functionalization was performed via the phosphoramidite chemistry through a synthesis directly programmed in the instrument.15 The SPS was performed on 150-200 mg of NP-CPG assembly using standard 10 µmol phosphoramidite coupling program. The silica nanoparticles were

functionalized

with

successive

incorporations

of

two

polyethylene

glycol

phosphoramidites and one 5’-aminohexyl phosphoramidite. The final loading of amine functions on the support was 6.50 ± 0.18 µmol.g-1 (calculated for the aminohexyl phosphoramidite incorporation). SPS led to nanoparticles coated by 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.


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Preparation of NPs with increasing amounts of G-CSF

G-CSF proteins were coupled to SiO2NP-DTPA via a simple and well-described EDC/sulfoNHS chemistry. Different stoichiometries of NPs and proteins were tested (NP/G-CSF: 1/3, 1/10, 1/34). The coupling reaction was analyzed by HPLC after 18 hours. A control sample of activated NP without protein was also injected (referred as 1/0). Surprisingly, two distinct populations of nanoparticles were observed. Non-functionalized NPs were eluted at 0.4 min, just after the dead volume of the column while the NP-DPTA/G-CSF were eluted at around 13 min after injection (Figure 1). The proportion of the two populations showed good correlation with the amount of protein engaged in the coupling step. The NP-DTPA/G-CSF conjugate peak (at 13 min) appeared together with the decrease of the non-conjugated NPs peak (at 0.4 min) when increasing the protein ratio. The maximal surface coverage of NPs was evaluated to be reached with the NP-DTPA/G-CSF-34 ratio as the signal of the free protein in solution (at 14 min) started to emerge on the chromatogram in presence of a higher amount of protein per NPs for coupling. The elution peak of the NP/G-CSF conjugate was very close to the peak of the free protein. This observation is correlated with results on the coupling of cytochrome c on gold nanoparticles by another group.16

Figure 1. HPLC chromatograms of NP-DTPA/G-CSF with increasing ratios of G-CSF. A break was introduced in the x axis for better visualization of the peaks. Note the differences in the scales of both x and y axes.



To assess the validity of this result, we calculated the theoretical value of the maximal number of protein molecules that could be grafted on the NP surface. We considered the dimensions of 4.5 x 2.6 x 2.6 nm for G-CSF as calculated by X-ray crystallography.17 The size of the silica nanoparticles was measured on TEM images. A mean diameter of 46.1 ± 6.5 nm was found (n = 100) (Figure 2). As about 60 % of the NP surface was coated with DTPA, a surface of 3988 ± 177 nm² may be accessible for protein grafting. We thus found that about 320 G-CSF proteins could be theoretically packed on one particle under a compact monolayer using the equation previously described by Farre et al.15. This theoretical value is 9-times higher than the apparent saturation observed by HPLC. The surface packing of the proteins is largely influenced by protein solvation and hydrodynamic diameter, steric repulsion, protein orientation, protein-protein interaction and NP surface charge. These parameters could affect the grafting. Consequently, the experimentally observed protein loading is far from the monolayer of closely packed protein. It is worth noting that our NPs multi-functionalization strategy also requires to maintain numerous active DTPA groups for further radiolabeling. At this stage, we preferred not to increase further the protein loading on the NP in order to keep intact the chelating properties of DTPA. Consequently, 34 proteins per NP ratio appeared to be a good compromise between a high loading of G-CSF and a subsequent efficient radiolabeling with indium 111. DLS characterization of the NPs showed an increase of the dynamic diameter after grafting of the protein as presented in Figure 3. An average diameter of 106±0.8 nm and 154±1.2 nm were recorded in water for NP-DTPA and NP-DTPA/GCSF34 respectively. Electrophoretic mobility studies were performed and zeta potential measurements were compared. NP-DTPA and NP-GCSF-34 zeta potential values were of 50.8±3.3 mV and -43.9±2.3 mV respectively. The change in zeta potential suggests that DTPA negative charges were partly masked by the proteins which have a global neutral charge in water (G-CSF isoelectric point is 6.1). Taken together with HPLC observations, DLS/zeta potential measurements confirm the efficacy of the NP surface coverage by the growth factors.


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Figure 2. Transmission Electronic Microscopy (TEM) images of NP/G-CSF-34.The average size estimated by TEM is 46.1 ± 6.5 nm.

