Self‐Stabilized Supramolecular Assemblies Constructed from PEGylated Dendritic Peptide Conjugate for Augmenting Tumor Retention and Therapy

Abstract Supramolecular self‐assemblies of dendritic peptides with well‐organized nanostructures have great potential as multifunctional biomaterials, yet the complex self‐assembly mechanism hampers their wide exploration. Herein, a self‐stabilized supramolecular assembly (SSA) constructed from a PEGylated dendritic peptide conjugate (PEG‐dendritic peptide‐pyropheophorbide a, PDPP), for augmenting tumor retention and therapy, is reported. The supramolecular self‐assembly process of PDPP is concentration‐dependent with multiple morphologies. By tailoring the concentration of PDPP, the supramolecular self‐assembly is driven by noncovalent interactions to form a variety of SSAs (unimolecular micelles, oligomeric aggregates, and multi‐aggregates) with different sizes from nanometer to micrometer. SSAs at 100 nm with a spherical shape possess extremely high stability to prolong blood circulation about 4.8‐fold higher than pyropheophorbide a (Ppa), and enhance tumor retention about eight‐fold higher than Ppa on day 5 after injection, which leads to greatly boosting the in vivo photodynamic therapeutic efficiency. RNA‐seq demonstrates that these effects of SSAs are related to the inhibition of MET‐PI3K‐Akt pathway. Overall, the supramolecular self‐assembly mechanism for the synthetic PEGylated dendritic peptide conjugate sheds new light on the development of supramolecular assemblies for tumor therapy.


Dissipative particle dynamics simulations
We constructed a coarse-grained (CG) model for dissipative particle dynamics (DPD) simulation to describe the supramolecular self-assembly process of PDPP. [1] There are five types of beads in this CG model: beads for PEG ("E"); beads for glutamic acid ("G"); beads for lysine ("L"); beads for pyropheophorbide a ("P"); and beads for solvent water molecules ("H"). The interaction parameter is a constant which describes the maximum repulsion between interacting beads, which are shown in Table 1. The coarse-grained model of PDPP is shown in Scheme S1, and the interaction parameters are presented in Table S1.
The interaction parameter between beads can be mapped to the Flory-Huggins χ parameter through Δ = 3.27 . The parameter χ can be calculated from Equation (1): Here, is the average molar volume of the bead, R is the gas constant, T is the absolute temperature, and are the solubility parameters for the chemical entity
The solvent was evaporated and the residue was dissolved in EtOAc. Then the mixture was washed with saturated NaHCO 3 , HCl (1 M) and brine. The solution was dried with MgSO 4 , and concentrated using a rotary evaporator. The residue was subjected to column chromatography purification (silica gel, CH 2 Cl 2 -CH 3 OH = 40: 1) to give a final yield of 74.6%.

Synthesis of Compound 2
6 After tert-butyl groups of Compound 1 (

Synthesis of Compound 4
After tert-butyl groups of The mixture was dialyzed in the dark with Milli-Q water using a dialysis membrane (MWCO = 8000), and then fractionated/purified by size-exclusion chromatography using a Superose 12 HR/16/30 column on an ÄKTA FPLC system (GE Healthcare) with sodium acetate buffer containing 30% acetonitrile (pH 6.5) as a mobile phase.
The mixture was dialyzed in the dark against Milli-Q water and lyophilized to give a final yield of 52.0%. 8

Drug loading
Ppa was dissolved in anhydrous DMSO and then diluted in different concentrations.
The absorbance values of PDPP at 667 nm were obtained and the drug loading was calculated according to the standard absorption curve. Drug loading content (DLC) = (weight of Ppa in PDPP / weight of PDPP)  100%.

Size, zeta potential and morphology
The size, polydispersity index (PDI), correlation coefficient and zeta potential of self-stabilized supramolecular assemblies (PDPP) were measured by DLS, and the

Colloidal stability
The colloidal stability of PDPP was assessed by monitoring the change in the size and PDI as a function of time. PDPP were added in H 2 O, PBS, H 2 O containing 10% of fetal bovine serum (FBS), and PBS containing 10% of FBS respectively, and these mixtures were incubated at 37℃ in a humidified chamber, and their sizes and PDI were monitored by DLS at a pre-determined time.

Critical assembly concentration (CAC)
The CAC of PDPP was determined by using pyrene as a fluorescence probe.
Typically, 20 μL of an acetone solution of pyrene (110 −4 mol L -1 ) was transferred into a 5 mL vial. After acetone was evaporated, 2 mL of an aqueous solution of PDPP was added into the vial to obtain the pyrene concentration of 510 -5 mol L -1 . The excitation spectra of pyrene at different PDPP concentrations were acquired (Emission: 668 nm, Excitation: 320-360 nm, slit width: 5 nm).

Photostability
In a 96-well plate, 100 μL aliquots of PDPP and Ppa in different solutions (DMSO, PBS and H 2 O) at a Ppa concentration of 10 μg mL -1 were arrayed in triplicate. The absorbance at 667 nm was read on a microplate reader (ThermoFisher SCIENTIFIC).
Each well was then irradiated with a 660 nm laser at 5 mW cm -2 at 1-min intervals for 10 min, and the absorbance value was read at pre-determined intervals. The absorbance values at 667 nm were then normalized to the pre-irradiation values and the normalized absorbance values were plotted versus the cumulative irradiation time.

