Detachment of ligands from nanoparticle surface under flow and endothelial cell contact: Assessment using microfluidic devices

Abstract Surface modification of nanoparticles is a well‐established methodology to alter their properties to enhance circulation half‐life. While literature studies using conventional, in vitro characterization are routinely used to evaluate the biocompatibility of such modifications, relatively little attention has been paid to assess the stability of such surface modifications in physiologically relevant conditions. Here, microfluidic devices were used to study the effect of factors that adversely impact surface modifications including vascular flow and endothelial cell interactions. Camptothecin nanoparticles coated with polyethylene glycol (PEG) and/or folic acid were analyzed using linear channels and microvascular networks. Detachment of PEG was observed in cell‐free conditions and was attributed to interplay between the flow and method of PEG attachment. The flow and cells also impacted the surface charge of nanoparticles. Presence of endothelial cells further increased PEG shedding. The results demonstrate that endothelial cell contact, and vascular flow parameters modify surface ligands on nanoparticle surfaces.


| I N TR ODU C TI ON
Numerous nanoparticles have been developed over the years for therapeutic applications where they hold great promise for effective therapeutic drug delivery, primarily in oncology. 1,2 These nanoparticles include polymeric systems, liposomes, micelles and nanocrystals, among others. [3][4][5][6] Regardless of the composition of nanoparticles, their surface is often modified to extend their blood circulation using polyethyleneglycol (PEG) for instance, or with targeting ligands to enhance tissuetargeting. [7][8][9][10] A large number of studies have demonstrated the ability of PEG to minimize opsonization and clearance by the reticulo-endothelial system sequestration. 9,11,12 At the same time, several targeting ligands including small molecules, peptides, and antibodies have been successfully used for targeting purposes. [11][12][13][14] A number of studies have focused on the design and characterization of surface coating of nanoparticles. 15,16 For example, studies have focused on characterization and optimization of PEG coating and surface ligand density. [17][18][19] These surface coatings are often developed based on in vitro studies and relatively little is known about their stability in vivo. In addition, close contact of these nanoparticles with endothelial cells in vivo may also adversely impact their stability. In this study, we systematically assess the role of vascular flow and The nanoparticles used in this study were rod-shaped camptothecin (CPT) nanocrystals, 20,21 modified with PEG which was physically or chemically tethered to the surface. Previous studies in our and other laboratories have demonstrated the utility of nanocrystals for therapeutic applications. [22][23][24][25][26][27] An additional variant of CPT nanocrystal, carrying PEG-folic acid (FA) conjugate was also investigated. These nanocrystalline camptothecin-based nanoparticles were selected as a test particle group primarily based on the previous research performed on these particles in our laboratory. Similar studies could be performed using other nanoparticle types. Two types of microfluidic devices were used; microvascular network devices (MNs) that mimic complex vasculature and fluid flow conditions observed in vivo or linear channel device's (LCs) which offer a simple and constant flow system.

| Preparation and analysis of camptothecin nanocrystals
All CPT nanocrystals were prepared using the solvent diffusion method as described previously 20 as shown in Figure 1  To incorporate PEG-FA into CPT nanoparticles, 5 ml of 0.8 mg/ml of CPT (Sigma Aldrich) in DMSO solution was pipetted dropwise into a 120 ml water mixture containing 1% w/w alpha-tocopherol (Sigma).
About 20% Acetonitrile-milliQ H 2 O (18.2 MXcm) eluent containing PEG-FA conjugate was then added dropwise to this solution and the overall mixture containing CPT, DSPE PEG2K Amine, and FA was stirred at 800 rpm with constant ultra-sonification at room temperature FIG URE 1 Schematic representation of three CPT nanoparticle scaffolds prepared and used in this study. In case of CPT 1 PEG, the lipid chain of DSPE-PEG is expected to noncovalently associate with the hydrophobic surface of CPT as shown in the schematic. In case of CPT-PEG, two configurations are likely; the amine group in DSPE-PEG-amine is chemically conjugated to the surface of CPT, thus exposing the lipid chain outside. DSPE-PEG-amine may also fold due to hydrophobic interactions between the lipid chain and hydrophobic CPT surface leading to anchoring of the lipid chain on the CPT surface thus exposing PEG in a loop. For CPT 1 PEG-FA, the DSPE-PEG-FA is expected to anchor on CPT by its hydrophobic tail, thus exposing FA outwards (228C) for 1 hr. After 1 hr, the CPT 1 PEG-FA nanocrystals were then centrifuged three times at 208C with milliQ water (18.2 MXcm) at 3,500 rpm. Presence of folic acid was quantified using absorbance at 290 and 370 nm, and CPT was quantified using fluorescence at 366/ 434 nm-both utilized a spectrophotometer (Tecan M220 Infinite Pro). DSPE PEG2K Amine was quantified using ICP-MS as used for CPT 1 PEG constructs.

