advances.sciencemag.org/cgi/content/full/6/6/eaay9937/DC1

Supplementary Materials for

Rapid transport of germ-mimetic nanoparticles with dual conformational

polyethylene glycol chains in biological tissues

Yiwei Yang, Falin Tian, Di Nie, Yuan Liu, Kun Qian, Miaorong Yu, Aohua Wang, Yaqi Zhang, Xinghua Shi*, Yong Gan*

*Corresponding author. Email: ygan@simm.ac.cn (Y.G.); shixh@nanoctr.cn (X.S.)

Published 7 February 2020, Sci. Adv. 6, eaay9937 (2020)

DOI: 10.1126/sciadv.aay9937

The PDF file includes:

Fig. S1. Characterization of NPs. Fig. S2. Schematic illustration and detection of HPEGC of LNS. Fig. S3. Distribution of logarithmic effective diffusivities for NPs with various PEG degrees at a time scale of 1 s. Fig. S4. Additional in vitro data. Fig. S5. Superiority of GMNP for oral delivery. Fig. S6. Superior tumor permeation of GMNP compared with the NR-2%PEG and the NR-6%PEG. Fig. S7. Mechanistic studies. Fig. S8. Protein corona-related and biodistribution studies. Fig. S9. The molecular models used in the CGMD simulation. Fig. S10. Diffusion and internalization simulations.

Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/6/6/eaay9937/DC1)

Movie S1 (.avi format). Diffusion pattern of GMNP in the mucus detected by STED. Movie S2 (.avi format). Diffusion pattern of SNS in the mucus detected by STED. Movie S3 (.avi format). Diffusion pattern of LNS in the mucus detected by STED. Movie S4 (.avi format). Diffusion pattern of NR-2%PEG in the mucus detected by STED. Movie S5 (.avi format). Diffusion pattern of NR-6%PEG in the mucus detected by STED. Movie S6 (.gif format). Simulation of NR with anisotropic PEG density diffusing in biological hydrogels.

Fig. S1. Characterization of NPs. (A) Distribution of adhesion forces on the surface of NPs

detected by COOH-modified AFM probe. Three independent tests were performed and more than

300 forces from 30 particles were analyzed for each group. (B) Hydrodynamic diameters of

PEGylated NPs. (C) Zeta potentials of PEGylated NPs. (D) PEG density measured by

fluorescamine-based assay. Data are means ± SEM.

Fig. S2. Schematic illustration and detection of HPEGC of LNS. (A) Schematic illustration of

detecting HPEGC via adhesion mapping in AFM. Naked NPs exerted negligible adhesion on

probes. For brush-like PEG chains at 6% degree, the probes could distinguish the surrounding

corona and core particle via both height and adhesion imaging. On contrary, dense brush-like

PEG chains for highly rigid HPEGC that exert low adhesion on the probes. When the peakforce

value was increased, the HPEGC could be distinguished from core NPs again. (B) LNS without

PEGylation showed limited adhesive forces and no corona. (C) With 2% PEGylation, negligible

corona of LNS could be found. (D) With 4% PEGylation, LNS showed uniform corona with

intermediate thickness similarly as SNS. (E) Thick hydrated PEG corona of LNS could be

detected at a 6% PEG content. (F) Highly rigid, low adhesive corona of LNS with 20%

PEGylation. The corona could be clearly observed again as the peakforce increased from 1.5 nN

to 5.0 nN. Representative images were presented. Scale bar: 100 nm.

Fig. S3. Distribution of logarithmic effective diffusivities for NPs with various PEG degrees

at a time scale of 1 s. Three independent experiments were carried out and more than 200

particles were analyzed for each group.

Fig. S4. Additional in vitro data. (A) SEM images of SNR and LNR. The size of SNR and LNR

was about 49 102 nm and 75 295 nm. (B) Hydrodynamic dimeters of SNR-6%PEG and LNR-

2%PEG. (C) Zeta potentials of SNR-6%PEG and LNR-2%PEG. Data are means ± SEM. (D)

Ensemble-averaged MSD of NPs diffusing in freshly obtained rat intestinal mucus. (E) Three-

dimensional view of the transport of NPs across multiple E12 barriers at different times. Neither

SNR-6%PEG nor LNR-2%PEG could overcome multiple barriers efficiently. Scale bar: 15 μm.

(F) The GMNP adhered to the membrane via the body side. Tumor spheroid permeation of (G)

non-PEGylated NPs and (H) NPs with 20% PEG degrees. Non-PEGylated NPs showed good

uptake efficiency in peripheral regions while poor diffusing ability limited their deep penetration.

In terms of NPs with highly rigid, hydrophilic HPEGC, poor cellular uptake severely restricted

them from internalizing the spheroids. Scale bar: 100 μm.

