Patchy Amphiphilic Dendrimers Bind Adenovirus and Control Its Host Interactions and in Vivo Distribution

The surface of proteins is heterogeneous with sophisticated but precise hydrophobic and hydrophilic patches, which is essential for their diverse biological functions. To emulate such distinct surface patterns on macromolecules, we used rigid spherical synthetic dendrimers (polyphenylene dendrimers) to provide controlled amphiphilic surface patches with molecular precision. We identified an optimal spatial arrangement of these patches on certain dendrimers that enabled their interaction with human adenovirus 5 (Ad5). Patchy dendrimers bound to the surface of Ad5 formed a synthetic polymer corona that greatly altered various host interactions of Ad5 as well as in vivo distribution. The dendrimer corona (1) improved the ability of Ad5-derived gene transfer vectors to transduce cells deficient for the primary Ad5 cell membrane receptor and (2) modulated the binding of Ad5 to blood coagulation factor X, one of the most critical virus–host interactions in the bloodstream. It significantly enhanced the transduction efficiency of Ad5 while also protecting it from neutralization by natural antibodies and the complement system in human whole blood. Ad5 with a synthetic dendrimer corona revealed profoundly altered in vivo distribution, improved transduction of heart, and dampened vector sequestration by liver and spleen. We propose the design of bioactive polymers that bind protein surfaces solely based on their amphiphilic surface patches and protect against a naturally occurring protein corona, which is highly attractive to improve Ad5-based in vivo gene therapy applications.


Materials, instruments, and animals
Chemicals were purchased from Sigma Aldrich unless otherwise stated.
Cell lines were obtained from ATCC unless otherwise specified. 1 H and 13 C NMR spectra were recorded on Bruker AMX250, AC300, AMX500, and AMX700 NMR spectrometers using the residual proton or the carbon signal of the deuterated solvent as an internal standard. MALDI-TOF mass spectra were measured using a Bruker Reflex II, which was calibrated against poly(ethylene glycol) (3000 g/mol). Samples for MALDI-TOF MS were prepared by mixing the analyte with the matrix (dithranol) in THF in a ratio of 1:50. In some cases, cationization by mixing the matrix with potassium trifluoroacetate (K) or silver trifluoroacetate (Ag) was performed. All reported MALDI-TOF MS measurements were within the experimental error, characteristic for the applied technique. Flow cytometry was performed with a Beckman-Coulter Gallios 3L10C instrument.
For in vivo studies, C57BL/6J mice were purchased from China Three Gorges University. Bicinchoninic acid (BCA) protein assay kit was purchased from Suzhou Comin Biotechnology. The enhanced chemiluminescence (ECL) kit and PVDF membrane were purchased from Millipore. Antibodies against EGFP, β-Actin, GAPDH and secondary antibody were all obtained from Cell Signaling Technology. Western blots were scanned and bands quantified using Tanon-5500 Chemiluminescent Imaging System.

General procedure for the synthesis of polyphenylene dendrimers (PPDs)
All dendrimers were synthesized by previously reported procedures. 1,2 PPD 1 was recovered as a faint yellow solid in 71% yield. 1

Ad5-based vectors
Virus vectors were prepared according to standard protocols 3 and stored at 80 ℃. All vectors were purified by double CsCl banding and subsequent desalting by PD-10 columns (GE Healthcare). Vectors were stored in 50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol. Vector titers were determined by OD260. 3 Vector purity was confirmed by SDS-PAGE and silver staining.

Light scattering
Light scattering was used to determine interaction between Ad5 and PPD 3 by means of measuring the polydispersity index (PDI) and the hydrodynamic diameter of the particles in case of a monodisperse suspension. A monodisperse suspension was characterized by a PDI ≤ 0.2 and a polydisperse suspension by a PDI ≥ 0.2. Complex formation was performed in a volume of 1 mL PBS with 10 11 Ad5 particles. PPD 3 was added in defined ratios to Ad5, then mixed and incubated for 10 min. After transfer to a cuvette, it was filled up with PBS to a total volume of 2 mL. All samples were tested for an appropriate scattering intensity before measurement (at least 5 × 10 4 ) and measured at an angle θ = 90°. For intensive cleaning of the cuvette, ethanol and acetone was used to avoid measurement errors by dust particles.

