Amphiphilic Dendrimers Control Protein Binding and Corona Formation on Liposome Nanocarriers

Amphiphilic polyphenylene dendrimers (PPDs) with distinct lipophilic and postively or negatively charged surface groups were adsorbed onto liposomes and their impact on protein adsorption in the blood plasma was studied. The PPD corona reduced binding of specific opsonins and increased the adsorption of proteins controlling cellular uptake based on their surface patches. The formation of a protein corona on the surface of nanoparticles is a cellular are that influence protein adsorption such as der 3 A of certain plasma proteins

Amphiphilic polyphenylene dendrimers (PPDs) with distinct lipophilic and postively or negatively charged surface groups were adsorbed onto liposomes and their impact on protein adsorption in the blood plasma was studied. The PPD corona reduced binding of specific opsonins and increased the adsorption of proteins controlling cellular uptake based on their surface patches.
The formation of a protein corona on the surface of nanoparticles is a critical factor that determines their biodistribution, cellular uptake pathways and immune response. There are many parameters that influence protein adsorption on nanocarriers such as their size, surface charges, hydrogen bonds and van der Waals interactions. 1,2 Liposomes are among the most applied nanocarriers, but their biological fate is hard to control. 3 A deeper understanding which surface groups facilitate binding of certain plasma proteins is still elusive. Polyethylene glycol surfaces usually reveal low protein adsorption and stealth-like properties. 1 However, instead of just limiting protein binding, it would be very attractive to design surface coatings that only adsorb certain proteins from blood plasma and thus, enhance cellular uptake or affect biodistribution. To understand and control protein corona formation, novel coatings mimicking features of proteins are designed providing amphiphilic patches with low nanometre dimensions due to hydrophilic, hydrophobic and charged surface groups at defined locations. Dendrimers are monodisperse macromolecules with customizable surface groups and nanometre dimensions. 4 However, many aliphatic dendrimers exhibit backfolding of their dendritic branches. Thus, surface groups are not exposed exclusively at the surface resulting in less defined peripheral patterns. 5 Widely used polycationic poly(amidoamine) (PAMAM) dendrimers interact with blood plasma proteins but they also reveal high cellular toxicities and trigger immune responses. 6 Herein, we apply polyphenylene dendrimers (PPDs) as coatings for liposomal nanocarriers to assess the impact of amphiphilic surface groups on plasma protein binding. The PPDs consist of a semi-rigid polyphenylene scaffold with no backfolding of dendritic branches. 7 Amphiphilic PPDs with alternating sulfonic acid and n-propyl groups provide cellular uptake as well as low cellular toxicity and mimic certain features of proteins. 8 In blood plasma, they form a novel PPD-corona at the surface of adenovirus 5 preventing the endogenous blood coagulation factor X from binding. 9 Liposomal nanocarriers coated with such similar PPDs reveal a considerably altered protein corona in blood serum. 10 Herein, PPDs with either positively or negatively charged amphiphilic or exclusively anionic peripheral groups were prepared and the impact on protein corona formation was studied. We envision controlling the fate of liposomal nanocarriers by the PPD surface in biological fluids such as blood ( Figure 1).  The synthesis of charged PPDs was performed according to the divergent growth approach (Scheme 1, Fig. S2-S5). Growth of the PPDs starts from an ethynylated core, which is reacted with tetraphenylcyclopentadienone (CP) building blocks in a [4+2] Diels-Alder cycloaddition. The CP determines the branching and the absolute surface pattern of the dendrimer. 