Fig. S5. Superiority of GMNP for oral delivery. (A) Transportation along and retention in the

rat small intestine of NPs. The moving pattern of the NR-2%PEG was similar to that of SNS and

LNS, while the NR-6%PEG with uniform, thick HPEGC exhibited a prolonged intestinal

retention effect. (B) Intestinal uptake examined under CLSM 1, 2, 3 and 6 h post intragastric

administration. After 6 h, a considerable amount of GMNP were still being absorbed at the

basolateral side of the intestine while NR-6%PEG with better retention effect showed poor

absorption at basolateral side. (White arrowheads indicated sustained absorption of GMNP.) (C)

Distribution of NPs in and intestinal uptake by duodenum at 1 h and (D) ileum at 6 h post i.g.

administration. (E) The release profile in PBS (n = 3). (F) PK profile after oral administration (n

= 6). Data are means ± SD. ***P < 0.001, one-way ANOVA plus Bonferroni’s test. (G)

Parameters of PK study. Data are means ± SD.

Fig. S6. Superior tumor permeation of GMNP compared with the NR-2%PEG and the NR-

6%PEG. (A) In vivo investigation of tumor permeation and retention effect in tumor-bearing

nude mice. (B) Tumor slices examined under CLSM at 8h post peritumoral injection. Apparently,

the NR-2%PEG showed negligible fluorescence, and although a considerable number of NR-

6%PEG aggregated in the near vascular regions, low uptake efficiency severely limited their

transport.

Fig. S7. Mechanistic studies. (A) Diffusing patterns of the NR-2%PEG and the NR-6%PEG in

intestinal mucus under STED microscopy. (B) Interactions between the tip of GMNP and the

cells. To avoid bias that the piles of GMNP on the AFM probe in Fig. 5C might interfere the

results, another method was adopted to fix the GMNP with exposure of its tip. Gold-coated AFM

probe was cut by FIB to expose the inner silica layer. Thio-PEG5k-silane (4%, w/w) was modified

onto the GMNP, and then the AFM probe was immersed in the solution of thio-terminated GMNP

(10 μg/mL). One particle was attached to the side of probe (as indicated by the yellow arrow) and

exposed its tip. We again investigated the interactions of rod tip with cell membrane via AFM and

the adhesion forces were similar to the previous data in Fig. 5C. Data are means ± SD.

.

Fig. S8. Protein corona-related and biodistribution studies. (A) SDS-PAGE analysis of

protein type coated on the particle surface after incubation with serum. There was no difference in

the amount or type of adsorbed protein. (B) BCA analysis indicated that three types of NPs had

similar amount of protein corona (n = 4). (C) Circulation of NPs. GMNP exhibited longer half-

life time than the isotropic counterparts, indicating the probability of different protein corona

effect during the circulation from in vitro assessment. (D) Bio-distribution of NPs at 24 h post i.v.

administration (n = 4). Data are means ± SEM, **P < 0.01, one-way ANOVA plus Bonferroni’s

test.

Fig. S9. The molecular models used in the CGMD simulation. (A) The model of the protein

network in mucus in the simulation. The network was composed 128 cross-linked polymer chains

(bright cyan chain) and 192 connect the nodes beads (purple beads). In the simulation, the node

beads were also applied to represent the hydrophobic region of the protein network in mucus. (B)

The model of the NR in the simulations. The different colors represented the different regions of

the NR (tip and body), which was used to divide the interaction parameter in the simulations. (C)

A typical initial configuration of the NR in the diffusing simulation. In this configuration, 27 NRs

with same surface property were randomly distributed throughout the polymer network. (D) The

model of the membrane patch composed by lipids and receptors. Red beads and green standed for

the head of the lipid and receptor molecule, respectively, and cyan bead was the tail of the lipids

and receptors. (E) A typical initial configuration for simulation the internalization of the NR. (F)

The interaction parameters for different particle types in the simulation of the NRs’

internalization.

Fig. S10. Diffusion and internalization simulations. (A) The representative MSD values for the

different types of NPs in the polymer network. (B) and (C) Effect of the pore size of the polymer

network in the molecular simulation on the diffusivity of NPs. The (B) MSDs and (C)

cooresponding diffusion coefficient for three types of NRs (with high PEG density

( 0.2, 0.2A B ), anisotropic PEG density ( 0.5, 1.2A B ) and low PEG density

( 0.8, 0.8A B )) when the pore size of the polymer network is tuned from10 to18

gradually. The red, green, and blue lines represent NR with high PEG density, anisotropic PEG

density, and ow PEG density, respectively. The dot line, solid line, and dash line represent MSD

values in pore size of 10 , 14 , and 18 , respectively. (D) The wrapping rate of the different

types of NPs varied with the simulation time. SNS, NR, LNS standed for the small spherical

nanoparticle, nanorod, and large spherical nanoparticle, respectively. w , m , ani , s represented

the weak, intermediate, anisotropic, and strong interactions between NPs and membrane,

respectively. In the simulation, 2.8, 2.8A Bw & 5.6, 5.6A Bs for each type of

NPs; 4.2, 4.2A Bm for SNS and LNS; 2.8, 5.8A Bani for anisotropic NR.