Nanoparticle Ligand-Decoration Procedures Affect in Vivo Interactions

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Nanoparticle ligand-decoration procedures affect in vivo interactions with immune cells Alexandros Marios Sofias, Trygve Andreassen, and Sjoerd Hak Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b00908 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 31, 2018

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Molecular Pharmaceutics

Nanoparticle ligand-decoration procedures affect in vivo interactions with immune cells Alexandros Marios Sofias1,*, Trygve Andreassen2, and Sjoerd Hak1 1Department

of Circulation and Medical Imaging, Faculty of Medicine and Health Sciences,

Norwegian University of Science and Technology (NTNU), Trondheim, Norway 2MR

core facility, Department of Circulation and Medical Imaging, Faculty of Medicine and

Health Sciences, Norwegian University of Science and Technology (NTNU), Trondheim, Norway *A.M.

Sofias: corresponding author at the Department of Circulation and Medical Imaging,

Faculty of Medicine and Health Sciences, NTNU, Medisinsk teknisk forskningssenter (MTFS), Olav Kyrres gate 9, 7030 Trondheim, Norway. E-mail addresses: [email protected] (A.M. Sofias), [email protected] (T. Andreassen), [email protected] (S. Hak). Abstract Ligand-decorated nanoparticles are extensively studied and applied for in vivo drug delivery and molecular imaging. Generally, two different ligand-decoration procedures are utilized; ligands are either conjugated with nanoparticle ingredients and incorporated during nanoparticle preparation, or they are attached to preformed nanoparticles by utilizing functionalized reactive surface groups (e.g. maleimide). Although the two procedures result in nanoparticles with very similar physicochemical properties, formulations obtained through the latter manufacturing process typically contain non-conjugated reactive surface groups. In the current study, we hypothesized that the different ligand-decoration procedures might affect the extent of interaction between nanoparticles and immune cells (especially phagocytes). In order to investigate our hypothesis, we decorated lipidic nanoparticles with a widely used cyclic Arg-Gly-Asp (cRGD) peptide using the two different procedures. As proven from in vivo experiments in mice, the presence of non-conjugated surface moieties results in increased recognition by the immune system. This is important knowledge considering the emerging focus on understanding and optimizing ways to target and track immune cells and the development of nanomedicine-based strategies in the field of immunotherapy.

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Molecular Pharmaceutics 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

Graphical abstract

Keywords Nanomedicine, active targeting, ligand conjugation, maleimide, immune system recognition, phagocytosis, cRGD


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1. Introduction Today’s approved nanomedicines, mostly utilised in oncology, rely on “passive targeting”; accumulation at pathological sites through the enhanced permeability and retention (EPR) effect 1. Although, these approved agents have been thoroughly characterized and the EPR effect is well established in small animal cancer models, a recent meta-analysis showed that on average only 0.7 % of an injected nanoparticle dose accumulates in tumors preclinically 2. Moreover, in patients, the EPR effect is complex and highly diverse 3, and there is doubt with respect to its actual clinical significance 4. An extensively studied alternative to passive targeting is so-called “active targeting”; the application of nanoparticles decorated with ligands recognizing molecular components of pathological tissue or cells (e.g. cell-surface receptors). Those ligand-conjugated nanoparticles have been examined, in their majority, qualitatively and there is lack in understanding their actual in vivo behavior. This was convincingly demonstrated by a very recent study, which quantitatively showed that cancer cell targeting with ligand-conjugated agents is challenging 5. Consequently, the promising pre-clinical potential of these agents has not been translated successfully into clinical practice

6

and no ligand-decorated

nanomedicines have been approved by FDA or EMA yet. However, despite the lack of clinical translation there is hope; as, a better understanding of the in vivo behaviour of these agents could enable the development of receptor / active targeting nanoparticles with predictable in vivo behavior and improved efficacy. One critical aspect of nanoparticle in vivo behavior, which remains unaddressed, is the effect of nanoparticle ligand-incorporation protocols on immune system recognition. Generally, two principally different ligand-decoration approaches are being used; targeting ligands are either pre-conjugated with nanoparticle ingredients and then incorporated during nanoparticle preparation

7,8,

or they are attached to preformed nanoparticles by utilizing reactive

functionalized groups on the nanoparticle surface (e.g. maleimide)

9–12.