11 Building block 1a with a sulfonate and n-propyl group was synthesized based on modified procedures from Stangenberg et al. 10,12 and was directly used for the dendrimer growth whereas 1b (Fig. S3) and 1c (Fig. S4) were further modified with the desired surface functionalities by Suzuki coupling reactions (Scheme 1). For the anionic dendrimer with peripheral sulfonate groups, intermediate 3 12 with two neopentylprotected sulfonic acid moieties was synthesized by C-C coupling of the pinacol boronic ester 2 to di-bromo-modified compound 1b. For cationic amphiphilic dendrimers, CP 1c with an iodo-and bromo-substituent was synthesized based on modified protocols from Zhang et al. (Fig. S4). 13 Terminal surface groups were attached to 1c by Suzuki coupling of npropyl functionalized phenyl derivative 4 and either meta-or para-substituted pyridine compounds 5 and 6 (Scheme 1). To achieve a positively charged pyridinium group on the dendrimer rim, the pyridine moieties were methylated. For the firstgeneration dendrimer, the methylation was performed prior to dendrimer synthesis (a priori) to obtain pyridinium modified CP 7a. When synthesizing a second-generation dendrimer, the pyridine group was methylated after dendrimer growth, due to incomplete dendrimer formation with already cationic building blocks at higher generations. In addition, the meta-pyridinyl group of 7b facilitated the methylation on the dendrimer surface. All PPDs are built around the core of 1,3,6,8tetraethynylpyrene (8). 14 The cycloaddition to achieve both neopentyl-protected sulfonic acid-based dendrimers were performed at 145 °C in o-xylene for 48 h. 8,12 After purification by silica gel column chromatography and recycling-gel permeation chromatography (GPC), the neopentyl protective groups of the sulfonic acids were thermally cleaved to obtain 10a and 10b. 8,12 Deprotected dendrimers were further purified via GPC in DMF. The positively charged amphiphilic dendrimer 10c was obtained by cycloaddition of pyrene core 8 and 7a in DMSO at 140 °C for 3 d. The product was precipitated and purified by dialysis. The second-generation dendrimer was synthesized from a first-generation pyrene-based PPD 9, 8 which was reacted with pyridine modified CP 7b in o-xylene at 160 °C for 3 d. After purification by GPC, the product was methylated. Cationic amphiphilic dendrimer 10d was obtained by precipitation in diethyl ether. NMR spectroscopy and MALDI-TOF or ESI mass spectroscopy confirmed the structure of 10a-10d. We coated liposomes and polystyrene nanoparticles with the PPDs and investigated protein corona formation in blood serum and plasma. To compare our results with our previous findings for highly branched amphiphilic PPDs, 10  Please do not adjust margins Please do not adjust margins monolayer by electrostatic interactions. 16 Since S8 did not bind to liposomes efficiently (Fig. S6B), we postulate that the hydrophobic interaction between n-propyl group and lipid tails is necessary to enhance binding to liposomes. In addition, secondgeneration dendrimer (PN)8 coated liposomes to a higher extent than (PN)4 resulting in a positive ζ-potential. Therefore, sufficient surface coverage of liposomes was only achieved for (PS)4 and (PN)8. To assess the impact of the n-propyl (P) group on the protein corona formation, we coated PS-NH2 nanoparticles with (PS)4 and S8. Due to the positive ζ-potential of aminofunctionalized PS particles, adsorption of negatively charged dendrimers is electrostatically driven. This was further supported by using positively charged (PN)4 and (PN)8, which did not show an efficient binding towards PS-NH2 (Fig. S6C). Thus, either blood serum or blood plasma was added to the dendrimercoated liposomes (lipo-dendrimer) or PS-NH2 nanoparticles. The protein adsorption was analyzed quantitatively by Pierce Assay (Fig. S7) and LC-MS/MS (Fig. 2, Fig. S8 and S9). For the blood serum preparation, fibrinogen and other blood clotting factors were removed by centrifugation, whereas blood plasma contained all proteins including clotting factors. For plasma, clotting was prevented by the addition of citrate as anticoagulant. Using the Pierce Assay, we could further confirm the binding efficiency of dendrimers to the particle surfaces by comparing the protein adsorption levels to uncoated nanocarriers (SI Fig. S7B-E). Furthermore, protein quantities increased for higher dendrimer concentrations, which did not largely affect the protein corona composition (SI, Fig. S12-S14). The heatmaps depict an overview of all blood proteins bound onto lipo-dendrimers (lipo-(PS)4 and lipo-(PN)8; Fig. 2A and Fig.  