NP-GCSF

NP-DTPA Size Distribution by Intensity

Size Distribution by Intensity 12 Intensity (Percent)

15 Intensity (Percent)

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


10

5

0

10 8 6 4 2 0

1

106± 0.8 nm

10

100

1000

Size (d.nm)

10000

1

154± 1.2 nm

Record 345: NP-DTPA GDC365 moy

10

100

1000

Size (d.nm)

Record 365: NP-GCSF GDC366 moy

Figure 3. Hydrodynamic diameter obtained by Dynamic Light Scattering (DLS) of the NP-DTPA and the NP/GCSF-34

Radiolabeling and stability tests NP-DTPA and NP-DTPA/G-CSF-X were labeled with 111InCl3 in citrate buffer 50 mM at pH 5 at room temperature. After steric exclusion chromatography purification, radiolabeled NPs were obtained with a high radiochemical purity exceeding 96%. Radiochemical yields of 3050% were obtained. Negligible radioactivity was lost from 111In-NP after incubation at 37 °C in PBS. Radiochemical purity was still greater than 95% in PBS after 48 h for NP-DTPA-111In and NP-DTPA-111In/G-CSF-X. Kinetic stability of radiolabeled NPs in the presence of a competitive chelator (Diethylenetriamine pentaacetic acid :DTPA) was also evaluated. At 48


10000


h post labeling, negligible radiochemical purity decrease was observed (less than 1%) with values varying from 95% (NP-DTPA-111In/G-CSF-3) to 97% (NP-DTPA-111In/G-CSF-34) indicating that labeled NPs with indium 111 were kinetically stable and suitable to perform in vivo quantitative biodistribution.

NFS-60 cell stimulation

To evaluate if the biological activity of G-CSF was affected by the nanoparticle conjugation, the stimulating effect of the conjugates were tested on murine myeloblastic NFS-60 cells, a G-CSF-dependent cell line. The cells were firstly incubated in a G-CSF deprived medium to synchronize cell cycles. Increasing concentrations of G-CSF or SiO2NP-DTPA/G-CSF-34 were then added to the cells. SiO2NP-DTPA with no G-CSF was used as control sample. After overnight incubation, the NFS-60 cells were analyzed by flow cytometry to evaluate cell cycle distribution. The proportion of cells in S-G2 phase is reported in Figure 4.

70.0

70.0

A

G-CSF 55.0

NP-DTPA S-G2 cells (%)

S-G2 cells (%)

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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B

NP-GCSF 55.0

40.0

40.0

25.0

25.0 0

0.1

0.5 1 G-CSF (ng/mL)

5

10

0

5.2E+10 1.6E+11 5.2E+11 1.6E+12 5.2E+12 Nanoparticles (NP/mL)

Figure 4. In vitro assay of G-CSF and SiO2NP-DTPA/G-CSF-34 activity on the stimulation of NFS-60 cells. The proportion of cells in S-G2 phase was evaluated by flow cytometry. Mean values of four experiments, error bars are the standard deviation. 4A : conventional GCSF; 4B: nanoparticles with (NP-GCSF) or without (NP-DTPA) functionalization

These results confirm that NFS-60 cells are dependent on G-CSF (Figure 4A) with a dosedependent response in terms of cell cycle entry. A plateau was reached at 10 ng/mL of soluble G-CSF in solution. Interestingly the cells were also strongly reactive to nanoparticles


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(Figure 4B). This was observed both with non-functionalized nanoparticles and with G-CSFcoupled nanoparticles indicating a possible intrinsic stimulating effect of the NP. Remarkably the amplitude of the effect was significantly greater with nanoparticles than with soluble GCSF. Of note the lowest concentration of nanoparticles evaluated (5.2 1010 NP/mL) contained an amount of G-CSF similar to that which provided a maximal effect when used as the soluble agent. Indeed the theoretical protein concentration in the 5.2 1010 NP/mL sample is estimated to be around 10 ng/mL, considering the equivalent number of protein per NP and the yield of the coupling reaction. These results suggest that nanoparticles and in particular G-CSF-coupled nanoparticles display G-CSF-activity in this preclinical model, and are much more potent than native soluble G-CSF. The stimulatory effect of nanoparticles per se on cell proliferation has already been described. Yao et al. have shown that gold nanoparticles stimulate proliferation and differentiation of murine osteoblast cells.18 Coté-Maurais et al. showed that fullerene nanoparticles increased IL2-induced T cell proliferation while silver nanoparticles tended to reduce proliferation.19

In vivo biodistribution

In order to evaluate tissue uptake, biodistribution of the radiolabeled NP-DTPA and NPDTPA/G-CSF-X were performed after i.v injection in the caudal vein of C57Bl6 mice at 30 min, 2h and 4-6h post injection (n=3 or 4 for each groups) and are presented in Figure 5. After i.v. administration of NP-DTPA-111In and NP-DTPA-111In/G-CSF-X, high levels of radioactivity were observed in the liver at 30 min as frequently observed for large NP. The liver uptake was found to be similar for functionalized and unfunctionalized NPs (~50-60 % of ID/g of tissue). At 24h post injection liver uptake of NP-DTPA-111In/G-CSF-34 was still over 50%ID/g of tissue. No significant lung accumulation was observed for any of the compounds at any time point, indicating a good colloidal stability since aggregation could lead to capillary blockage in the lungs. Furthermore, the brain uptake was very low at all time points for NP-DTPA-111In and NP-DTPA-111In/G-CSF-X which suggest the radiolabeled NPs could not cross the blood-brain barrier. Spleen uptake was significantly higher (Student t test, p

Granulocyte-Colony Stimulating Factor Nanocarriers for Stimulation of

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