| CPT-PEG nanoparticles (PEG chemically conjugated to CPT)
To prepare CPT-PEG, unmodified Camptothecin nanocrystal surfaces were activated with carbonyldiimidazole (CDI, MR 1:10, Sigma) in pH 7.4 1x PBS buffer for 5 min. The activated particles were then centrifuged at 5,000 rpm, for 30 min at 208C and washed three times using DI water. They were then mixed with DSPE PEG2K Amine (PEG-NH 2 , MR 1:3) in 1x PBS pH 7.4 and left for overnight coupling at 48C. Finally, the unreacted PEG-amine was removed after three washes with DI water.
In some experiments, FA was directly conjugated to CPT (CPT-FA).
To prepare these particles, 600 ml of 0.5 mg/ml of unmodified CPT nanocrystals were added to 400 ml of the CDI stock solution. This solution was allowed to rotate for 15 min at room temperature. CDIactivated CPT nanocrystals were spun down at 5,000 rpm for 15 min at room temperature. Pellets were collected, washed with DI water, and resuspended in a 0.8 mg/ml solution of Folic Acid in MQH 2 O at pH 5. Folic acid and CDI activated CPT nanocrystals were incubated together and rotated overnight at 48C. CPT-FA were then spun down at 5,000 rpm, 30 min at 208C and washed two times at these conditions in MQH 2 O. CPT and FA presence were determined via absorbance at 366 and 290 nm respectively using independent CPT and FA standard curves on the Tecan M200 PlateReader.
Morphologies of CPT 1 PEG, CPT-PEG, and CPT 1 PEG-FA nanocrystals were analyzed using a scanning electron microscope (SEM).
Surface charges of all nanocrystalline scaffolds suspended in 1x PBS pH 7.4 were measured as zeta potential (ZP) using a Nanoseries-Zetasizer (Malvern).

| R E SU LTS
All CPT nanocrystals were flown through MNs and LCs at 4 ll/min.  Figure 5B). The changes observed in MNs were qualitatively similar to those observed in LCs ( Figure 5C,D). Taken together these matching trends support the core significant findings of this work which depict the reversal of detachment patterns upon the addition of a ligand which is chemically visible to the endothelial cell surface.

| D I SCUSSION
This study used a combination of microfluidic devices to probe the effects of flow and endothelial cell contact on ligand shedding from nanoparticles. Camptothecin nanocrystals were used as a test particle system for use within the microfluidic devices. Several studies report the use of microfluidic devices for studying tumor microenvironments, target-screening, and cancer metastases modeling. [33][34][35][36][37][38][39][40] This study aims to use microfluidic devices to specifically probe the combined effects  42,43 In the presence of cells, PEG shedding was significantly reduced possibly due to reduced interactions of PEG with the cells. The most striking differences were found for PEG-FA-coated nanoparticles. In the absence of cells, CPT 1 PEG-FA was the most resilient coating, exhibiting little shedding. In the presence of cells, this was significantly reversed, leading to extensive shedding, likely due to direct interactions of FA with endothelial cells.
All particles exhibited a significant change in ZP due to passage through the device ( Figure 5A Some of the ligand on the particle may actually potentially be simply loosely adsorbed. While we removed unbound ligand by centrifugation after synthesis, it is possible that some ligands are physiosorbed on the nanoparticle, do not come off during centrifugation, but come off with the flow. Further, note that the ligands are attached to the nanoparticle surface which itself is susceptible to dissolution. Hence, it is possible that flow loosen the attached ligands which are then completely removed during centrifugation post-flow. In other words, centrifugation may enhance the measured effect of flow in the device. In view of this possibility, we suggest that emphasis should be placed on the trends rather than the actual fraction deemed desorbed from the analysis. It is also possible that the shedding of the ligand in the devices is determined by some specific critical locations of high shear or cellular contact, rather than average uniform shear throughout the device. The extent of ligand-shedding will depend on several nanoparticle parameters including material, size, shape, and deformability. Fragility of the particles may also play a role. Conjugation chemistry, especially the length of the linker and the strength of the covalent bond will also impact the degree of shedding. Finally, the chemistry of the ligand, especially the strength of interaction with the vascular wall as well as that with the nanoparticle surface will also impact the extent of detachment. Specifically, flow of the particles in the devices exposes to the high surface area of the vascular wall, which depending on its chemistry, may induce redistribution of the ligand depending on the chemistry. Studies should be performed in future to fully understand the extent of ligand detachment. While the extrapolation of results presented here to other nanoparticles should be done with caution, the findings clearly demonstrate the necessity of assessing these issues during translation of nanoparticles from in vitro to in vivo studies.