S8) as well as PS-dendrimers (PS-(PS)4 and PS-S8) (SI Fig. S9) in blood plasma and serum. First, we observed a similar alteration in the adsorption of certain proteins from uncoated liposomes to lipo-dendrimers regardless of the charge of the surface group ((PS)4 vs (PN)8; Fig. 2B). Both dendrimer surfaces lead to a significant reduction of the opsonins immunoglobulin γ-2 (Ig γ-2) and complement C3 levels in comparison to uncoated liposomes ( Fig. 2C and D). Opsonins are recognized by immune cells, which are part of the mononuclear phagocytic system (MPS), and they mediate cellular uptake of nanocarriers into phagocytic cells. 1,17 Thus, these opsonins enhance blood clearance and reduce the interaction with targeted cells. 18 In contrast, we observed an enhancement in clusterin binding from uncoated to covered liposomes (Fig. 2E). Clusterin, also termed Apolipoprotein J, decreased unspecific cellular uptake of PEGylated nanocarriers in vitro and functioned as a dysopsonin for macrophages. 19 In general, apolipoproteins bind to a higher extent to hydrophobic nanocarriers. 20 Thus, we assume that the clusterin binding is related to the hydrophobic character of PPDs. Furthermore, fibrinogen was adsorbed on all lipo-dendrimers (Fig. 2F). Previously, the pre-coating of PS nanoparticles with IgG-depleted plasma furnished a high enrichment of fibrinogen and a reduced cellular uptake in macrophages. 21 In addition, the dendrimer coating gave rise to accelerated vitronectin binding (Fig. 2G). These findings were consistent with our previous studies for PPDs and dendrons with higher density of sulfonic acid and n-propyl groups (Fig.  S10). 10 Vitronectin was reported to mediate a selective uptake of lipoplexes into cancer cells with overexpressing αvβ3 vitronectin receptors. 22 We also found fibrinogen and vitronectin binding for PS-(PS)4 and PS-S8 (SI Fig. S11C and D) emphasizing that the binding of these proteins is attributable to the hydrophobic PPD scaffold. Second, we also observed remarkable differences in the protein corona that might be caused by the surface charges (Fig. 3A).
Binding of serum albumin (HSA) for lipo-(PN)8 was enhanced whereas lipo-(PS)4 only showed a very low HSA adsorption (Fig.  3B). HSA is reported to serve as dysopsonin leading to higher blood circulation times. 23 PS-(PS)4 and PS-S8 also displayed a reduction of HSA in the protein corona compared to uncoated nanoparticles (Fig. S11B). This suggests that the positively charged pyridinium group is involved in the interaction of lipo-(PN)8 with HSA. In contrast, inter-α-trypsin inhibitor (IαI) H4 was increased for all negatively charged liposomes and nanoparticles (lipo-(PS)4 (Fig. 3C), PS-(PS)4 and PS-S8 (Fig. S11E)) and IαI is known to interact with the highly negatively charged hyaluronic acid. 24 Additionally, lipo-(PS)4 adsorbed β2-glycoprotein 1 also known as Apolipoprotein H (ApoH; Fig. 3D), which is consistent with our previous findings. 10 ApoH binds to negatively charged phospholipids and mediates cellular uptake into mesenchymal stem cells. 25 For PS-S8, we observed an even higher ApoH adsorption, suggesting that this interaction is favoured by the peripheral sulfonates (Fig. S11F).
In conclusion, we synthesized PPDs with different surface patterns in terms of amphiphilicity and charges with the advantage of shape persistence leading to surface structures with nano-site perfection. We could show that surface charges and hydrophobicity of PPDs alter the protein corona on liposomes. For all PPDs, a reduction of opsonization proteins and enhancement of proteins, which might control selective cellular uptake, were observed. Thus, we demonstrated that the protein corona of nanoparticles is modulated through PPD coating, which opens new avenues to control their biodistribution in vivo. T.W. and K.M. thank the Volkswagen Foundation (Project No. 88396) for financial support. The project is supported by the German Research Foundation (DFG) -Project number 213555243 -SFB 1066. We thank P. Schiel, M. Gai and K. Klein for technical and S. Schuhmacher for graphical assistance.

Conflicts of interest
There are no conflicts to declare.