Even though this

second manufacturing process is widely used, for efficient ligand conjugation the use of a molar excess of reactive groups as compared to ligands is required

13,

resulting in the

presence of non-conjugated reactive surface groups in administered nanoparticle formulations. Since this may increase recognition by the immune system, this study attempts to elucidate to what extent the chosen ligand-conjugation procedure affects nanoparticle interaction with phagocytic cells.



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To conduct our study we selected cyclo[Arg-Gly-Asp-D-Phe-Lys(Ac-SCH2CO)], (referred to as cRGD) conjugated

14

lipid-based nanoparticles based on their extremely wide use (over

500 manuscripts since 2011; source scopus.com; searched term: “RGD” AND “Nanoparticles” OR “Liposomes” in “Abstract”) as well as our experience with these agents, both in in vitro and in vivo studies

9,15,16.

cRGD is widely used for targeting αvβ3 integrin; a receptor expressed

amongst others by angiogenic vascular endothelial cells, for example in inflammatory conditions or solid tumors

17.

Regarding lipidic nanoparticles they are among the most well-

studied type of nanoparticles for biomedical applications and about half of clinically approved nanoformulations are lipid-based (e.g. Doxil, Myocet, Daunoxome, Marqibo)

18.

We

hypothesized that a nanoparticle surface with and without remaining reactive groups would interact differently with the phagocytic system. As proven by studying nanoparticle uptake in immune cells, we confirmed our hypothesis and demonstrated that remaining reactive surface moieties increase recognition by phagocytes in vivo. Since this alternates targeting efficiency and specificity, it is an important realization in the development of future ligand-conjugated nanomedicine. 2. Materials and methods 2.1.

Materials and equipment

1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2000-DSPE), and 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (malPEG2000-DSPE) were purchased from Avanti Polar Lipids, Inc. 1,2-Dioleoyl-sn-glycero-3phosphoethanolamine labeled with Atto 633 (Atto633-DOPE), soybean oil, and solvents were purchased from Sigma-Aldrich. Cyclo[Arg-Gly-Asp-D-Phe-Lys(Ac-SCH2CO)] (cRGD) and Cyclo[Arg-Ala-Asp-D-Phe-Lys(Ac-SCH2CO)] (cRAD) peptides were purchased from Peptides International. cRGD-PEG2000-DSPE was purchased from SyMO-Chem (Eindhoven, The Netherlands). For composing de-acetylation buffer hydroxylamine, HEPES, and EDTA were purchased from Sigma-Aldrich. Brilliant Violet 421™ anti-mouse/human CD11b Antibody (Clone M1/70), Brilliant Violet 510™ anti-mouse Ly-6G Antibody (Clone 1A8), PE anti-mouse F4/80, APC/Fire™ 750 anti-mouse Ly-6C Antibody (Clone HK1.4), Alexa Fluor 488 antimouse/rat CD61, PE anti-mouse CD51, and TruStain fcX™ (anti-mouse CD16/32) Antibody (Clone 93) were purchased from Biolegend. 7-Aminoactinomycin D viability test (7-AAD) was purchased from ThermoFisher Scientific. For nanoparticle downsizing, a Heat SystemsUltrasonics W-225R ultra-sonicator was used. For nanoparticle dialysis against HBS, Spectra/Por Float-A-Lyzer G2, 100 kDa MWCO were purchased from Spectrum Laboratories.


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Dynamic light scattering was performed using a Malvern Zetasizer Nano ZS. NMR spectra were recorded at 300 K on a Bruker Biospin Avance III 600 MHz instrument, fitted with a QCI cryoprobe. For efficient water suppression, a 1D NOESY pulse sequence was applied (noesygppr1d) with mixing time 10 ms, 64k data points (TD), spectral width 20 ppm, 256 scans and a relaxation delay of 4 s. Cell populations and nanoparticle uptake by immune cells were determined by using Gallios Beckman Coulter flow cytometer and the data was analysed using Kaluza software. 2.2.

Animals

Balb/c mice aged 12-16 weeks were purchased from Janvier laboratories. The mice were kept under pathogen-free conditions at 20 oC, 50 to 60 % humidity, and were allowed food and water ad libitum. All procedures were approved by the Norwegian Animal Research Authorities. 2.3.

