Coarse-grained molecular simulation of extracellular vesicle squeezing for drug loading

Abstract

In recent years, extracellular vesicles have become promising carriers as next-generation drug delivery platforms. Effective loading of exogenous cargos without compromising the extracellular vesicle membrane is a major challenge. Rapid squeezing through nanofluidic channels is a widely used approach to load exogenous cargoes into the EV through the nanopores generated temporarily on the membrane. However, the exact mechanism and dynamics of nanopore opening, as well as cargo loading through nanopores during the squeezing process remains unknown and it is impossible to visualize or quantify it experimentally due to the small size of the EV and the fast transient process. This paper developed a systemic algorithm to simulate nanopore formation and predict drug loading during extracellular vesicle (EV) squeezing by leveraging the power of coarse-grain (CG) molecular dynamics simulations with fluid dynamics. The EV CG beads are coupled with implicit the fluctuating lattice Boltzmann solvent. The effects of EV properties and various squeezing test parameters, such as EV size, flow velocity, channel width, and length, on pore formation and drug loading efficiency are analyzed. Based on the simulation results, a phase diagram is provided as a design guide for nanochannel geometry and squeezing velocity to generate pores on the membrane without damaging the EV. This method can be utilized to optimize the nanofluidic device configuration and flow setup to obtain desired drug loading into EVs.

Figure 1:

Drug loading through EV

Figure 2:

Simulation setup (a) XY view, (b) orthogonal view, (c) CG model of EV, (d) and (e) simulation box with EV

Figure 3:

Comparison of our simulation result with literature^. (a) Variation of DI with various friction coefficients($\zeta$). $\zeta =2$ shows agreement with Seifert et al. (b) Variation of DI with various Ca. Calculated value shows good agreement with literature

Figure 4:

EV squeezing at a high flow velocity of 500 mm/sec. (a) Visualization of the simulation channel from XZ plane (b) molecular representation of EV from XY plane. (I) EV at entrance location $X_s$, (II) EV at middle of squeezing channel $X_m$, (III) EV at exit location $X_e$.

Figure 5:

Squeezing of EV under various flow velocities. (a) XY plane top view (b) XY plane bottom view; (I) $V_1=300\mathrm{m}\mathrm{m}/\mathrm{s}\mathrm{e}\mathrm{c}$, (II) $V_2=500\mathrm{m}\mathrm{m}/\mathrm{s}\mathrm{e}\mathrm{c}$ and (III) $V_3=700\mathrm{m}\mathrm{m}/\mathrm{s}\mathrm{e}\mathrm{c}$.

Figure 6:

Simulation result under various flow velocities. (a) horizontal diameter, (b) vertical diameter, (c) Center of Mass (COM) velocity, and (d) Pore area vs. channel location (e) Drug loading over time.

Figure 7:

EV shape during squeezing at $V_2\approx 500\mathrm{m}\mathrm{m}/\mathrm{s}\mathrm{e}\mathrm{c}$ and L=6D. (a) No pore under constriction width $W_{sq}=.8\mathrm{D}\mathrm{\sigma }$; (b) circular pore for constriction width $W_{sq}=.7\mathrm{D}\mathrm{\sigma }$; (c) EV damage for constriction width $W_{sq}=.6\mathrm{D}\mathrm{\sigma }$.

Figure 8:

EV squeezing simulation result under various channel widths. (a) horizontal diameter, (b) vertical diameter, (c) Center of Mass (COM) velocity, (d) Pore area vs. channel location, (e) Drug loading over time.

Figure 9:

EV shape during squeezing at ${V_2}_{}\approx 500mm/sec$ and L=6D for various EV sizes. (a) EV diameter $D=60\sigma$, (b) EV diameter $D=70\sigma$, (c) EV diameter $D=80\sigma$

Figure 10:

EV squeezing simulation result for EV of various diameters. (a) horizontal diameter, (b) vertical diameter, (c) Center of Mass (COM) velocity, (d) Pore area vs. channel location, and (e) Drug loading over time.

Figure 11:

EV shape and pore formation during squeezing at : $V_{}\approx 500mm/sec$ and $W_{sq}=0.7\mathrm{D}$ for various channel lengths (a) Very few pores for constriction length $L_{sq}=1\mathrm{D}$, (b) medium size pores for constriction length $L_{sq}=2\mathrm{D}$, (c) small and large pore for constriction length $L_{sq}=3\mathrm{D}$.

Figure 12:

EV squeezing simulation results under various channel lengths. (a) horizontal diameter, (b) vertical diameter, (c) Center of Mass (COM) velocity, and (d) Pore area vs. channel location (e) Drug loading over time.

Figure 13:

Phase diagram of pore formation status under different EV velocities and constriction widths. Damage zone is where permanent damage or rupture happens on EV. No pore zone is where there is no pore formation on EV. Safe zone is the suggested status where transient pores are formed on EV enabling drug loading.

Figure 14:

Summary of maximum pore area for EV squeezing under various conditions.