Transmission electron microscopy (TEM)
For each sample, complex formation with different ratio of PPD 3 was prepared.
The following procedure was performed similarly to Chen et al. 4 The incubation mixtures were incubated for 10 min and immediately one grid per sample was

Preformed PPD3/Ad5 complexes are essential for increased transduction.
The PPD3 and Ad5 was pre-incubated at different times and the transduction efficiencies were tested ( Figure S3). Within 20 min, the transduction efficiency increased according to the increasing of PPD3/Ad5 pre-incubation time.
Prolonging the incubation time by more than 20 min resulted in no additional increase in transduction efficiency. Thus, the 20 min pre-incubation is optimal for sufficient complex formation, and this condition has also been applied in the following experiments. To further prove that the complex formation by preincubating PPD3 and Ad5 together is essential, PPD3 and Ad5 were added to SKOV-3 cells by various application procedures. Figure     and without PPD3 at 10 7 M. Ad5 with/without PPD3 were pre-incubated 1 h at 37 °C 5% of CO 2 . These experimental conditions were applied to all cell lines. The experiments were performed in duplicate. The error bars present the standard deviation.

Testing the wide type Ad5 (wtAd5) activity when complexed with PPD3
In this experiment we used replication-competent wild-type viruses instead of replication-defective vectors. Upon entry the viruses replicated in the cells, specific particles were formed and the cells were lysed. Therefore, to study cell viability after treatment with wtAd5, the CellTiter-Glo (Luminescent Cell Viability Assay by Promega) was applied. In brief, metabolic activity of the cells was

Real-time quantitative PCR (qPCR) analysis
We The results were consistent with western blot. We also observed significantly decrease of EGFP DNA in liver when mice were infected by PPD3/Ad5 complex instead of raw Ad5. Figure S8. Quantification of EGFP DNA levels in the liver with qPCR (n = 3) after 7 days and 14 days. * represents p-value ≤ 0.05.

Hematoxylin and eosin staining:
For hematoxylin and eosin staining, thin sections of the embedded tissues were then stained with hematoxylin and eosin. After staining, the sections were dehydrated in ascending grades of ethyl alcohol, cleared with xylene, and covered with a coverslip.

Kinetic binding analysis
The interaction between PPD3 and Ad5 was studied by Bio-Layer Interferometry assays (BLI) from Octet96 (Pall ForteBio, CA, USA). In order to receive a significant signal for this binding event, we immobilized PPD3 dendrons at the sensor surface and applied Ad5 as binding molecule. To immobilize PPD at the surface of streptavidin-coated biosensors, we used a biotinylated dendrimer branch of PPD3 with the same surface structure. The basic experiment contains four steps: Step 1: Hydration of the biosensor to record the baseline.
Step 2: Immobilization of PPD3 dendrons on the streptavidin (SA) biosensor. Step 3: Washing and establishing the baseline.
Step 4: Association of the Ad5.
Step 5: Dissociation ( Figure S10). A significant interaction signal was seen even in the presence of only 2 pM Ad5.
The K D (equilibrium dissociation constant) determined by this method is 1.27 × 10 12 M. We believe that this very strong binding could be a result of multivalent interactions between the large virus particles providing large numbers of binding sites and the sensor surface densely coated with PPD3 dendrons.
These results clearly support that there is a strong binding between PPD3 and Ad5 viruses.

Liquid chromatography coupled to mass spectrometry (LC-MS) analysis.
Proteins were digested as previously described. 8 Figure S12. Hard protein corona analysis of polystyrene nanoparticles (PS-NP) and dendrimer (PPD3) coated nanoparticles after serum incubation. 1 µg of protein was applied to the SDS-PAGE (reducing conditions). One representative SDS-PAGE is shown. The experiment was repeated three times. Figure S13. Hard protein corona analysis of polystyrene nanoparticles (PS-NP) and PPD3 coated nanoparticles after plasma incubation. 1 μg of protein was applied to the SDS-PAGE (reducing conditions). One representative SDS-PAGE is shown. The experiment was repeated three times.