Nanoparticle synthesis

For nanoparticle synthesis we used a similar method as previously described 9. In brief, for non-maleimide cRGD nanoparticles and for all bare nanoparticles (Table 1; formulations 1, 3, 4, 5, 7, 8), one-step synthesis was used. All lipids, and in case of the nanoemulsions the oil as well, were dissolved in 4 : 1 chloroform : methanol and the lipidic mix was dripped into preheated isotonic HEPES buffered saline (HBS) of pH 7.4, at 70 oC, under vigorous stirring at 700 rpm. The aqueous/organic solution remained under stirring for 5 minutes at constant temperature, 70

oC,

to completely remove the organic solvents. The obtained crude

nanoparticles were downsized using ultra-sonication (30 Watt and 20 kHz). The sonication duration was regulated proportionally to the total lipid and oil mass of each formulation: 15 min / 25 % duty cycle for 20 µmole lipids for liposomes, and 25 min / 50 % duty cycle for 20 µmole lipids for nanoemulsions. For all cRGDmal (Table 1; formulations 2, 6) or control cRADmal nanoparticles two-steps synthesis was used. Following the same procedure as above, maleimide functionalized nanoparticles were formed in isotonic HBS at pH 6.7 to enable successful peptide-maleimide conjugation. The peptides were dissolved at 2.5 mg/ml in ultrapure water and the acetyl-protected thiol group on the peptides was de-acetylated at pH 7 for 60 min. The activated peptide was added to the pre-formed nanoparticles at a 2:1 maleimide:peptide molar ratio, and the mixture was placed at 4 °C to react overnight. The maleimide nanoparticles were dialysed against HBS of pH 7.4 to remove unconjugated peptide and to obtain physiological pH. After preparation, the maleimide nanoparticles were



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stored at 4 °C for 5 days to assure hydrolysis of non-reacted maleimide groups. Fig. 1 shows cartoons describing the two nanoparticle synthetic methods.

Fig. 1. Cartoons for nanoparticle synthetic methods. (A) One-step synthesis; (B) Two-steps synthesis.

2.4.

Nanoparticles characterization

All injected nanoparticles were characterized for their hydrodynamic diameter, size distribution (dispersity), and zeta potential using dynamic light scattering. All nanoparticles were diluted 1:100 v/v in dH2O enabling a count rate of 300 kcps. Size, size distribution and zeta potential were determined by averaging 7 measurements for each nanoparticle. To determine cRGDconjugation efficiency for formulations 2 and 6 (Table 1), we measured cRGD concentrations in the maleimide containing formulations with NMR spectroscopy. We mixed 10 vol % D2O into the nanoformulations and placed the samples in 5 mm NMR tubes in the spectrometer. The spectra have been scaled to highlight the aromatic signals from the phenyl group in cRGD [cyclo(Arg-Gly-Asp-D-Phe-Lys)] at 7.15-7.35 ppm. The integral of this signal was used to determine cRGD concentration in all samples, using the sample with known cRGD concentration as an external reference and applying the PULCON principle

19.

To determine

conjugation efficiency, we divided the measured cRGD concentration in the formulations by the added cRGD concentration at the start of nanoparticle preparation. 2.5.

In vivo experiments

Nanoparticle uptake / interaction with circulating immune cells was evaluated ex vivo. Mice were anesthetized by a subcutaneous injection of a mixture of fentanyl (0.05 mg/kg), medetomidine (0.5 mg/kg), midazolam (0.5 mg/kg) and water (2:1:2:5) at a dose of 0.1 ml per