Molar extinction coefficient (ε)
The molar extinction coefficient of PDPP was measured by a UV/Vis spectrophotometer. PDPP was dissolved in DMSO and then diluted to different concentrations (0.01875, 0.0375, 0.075, 0.15, 0.3, 0.6, and 1.210 -6 mol L -1 ). The absorbance values of PDPP samples at 668 nm were read and the extinction coefficient was calculated according to the Beer-Lambert law.

Fluorescent quantum yield (FQ)
PDPP and Ppa were dissolved in anhydrous toluene (610 -6 mol L -1 ). The peak area of the fluorescence emission spectrum of PDPP and Ppa was calculated (Excitation: 660 nm, Emission: 600-750 nm, slit width: 5 nm). The absorbance value of PDPP and Ppa at 600 nm was read. The fluorescent quantum yield was calculated from Equation (2): Where A is the absorbance at an excitation wavelength of 660 nm, and P is the peak area of the fluorescence emission spectrum. S refers to the sample and R to the reference. The FQ (Φ Δ ) of each sample is calculated from the reference Φ Δ value, 0.30 for Ppa.

Singlet oxygen quantum yield (SOQ)
The amount of singlet oxygen generated from PDPP and Ppa was detected using 9, Where A is the absorbance at 660 nm (the excitation wavelength), and K is the gradient of the DMA intensity curve. S refers to the sample and R to the reference.

In vitro ROS generation
The intracellular ROS generation was analyzed by using a peroxide-sensitive Beijing, China). According to the manufacturer's instruction, cells (110 6 cells per well) were treated SSAs and Ppa at the same concentration of 1 μg Ppa mL -1 . After 24 h incubation, the spent medium was discarded and fresh one added, and each well was then irradiated using a 660 nm laser with 1 J cm −2 . After cells were incubated with 1.0 μM DCFH-DA at 37℃ for 40 min, they were harvested and suspended in 100 μL of PBS for flow cytometer analyses. 20,000 events were collected and Flowjo software was used for quantifying the intensity of fluorescence.

Cytotoxicity
The CCK-8 assay was used to examine the photodynamic therapeutic effect of SSAs on cell viability against three cell lines. The cytotoxicity of SSAs and PDP without laser irradiation was also measured by CCK-8 assay. 510 3 L02 normal cells per well were seeded in a 96-well plate, and then treated by Ppa and SSAs for 24 h incubation or PDP for 24 h and 48 h incubation.
Cell viability of each group was obtained through the same process.

In vitro cellular uptake
To determine cellular uptake and intracellular distribution of SSAs, 4T1 cells (210 4 ) were seeded on each glass coverslip to allow attachment, and incubated with For TEM, 4T1 cells cultured in 6 cm dishes were incubated with Ppa and SSAs.
After incubation, cells were rinsed and fixed with 5% glutaraldehyde in sodium cacodylate buffer at room temperature, and then harvested for acquiring TEM images. [2] 1. antitumor studies.

Erythrocytes morphologies and aggregation
Erythrocytes were collected from female BALB/c mice, and erythrocytes suspension was prepared by centrifuging the citrated whole blood at 1000 g for 5 min at room temperature. After the plasma and buffy coat layers were removed, the The dried erythrocytes samples were coated with gold and analyzed under a SEM.

Hemolysis of erythrocytes
Erythrocytes were diluted with PBS to prepare the erythrocyte suspension. Microplate Reader at 540 nm, and hemolysis rate was determined using the following

Equation (4):
Hemolysis rate (%)= Where A S is the absorbance of the sample, A 100 is the absorbance of lysed erythrocytes in H 2 O (a positive control), and A 0 is the absorbance of 0% hemolysis in PBS (a negative control).

Skin photosensitization
The backs of healthy BALB/c mice were depilated 3 days before treatment to ensure that all injures had recovered. The shaved mice were randomly divided into 4 groups: (a) SSAs + laser irradiation; (b) Ppa + laser irradiation; (c) laser irradiation; (d) control (without any treatment). The dose of Ppa was 5 mg Ppa kg -1 . Right after administration, the anesthetized mice were irradiated by a 660 nm laser (0.26 W cm -2 , 10 min, 108 J cm -2 ). The spleen was covered with tinfoil to avoid the expose to the laser. The skin on the back of the mice was photographed on day 0, day 1, day 2 and day 3 after irradiation. The degree of skin response was examined and scored according to Table S5, and Paraffin sections of the skin were stained with hematoxylin and eosin (H&E) according to the manufacturer's instructions. [3]

In vivo pharmacokinetics
Healthy pathogen-free BALB/c mice (5-6 weeks, female) were used to determine the pharmacokinetics of SSAs (n = 7). The mice were intravenously injected with Ppa Plasma was diluted to 50-fold with DMSO for fluorescent measurements using a BioTek plate reader. The pharmacokinetic parameters were calculated using a two-compartment model by WinNonlin 5.2 software.

In vivo fluorescent imaging
After BALB/c nude mice developed established tumors (100 mm 3  were sacrificed on day 61 after treatment.