Distinct Proteins in Protein Corona of Nanoparticles Represent a Promising Venue For

Endogenous Targeting - Part I: In vitro Release and Intracellular Uptake Perspective

Aya Ahmed Sebak1a*, Iman Emam Omar Gomaa2, Aliaa Nabil ElMeshad3, Mahmoud Hussein

Farag1, Ulrike Breitinger1b, Hans-Georg Breitinger1b, Mahmoud Hashem AbdelKader4,5

Supplemental Materials

1 Results and discussion:

1.1 Western Blot (WB) analysis

Two bands of ANXA1 were observed in the medium control (Figure S1), corresponding to two isoforms

of the protein. Two isoforms of ANXA1 were previously reported in rats by Qin et al.1

Figure S1: Western Blot of the medium control of ANXA1. ROTI®Mark TRICOLOR XTRA (10–310 kDa) is used as a

standard protein marker.

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1.2 In vitro release of the loaded cargo, FITC dye, from uNPs and cNPs:

Figure S2: In vitro release of FITC from uNPs and cNPs in different release media: The statistical analysis of the

difference in cumulative released % of FITC after 24 h between uNP1 & cNP1 (a), uNP2 & cNP2 (b), uNP3 & cNP3

(c) and uNP4 & cNP4 (d). (****), (***), (**) and (*) represent (P value < 0.0001), (P value < 0.001), (P value = 0.001

to 0.01) and (P value = 0.01 to 0.05) respectively.

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1.3 Mechanism of Endocytic uptake of uNPs and cNPs:

Sodium azide, NaN3, treatment prior to NPs application caused a significant decrease in cellular

uptake in all formulations (Figure S2) suggesting an energy-dependent endocytic mechanism

because NaN3 is regarded as an ATP depleting agent 2. The extent of decrease in the uptake was

more prominent in the SR conditions in the case of uNPs and in the SF conditions in the case of

cNPs. MβCD pretreatment caused a significant decrease in the NPs accumulation in the case of

hybrid uNPs only, uNP3 and uNP4, especially in the presence of MS. MβCD can extract steroids

out of the plasma membrane inhibiting cholesterol-dependent endocytic processes 3.

Moreover, utilization of sucrose, a clathrin-mediated endocytic inhibitor, caused a reduction in

the extent of the uptake in uNPs only independent of the presence of MS 4. High extent of

clathrin-mediated endocytosis has been reported as a main endocytic uptake pathway for

PLGA-based NPs in the size range of 200 nm 5–7. While, the absence of sufficient reduction in the

uptake of cNPs in the presence of sucrose suggests that peptide conjugation could change the

uptake pathway effecting a change in the overall accumulation of NPs as reported earlier 8. In a

similar manner, nystatin pretreatment reduced the extent of NPs’ uptake in all formulations.

But, the presence of serum reduced the extent of the reduction in the uptake in the case of

cNPs. This suggests a role of caveolin-mediated endocytosis in the accumulation of NPs in the

cells 4. The difference in the extent of decrease in NPs’ uptake in SF and SR conditions in the

presence of NaN3 and nystatin pretreatment suggests a role of serum proteins on changing the

uptake pathway of the NPs 9,10.

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Figure S3: Relative Intracellular Uptake of uNPs and cNPs Upon Blocking of The Endocytic Pathways in uNP1 &

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cNP1 (a&b), uNP2 & cNP2 (c&d), uNP3 & cNP3 (e&f) and uNP4 & cNP4 (g&h). (****), (***), (**) and (*) represent

(P value < 0.0001), (P value < 0.001), (P value = 0.001 to 0.01) and (P value = 0.01 to 0.05) respectively.

1.4 Evaluation of the role of selected serum and cellular proteins on the in vitro

intracellular uptake of uNPs and cNPs:

First Approach; B16F10 melanoma cells pretreated with the antibodies:

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Figure S4: Relative Intracellular Uptake of uNPs and cNPs upon pretreating B16F10 melanoma cells with

antibodies against selected serum and cellular proteins; uNP1 & cNP1 (a&b), uNP2 & cNP2 (c&d), uNP3 & cNP3

(e&f) and uNP4 & cNP4 (g&h). (***), (**) and (*) represent (P value < 0.001), (P value = 0.001 to 0.01) and (P value

= 0.01 to 0.05) respectively.

Second Approach; NPs premixed with the antibodies:

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Figure S5: Relative Intracellular Uptake upon premixing the uNPs and cNPs with antibodies against selected serum

& cellular proteins; uNP1 & cNP1 (a&b), uNP2 & cNP2 (c&d), uNP3 & cNP3 (e&f) and uNP4 & cNP4 (g&h).

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