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10 gram of body weight. Then, they were intravenously injected with 10 mM cRGD / cRAD liposomes / nanoemulsions (n=3 per nanoparticle) or bare liposomes / nanoemulsions (n=1 per nanoparticle) of a total amount of 2 µmol lipids in a 200 µl volume. Right after injection, all mice were subcutaneously injected with antidote of atipemazol (2.5 mg/kg), flumazenil (0.5 mg/kg) and water (1:1:8) at a dose of 0.1 ml per 10 g of body weight for reversing the anaesthesia completely. At 2 h post-injection, mice were sacrificed and approximately 500 µl blood was collected from the heart. Red cells were lysed and the obtained immune cells were re-suspended in 500 µl FACS buffer (PBS, supplemented with 2 % fetal calf serum and 2 mM EDTA). Two parts of 100 µl each were incubated with 1.0 µg TruStain fcX™ (anti-mouse CD16/32) antibody for 10 minutes. Those two parts were incubated for 30 minutes with either a) CD11b (2 µl), Ly6G (2 µl), F4/80 (2.5 µl) and Ly6C (2 µl), or b) CD11b (2 µl), Ly6G (2 µl), CD61 (β3 integrin marker) (1.5 µl), and CD51 (αv integrin marker) (2.5 µl) antibody cocktails. Subsequently, cells were washed and centrifuged twice at 400 g for 5 min and resuspended in 200 µl FACS buffer. Finally, 1 µl 7-AAD (life/dead marker) was added in each sample, and flow cytometry was performed for each sample until 100000 total counts were collected. The 405 nm laser was used to excite Brilliant Violet 421 and Brilliant Violet 510 fluorophores and the fluorescence was detected using a 450/50 nm and a 550/40 nm bandpass filter respectively. The 488 nm laser was used to excite Alexa Fluor 488, PE and 7-AAD and the fluorescence was detected using a 525/40, a 582/15 and a 620/30 nm bandpass filter respectively. The 633 nm laser was used to excite Atto633 and APC/Fire™ 750 and the fluorescence was detected using a 660/20 nm bandpass filter and a 755 nm long pass filter, respectively. For compensating fluorophore detection, single color samples were run under the same laser voltage conditions. The flow cytometry data were analysed using the Kaluza software. Cellular fragments and debris were gated out of the analysis by utilising forward and side angle light scatter signal. As negative control for the uptake experiment, the blood from a non-injected mouse was used.



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3. Results 3.1.

Nanoparticle characterization

We synthesized four liposomal formulations and four nanoemulsions. The liposomes (Table 1, 1-4) were approximately 100 nm in hydrodynamic diameter and had a low dispersity (PdI < 0.14). The nanoemulsions (Table 1, 5-8) were slightly bigger with hydrodynamic diameters around 130 nm and low dispersity too (PdI < 0.1). Further details about nanoparticle exact composition, size, and PdI are given in table 1. To assess conjugation efficiency, we measured cRGD concentrations in the cRGDmal liposomes and cRGDmal nanoemulsions. We found the conjugation efficiency to be approximately 40 % and 60 % for liposomes and nanoemulsions respectively, resulting in 0.86 and 1.38 cRGD mol % in the formulations. NMR spectra of cRGDmal liposome, cRGDmal nanoemulsion and cRGD are shown in Fig. 2. Table 1. Composition, size, size distribution, zeta potential, and cRGD conjugation efficiency for nanoparticles administered in vivo.

Liposomes (1) cRGD

(2) cRGD

(3) mal

Bare

Nanoemulsions (4) Bare

mal

(5) cRGD

(6) cRGD

(7) mal

Bare

(8) Bare

mal

DSPC (mol %)

62

62

62

62

62

62

62

62

Cholesterol (mol %)

33

33

33

33

33

33

33

33

PEG

-DSPE (mol %)

4

-

5

-

4

-

5

-

mal-PEG

-DSPE (mol %)

-

5

-

5

-

5

-

5

-DSPE (mol %)

1

-

-

-

1

-

-

-

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

-

-

-

-

2.5

2.5

2.5

2.5

Size (nm)

104.5

114.4

92.3

107.9

139.7

140.1

114.8

130.3

Dispersity

0.136

0.133

0.056

0.118

0.095

0.073

0.061

0.086

Zeta potential (mV)

-17.38

-17.56

-13.42

-15.17

-30.47

-39.92

-37.04

-32.15

Standard deviation (mV)

± 4.37

± 7.39

± 4.33

± 2.08

± 1.89

± 6.05

± 2.44

± 8.44

n/a

37.2 %

n/a

n/a

n/a

58.9 %

n/a

n/a

2000

cRGD-PEG

2000 2000

Atto633-DOPE (mol %) Soybean oil (mg / µmol lipids)

cRGD conjugation efficiency


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Fig. 2. Proton NMR spectra of (A) cRGDmal liposomes, (B) cRGDmal nanoemulsions, and (C) cRGD sample. Spectra have been scaled and cropped to clearly show the phenyl signals at 7.15-7.35 ppm used for quantification.

3.2.

