Functional cRGD-Conjugated Polymer Prodrug for Targeted Drug Delivery to Liver Cancer Cells

To overcome the limitation of conventional nanodrugs in tumor targeting efficiency, coupling targeting ligands to polymeric nanoparticles can enhance the specific binding of nanodrugs to tumors. Cyclo(Arg-Gly-Asp-d-Phe-Lys) (abbreviated as c(RGDfK)) peptide has been widely adopted due to its high affinity to the tumor marker αvβ3 integrin receptor. In this study, we develop a cRGD peptide-conjugated camptothecin (CPT) prodrug, which enables self-assembly of nanoparticles for precise targeting and enrichment in tumor tissue. We first synthesized a camptothecin derivative (CPT-ss-N3) with a reduction-sensitive bond and simultaneously modified PEG to obtain cRGD-PEG-N3. After ring-opening polymerization of the 2-(but-3-yn-1-yolxy)-2-oxo-1,3,2-dioxaphospholane (BYP), an amphiphilic polymeric prodrug, referred to as cRGD-PEG-g-(PBYP-ss-CPT), was obtained via copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction. The self-assembly in buffer solution of the cRGD-functional prodrug was studied through DLS and TEM. The in vitro drug release behavior of cRGD-PEG-g-(PBYP-ss-CPT) nanoparticles was investigated. The results show that the nanoparticles are reduction-responsive and the bonded CPT can be released. Endocytosis and MTT assays demonstrate that the cRGD-conjugated prodrug has better affinity for tumor cells, accumulates more intracellularly, and is therefore, more effective. The in vivo drug metabolism studies show that nanoparticles greatly prolong the retention time in circulation. By monitoring drug distribution in tumor and in various tissues, we find that free CPT can be rapidly metabolized, resulting in low accumulation in all tissues. However, cRGD-PEG-g-(PBYP-ss-CPT) nanoparticles accumulate in tumor tissues in higher amounts than PEG-g-(PBYP-ss-CPT) nanoparticles, except for the inevitable capture by the liver. This indicates that the nanomedicine with cRGD has a certain targeting property, which can improve drug delivery efficiency.


Synthesis of Polyphosphoester (PBYP)
The polymer PBYP was prepared by ring opening polymerization of 2-(but-3-yn-1-yolxy)-2-oxo-1,3,2-dioxaphospholane (BYP). The reaction was carried out as follows: 8 mL of CH2Cl2 was added to a pre-dried 50 mL branched flask under nitrogen. After adding the initiator isopropanol (8.30 mg, 0.14 mmol) and catalyst 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) (63.0 mg, 0.41 mmol) in turn, the monomer BYP (1.70 g, 9.66 mmol) was added rapidly by differential method using a syringe, and the reaction was carried out under nitrogen protection at 30 C for 30 min. The mixed product was concentrated and precipitated three times in a mixture of ice anhydrous ether and anhydrous methanol (20:1, v/v). The precipitated solution was aspirated and the product was dried in a vacuum drying oven for 24 h to obtain pure pale yellow viscous solid PBYP. (1,36 g, yield: 80.1 %).

One-Pot Synthesis of cRGD-PEG-g-(PBYP-ss-CPT) Polymeric Prodrug
The amphiphilic targeted polymer cRGD-PEG-g-(PBYP-ss-CPT) was synthesised via CuAAC reaction. 4 Briefly, to a Schlenk tube, a mixture of PBYP was added to the mixture before freezing vacuum for 3 min. After the mixture thawed, CuBr (16.91 mg, 0.12 mmol,) was also added to the Schlenk tube. The reaction mixture was stirred magnetically at 35 C under nitrogen. After 12 h of reaction, the S-5 solution was terminated by quickly cooling the Schlenk tube in an ice-water bath, followed by dialysis (MWCO 3500) against DMF and deionized water for 48 h to remove copper ions. The solution was collected and then freeze-dried under vacuum to obtain the product cRGD-PEG-g-(PBYP-ss-CPT). (57.2 mg, yield: 54.9%).

Characterization
1 H NMR spectra were recorded on a 300 MHz spectrometer (INOVA-300, Varian), using CDCl3 or D2O as the solvents and tetramethylsilane (TMS) as the internal standard. The number-average molecular weights (M ̅ n) and dispersity (Đ) of PBYP and PEG-g-(PBYP-ss-CPT) were analyzed by gel permeation chromatography (GPC) instrument (HLC-8320, Tosoh) using polystyrene as the standard and DMF including 0.1 wt% LiBr as the eluent. The ultraviolet-visible (UV-vis) absorption spectra were conducted on a UV-vis spectrophotometer (UV-vis 1900, Shimadzu), and fluorescence spectra were recorded on a fluorescence spectrophotometer (Cary Eclipse, Agilent). The self-assembly behavior of the polymer nanoparticles and morphological changes under different conditions were explored by DLS and TEM.
Various methods were used to analyze the in vitro and in vivo effects of nanoparticles.