In vivo nanoparticle interactions with immune cells

3.2.1. Detecting phagocytic cell populations We obtained blood samples 2 hours post nanoparticle injection, lysed the red cells and stained the immune cells of interest with appropriate antibody panels. The sample preparation procedure resulted in high and reproducible viability, 92.2 % ± 8.5 % (Fig. 3A-B). The CD11b positive immune cells were sub-divided into the three major phagocyte populations, i.e. neutrophils, Ly6Clow monocytes / macrophages (Mo/MΦ), and Ly6Chigh Mo/MΦ, (Fig. 3C-D). The immune cell gating was defined after plotting the cells for F4/80 over Ly6C (Fig. 3D). By utilizing the chosen antibody panels we were able to distinguish the three phagocyte populations clearly (Fig. 3D). As expected, neutrophils (gated as CD11bhighLy6GhighF4/80low) formed the dominant population among CD11bhigh immune cells, 89.2 % ± 4.9 %, while Mo/MΦ (gated as CD11bhighLy6GlowF4/80highLy6Clow or CD11bhighLy6GlowF4/80intLy6Chigh) were significantly fewer, Ly6Clow Mo/MΦ 5.1 % ± 3.5 % and Ly6Chigh Mo/MΦ 5.7 % ± 1.9 %.



Fig. 3. Flow cytometry data and gating procedure. (A) Forward to side angle light scatter for all detected cells. Very low forward and side scattering indicate cellular fragments and debris and were excluded from analysis. (B) By using the 7-AAD live/dead marker we identified live (7-AAD negative) cells. High viability was observed: 92.2 % ± 8.5 % (n=16). (C) Immune cells were gated based on their high CD11b levels. Ly6G antibody staining allowed to distinguish the neutrophil population. (D) Sub-division of CD11bhigh immune cells into the three major populations by utilizing F4/80 and Ly6C monoclonal antibodies. Approximately 9 out of 10 cells were neutrophils (i), 89.2 % ± 4.9 % (n=16), while only 1 out of 10 cells was determined as monocyte / macrophage (Mo/MΦ), Ly6C- Mo/MΦ (ii) 5.1 % ± 3.5 % (n=16), Ly6C+ Mo/MΦ (iii) 5.7 % ± 1.9 % (n=16).

3.2.2. Nanoparticle association with CD11bhigh phagocytic immune cells For assessing the nanoparticle uptake by phagocytes, the three CD11bhigh phagocytic cell populations (Fig. 3D) were plotted together in side scatter (SSC) versus nanoparticle uptake (Atto633 signal) density-plots. The logarithmic x-axis (Atto633 signal) was divided in three sections (Fig. 4A-B). The first section, x ∈ (0-2]; no uptake (-), was separated from the second section, x ∈ (2-100]; uptake (+), after comparing the Atto633 signal with the autofluorescence in an equivalent density plot from a non-injected mouse (Fig. 4B). The distinction between the second (uptake) and third section, x ∈ (100-1024]; high uptake (++), was selected arbitrarily. The most important observation arises from the comparison of the non-maleimide (Fig. 4A; top four) with the maleimide formulations (Fig. 4A; bottom four). CD11bhigh phagocytes


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exhibited higher uptake for nanoparticles when maleimide was present in the formulation. Specifically, 44 % of the CD11bhigh phagocytes was highly positive (++) for cRGD liposomes with maleimide, but only 6 % of them was highly positive (++) for cRGD liposomes without maleimide. This pattern was similar for bare liposomes, as 31 % of the CD11bhigh phagocytes was highly positive (++) for bare liposomes with maleimide, but 0 % highly positive (++) for bare liposomes without maleimide. For nanoemulsions, the extent of uptake by CD11bhigh phagocytes followed the same pattern. In other words, nanoparticle association with CD11bhigh phagocytes clearly increased when maleimide was present in the formulations, demonstrating increased immune recognition and phagocytosis of maleimide containing formulations to occur. A second observation shows that all four cRGD nanoparticles (Fig. 4A; left four) were taken up by phagocytic cells to a higher extent than the nanoparticles without cRGD peptide (Fig. 4A; right four). For all four cRGD nanoparticles, 99 % (average) of the phagocytes had taken up nanoparticles, while only 36 % (average) of the phagocytes engaged non-cRGD formulations. This result was in accordance with the observation that CD11bhigh phagocytes co-express αv and β3 integrin sub-units (Fig. 4C), together making up the target for cRGD, explaining their increased affinity for cRGD nanoparticles. Finally, to confirm that the enhanced uptake of the cRGD formulations was due to interaction with the integrins, we assessed cellular uptake of nanoparticles conjugated with cRAD control peptide (Fig. 4D-E, liposomes: 112.6 nm, PdI: 0.078 and nanoemulsions: 152.6 nm, PdI: 0.090, zeta-potential and peptide conjugation efficiency assumed similar to the cRGD formulations). Indeed, cRADmal nanoparticles associated with CD11bhigh cells to much lower extent than cRGD-decorated analogues and to similar extent as baremal formulations. The association with CD11bhigh immune cells of cRGD, cRAD, and bare nanoparticles with and without maleimide are synopsized in Fig. 5.



Fig. 4. Nanoparticle uptake by CD11bhigh phagocytic cells assessed with flow cytometry. (A) Side scatter versus nanoparticle association (Atto633 signal) density plots for eight injected nanoparticles with and without cRGD and maleimide. The extent of uptake was divided into three groups: no uptake (-) marked with light grey color, uptake (+) marked with grey color and high uptake (++) marked with dark grey color. Both the presence of cRGD as well as the presence of maleimide resulted in an increased association with the CD11bhigh phagocytic cells. (B) Negative control flow cytometry side scatter versus Atto633 signal density plot from a non-injected mouse to determine autofluorescence in the Atto633 channel. (C) CD11bhigh phagocytic cells co-express the αv and β3 integrin subunits, explaining their affinity for cRGD-nanoparticles. (D) cRADmal liposomes and nanoemulsions as control for cRGDmal nanoparticles associated with immune cells to a similar extent as bare maleimide containing formulations. (E) Direct comparison between cRGD and cRAD maleimide containing formulations show the important role of cRGD for targeting CD11bhigh phagocytic cells.


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Fig. 5. Summary of the nanoparticle association with CD11bhigh immune cells observed in this study. (A) A table presenting the percentages of cells displaying no uptake (light grey), uptake (grey), and high uptake (dark grey) after injection of the different formulations. (B) The cumulative percentages of positive cells according to the table in A, visualizing the increased association of the CD11bhigh phagocytic cells with maleimide containing nanoparticle formulations.

4. Discussion The main goal in this study was to evaluate how specific ligand-functionalization protocols affect the interactions between nanoparticles and immune cells in vivo. We decorated lipidic nanoparticles with cRGD via two widely used approaches; a one-step method, by incorporating pre-conjugated cRGD-lipid, or a two-step method where cRGD was conjugated to already formed nanoparticles containing maleimide on their surfaces. Due to inefficient conjugation, a common phenomenon in formulations obtained through the two-step process is the presence of non-reacted functional groups on nanoparticle surfaces in administered formulations. This was also the case in the current study and using ex vivo flow cytometry we demonstrated that these remaining reactive moieties result in increased recognition and phagocytosis by circulating immune cells. In order to validate the association of residual maleimide with the increased phagocytosis, we conducted the same experiment not only with cRGD-decorated nanoparticles, but also with bare nanoparticles with and without maleimide. Furthermore, we observed that the presence of cRGD in the nanoparticles increased nanoparticle uptake by circulating phagocytes as compared to control cRAD as well as nonconjugated agents, presumably as a result of αv and β3 integrin sub-units expression on these cells. A graphical summary of our observations is provided in Fig. 6.



In the current study, we utilized two different lipid-based nanoparticles, which are among the most commonly used and well characterized nanoparticles in the field of nanomedicine. Although it is well-established that nanoparticle material and properties such as size, shape, and surface charge affect “nanoparticle in vivo behaviour” and interactions with cells, we observed very similar effects of maleimide residues on phagocyte recognition for both lipidic agents. Moreover, targeting ligands (such as cRGD) are used on a wide variety of formulations and generally result in very similar effects on nanoparticle in vivo behaviour, irrespective of nanoparticle characteristics

20–22.

Therefore, we expect maleimide residues to increase

phagocyte recognition of different nanoparticle platforms than studied here as well. Additional to nanoparticle properties, the administered dose is also a parameter which may affect nanoparticle-phagocyte interactions. For example, varying dose could alter the percentage of administered material associating with phagocytes. However, since we selected a dose (100 µmol of lipid/kg) in the range of typically used preclinical Doxil doses (20-200 µmol of lipid/kg) 23,24,

the effects noted can be considered relevant in typical therapeutic studies.