MALDI-TOF MS Measurement
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra were recorded on an AXIMA Performance-MALDI TOF/TOF mass spectrometer (Shimadzu Biotech Manchester, UK) equipped with a N2 laser emitting at 337 nm. All spectra were measured in the linear mode. The optimized mass spectra S-6 were obtained with anthratriol as the matrix. Typically, the samples were dissolved in a 5 mg mL -1 matrix with 50% acetonitrile-0.1% trifluoroacetic acid (TFA) and crystallized in a MALDI target. The typical spot size on the standard 384 spot stainless-steel targets was about 2 mm. 250-500 laser shots were acquired for each mass spectrum, and the spectra were obtained at a laser power that was optimized to maximize resolution and peak intensity.

Self-assembly Behavior
The critical aggregation concentration (CAC) of PEG-g-(PBYP-ss-CPT) nanoparticles was measured by the fluorescence probe method using pyrene as the fluorescence probe. Briefly, to group vials 50 L of pyrene solution in acetone (610 -7 M) was added and acetone was removed under vacuum conditions. Then 5 mL polymer aqueous solutions of different concentrations were added, followed by ultrasonication for 30 min and stirring for 48 h at room temperature. A fluorescence spectrophotometer was used to analyze the intensity of pyrene. The excitation was set at 335 nm, while emission spectra were recorded with a 5 nm slit width over a wavelength from 350 to 550 nm. The intensity ratio (I383/I372) of the peak (383 nm, I383) to the peak (372 nm, I372) from the emission spectra was analyzed as a function of the logarithmic concentrations of polymeric prodrug. The intersection was determined as the CAC value.

In vitro Enzymatic Degradation
The degradation of polymers as drug carriers is an indispensable property. To S-7 verify the enzymatic degradation performance of the polyphosphoester backbone, 1 H NMR was used to test the degradation products at different times in the presence of phosphodiesterase I (PDE I). In detail, 30 mg of PEG-g-PBYP was dissolved in 15 mL of buffer solution containing 0.5 mg mL -1 phosphodiesterase I (PDE I) and 5 mg mL -1 MgCl26H2O, which was then divided into three portions and immersed in a thermostatic shaker at 37 C. Each portion was freeze-dried at a predetermined time point and then subjected to 1 H NMR analysis.

In vitro CPT Release from cRGD-PEG-g-(PBYP-ss-CPT)
The in vitro CPT release behavior of cRGD-CPT NPs was studied in the following process. First, 15 mg of cRGD-PEG-g-(PBYP-ss-CPT) prodrug was ultrasonically

In Vitro Hemolysis Activity
The blood compatibility of free CPT and PEG-g-(PBYP-ss-CPT) was evaluated by spectrophotometry technique according to previous research. 5,6 The Second Affiliated Hospital of Soochow University offered the mouse blood. Typically, physiological saline (2 mL) was added into blood sample (1 mL). After gentle blowing with pipettes, red blood cells (RBCs) were isolated from serum by S-9 centrifugation at 500 g for 10 min. The RBCs were further washed three times with PBS and then diluted to 10 mL in PBS. Then, diluted RBC suspension (0.5 mL) was added into 0.5 mL of free CPT and PEG-g-(PBYP-ss-CPT) at different concentrations

Cellular Uptake of cRGD-PEG-g-(PBYP-ss-CPT) Micelles
The cellular uptake and intracellular release behaviors of free CPT and CPT prodrug in HepG2 cells were investigated by the confocal laser scanning microscope (Zeiss, LSM 800). Typically, HepG2 cells were seeded in a Φ=20 mm confocal dish at a density of 2×10 5 cells per well and cultured in high glucose DMEM culture medium at 37 °C under a 5% CO2 atmosphere for different times. Afterward, the culture medium was removed. The cells were washed with PBS three times and stained with Lyso-Tracker Red (0.1 μL mL -1 ) for 1 h, followed by washing with PBS three times. The culture medium was removed within the designed time, and fresh S-11 culture medium containing sample was added for further culture. The concentration of CPT in all newly added media was 13 mg L -1 . The images were then captured at excitation wavelengths of 589 nm (red) and 363 nm (blue).

Flow cytometry
HepG2 cells were inoculated at a density of 2×10 5 cells cm -2 in 6-well plates and incubated in 2 mL of DMEM containing 10% FBS for 12 h. Subsequently, the old medium was removed at different time points and DMEM containing 10% sample was added and incubated for 4 h and 6 h, respectively. After removing the medium and subsequently washing with PBS solution thrice, the cells were collected for flow cytometry quantitative analysis (BD FACSCalibur, USA) with an excitation at 365 nm.

Pharmacokinetics and In vivo Biodistribution of Nanoparticles
Pharmacokinetics and in vivo biodistribution of nanoparticles studies were performed according to the previous reports. 7 where Ix is the fluorescence intensity of the extract at different blood sampling times, I0 is the fluorescence intensity of the blood extract before administration, m is the mass of blood (g), Is is the fluorescence intensity of the injected sample diluted 1000 times, and V is the single injection volume (μL). The blood extraction time was used as the horizontal coordinate and the drug level (%ID/g) was used as the vertical coordinate to make a dotted line graph of the blood circulation of the drug in mice.
Eighteen mice were selected and divided into 3 groups of 3 mice each, and the mice were injected with free CPT, prodrug NPs and cRGD-CPT NPs in the tail vein at a dose of 5 mg kg -1 . The mice were sacrificed at 24 h and 48 h, respectively, and the following organs were removed: heart, liver, spleen, lung kidney and tumor. The