Using NMR spectroscopy, we observed approximately 40 % and 60 % conjugation efficiency for cRGD to maleimide on liposomes and nanoemulsions respectively, demonstrating the imperfect conjugation obtained in this procedure. These observations are in line with a study published recently

13

showing that a high ligand conjugation efficiency to maleimide can only

be achieved at 2:1 or higher molar excesses of maleimide. Since investigators typically focus on ligand conjugation efficiency, rather than on the presence of non-functionalized moieties on nanoparticle surfaces, an excess of reactive surface groups is often used 11,12. Hence, the presence of remaining reactive groups on nanoparticle surfaces is a common phenomenon. Variations in the concentrations of these remaining reactive moieties as well as ligand surface densities presumably affect the extent of nanoparticle recognition by phagocytes. The obvious solution to avoid these variations would be to utilize a one-step synthesis. If one is dependent on a two-step synthesis, using a large molar access of ligand to conjugate all reactive moieties may provide control over these two parameters. Nanoparticle interactions with circulating immune cells were evaluated by examining the percentages of nanoparticle (Atto633 dye) positive cells in flow cytometry. For sorting cells into different populations, FSC, SSC, and four common antibodies, i.e. CD11b, Ly6G, Ly6C, F4/80, were used. The use of these four antibodies allowed a robust division of the phagocytic cells into 3 distinct sub-populations (Fig. 3D). Interestingly, by projecting the gating back to the CD11b / Ly6G density plot, it is evident that those three sub-populations are already separated with high accuracy (Fig. 3C). This can be quite useful in complex flow cytometry


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panels, when several antibodies have to be used in a protocol with limited space for leukocyte staining. In that occasion, neutrophils can be gated as CD11bhighLy6GhighSSCint, Ly6Clow Mo/MΦ as CD11bhighLy6GintSSChigh, and Ly6Chigh Mo/MΦ as CD11bhighLy6GlowSSClow. One reason to study circulating rather than tissue resident immune cells is the ease with which large numbers of phagocytes can be obtained from blood. Another important reason is the realization that circulating phagocytes interact extensively with intravenously administered formulations and can represent a significant barrier for extravascular nanoparticle delivery 25. Hence, our approach not only allows for studying immune system recognition of nanomedicines, but it may also be applied for optimizing nanoparticle design with respect to circulating phagocyte evasion. The nanoparticles were decorated with the cRGD peptide, which is widely used for targeting αvβ3 integrin expressed on angiogenic endothelial cells

26,27.

We observed that the αv and β3

integrin subunits are co-expressed also on the surface of CD11bhigh cells (Fig. 4C) explaining the increased cRGD nanoparticle interaction with these cells as compared to non-cRGD nanoparticles. We assessed co-expression of the integrin subunits rather than expression of the dimeric αvβ3 integrin receptor since no murine αvβ3 integrin specific antibody exists. In addition, we observed that the different manufacturing process used here did not affect nanoparticle physiochemical properties (same size, dispersity, and zeta potential), but obviously, it did alternate the way that the nanoparticles interact with the phagocytes. The maleimide residues may alter the immunobiological properties of the nanoparticle by diminishing the shield provided by the PEG coating, potentially affecting the composition of the particle protein corona

28.

The understanding of nanoparticle-phagocyte interactions has

become a hot topic as the field of cancer immunotherapy is rapidly growing. At the same time, the necessity to target and track immune cells becomes more and more important

29,30.

Investigators focus on understanding and optimizing ways that will allow us to succeed in enhancing

immunomodulation,

decreasing

immunotoxicity,

reverse

tumor-mediated

immunosuppression, and improve the precision on activating specific immune system pathways

29–33.

Eventually, understanding nanoparticle targeting mechanisms and in vivo

behavior patterns, will enable their utilization in cancer immunotherapy. We demonstrated that each parameter in designing nanoparticles is crucial, especially in therapies aiming to target immune cells.



Fig. 6. Suggested cRGD nanoparticles uptake mechanism by phagocytes. The presence of hydrolyzed maleimide groups on nanoparticle surfaces increases nanoparticle recognition by the immune system, resulting in enhanced phagocytosis. Consequently, cRGD nanoparticles manufactured via the “two-step method” (maleimide method) are taken up to a higher extent than cRGD nanoparticles manufactured via the “one-step method” (cRGD-PEG2000DSPE pre-synthesis).

5. Conclusion To conclude, we tackled the topic of nanoparticle characterization from a new angle based on immunological observations. Despite the fact that two different manufacturing processes produced nanoparticles with very similar physicochemical properties, we showed that nonreacted functional moieties on nanoparticles obtained via the two-step (maleimide) method significantly increased their recognition and phagocytosis by circulating immune cells. This is important knowledge, especially when considering the emerging focus on understanding and optimizing ways to target and track immune cells and the development of nanomedicine-based immunotherapeutic strategies. 6. Acknowledgements Authors would like to acknowledge funding from Central Norway Regional Health Authority (Helse Midt-Norge). The NMR experiments were performed at the MR Core Facility, NTNU, funded by the Faculty of Medicine and Health Sciences at NTNU and Central Norway Regional Health Authority. 7. Competing interests


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Fig. 1. Cartoons for nanoparticle synthetic methods. (A) One-step synthesis; (B) Two-steps synthesis. 240x119mm (150 x 150 DPI)


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Fig. 2. Proton NMR spectra of (A) cRGDmal liposome, (B) cRGDmal nanoemulsions, and (C) cRGD sample. Spectra have been scaled and cropped to clearly show the phenyl signals at 7.15-7.35 ppm used for quantification. 153x124mm (150 x 150 DPI)



Fig. 3. Flow cytometry data and gating procedure. (A) Forward to side angle light scatter for all detected cells. Very low forward and side scattering indicate cellular fragments and debris and were excluded from analysis. (B) By using the 7-AAD live/dead marker we identified live (7-AAD negative) cells. High viability was observed: 92.2 % ± 8.5 % (n=16). (C) Immune cells were gated based on their high CD11b levels. Ly6G antibody staining allowed to distinguish the neutrophil population. (D) Sub-division of CD11bhigh immune cells into the three major populations by utilizing F4/80 and Ly6C monoclonal antibodies. Approximately 9 out of 10 cells were neutrophils (i), 89.2 % ± 4.9 % (n=16), while only 1 out of 10 cells was determined as monocyte / macrophage (Mo/MΦ), Ly6C- Mo/MΦ (ii) 5.1 % ± 3.5 % (n=16), Ly6C+ Mo/MΦ (iii) 5.7 % ± 1.9 % (n=16). 178x175mm (150 x 150 DPI)


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Fig. 4. Nanoparticle uptake by CD11bhigh phagocytic cells assessed with flow cytometry. (A) Side scatter versus nanoparticle association (Atto633 signal) density plots for eight injected nanoparticles with and without cRGD and maleimide. The extent of uptake was divided into three groups: no uptake (-) marked with light grey color, uptake (+) marked with grey color and high uptake (++) marked with dark grey color. Both the presence of cRGD as well as the presence of maleimide resulted in an increased association with the CD11bhigh phagocytic cells. (B) Negative control flow cytometry side scatter versus Atto633 signal density plot from a non-injected mouse to determine autofluorescence in the Atto633 channel. (C) CD11bhigh phagocytic cells co-express the αv and β3 integrin sub-units, explaining their affinity for cRGDnanoparticles. (D) cRADmal liposomes and nanoemulsions as control for cRGDmal nanoparticles associated with immune cells to a similar extent as bare maleimide containing formulations. (E) Direct comparison between cRGD and cRAD maleimide containing formulations show the important role of cRGD for targeting CD11bhigh phagocytic cells. 318x345mm (150 x 150 DPI)



Fig. 5. Summary of the nanoparticle association with CD11bhigh immune cells observed in this study. (A) A table presenting the percentages of cells displaying no uptake (light grey), uptake (grey), and high uptake (dark grey) after injection of the different formulations. (B) The cumulative percentages of positive cells according to the table in A, visualizing the increased association of the CD11bhigh phagocytic cells with maleimide containing nanoparticle formulations. 287x166mm (150 x 150 DPI)


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Fig. 6. Suggested cRGD nanoparticles uptake mechanism by phagocytes. The presence of hydrolyzed maleimide groups on nanoparticle surfaces increases nanoparticle recognition by the immune system, resulting in enhanced phagocytosis. Consequently, cRGD nanoparticles manufactured via the “two-step method” (maleimide method) are taken up to a higher extent than cRGD nanoparticles manufactured via the “one-step method” (cRGD-PEG2000-DSPE pre-synthesis). 262x151mm (150 x 150 DPI)



Graphical abstract 262x151mm (150 x 150 DPI)


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Nanoparticle Ligand-Decoration Procedures Affect in Vivo Interactions

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