cRGD Nanoparticles Loaded with Microbubbles for Enhancing Anti-Proliferation and Anti-Angiogenesis

Background: The inhibition of angiogenesis leads to the degeneration of tumors; thus, anti-angiogenesis therapy is a strategy for the treatment of tumors. Accumulating documents has demonstrated effective anti-angiogenic strategies should simultaneously inhibit endothelium-dependent vessels (EDV) and tumor cell-mediated vasculogenic mimicry (VM). Although cRGD peptide enabling to bind with a v b 3 integrin can specically inhibit both EDV and VM, however peptide-based drug delivery platforms have not obtained the expected therapeutic ecacy due to their rapid degradation and low tumor accumulation. Results: Herein, we developed cRGD nanoparticles (cRGD Nano) loaded microbubbles (MB) to enhance their anti-EDV and anti-VM ability for endothelial cells and ovarian cancer cells due to the sonoporation effect from ultrasound (US). Heparin as backbone conjugated with cRGD to prepare cRGD-H conjugate and then loaded with biotin thereby following dialysis to fabricate cRGD Nano. Subsequently, the cRGD Nano were incorporated with MB via avidin-biotin linkage to fabricate cRGD Nano-MB. The enhanced anti-angiogenic effect can take prot of cRGD from cRGD Nano and sonoporation effect from US combined with MB. cRGD Nano has been characterized, and cRGD Nano-MB combined with US exhibited the best intracellular uptake ability than cRGD Nano and cRGD Nano-MB without US by ow cytometry and confocal laser scanning microscopy. Also, cRGD Nano-MB combined with US could eciently discourage the tube formation of endothelial cells and cancer cells, and presented excellent signicant anti-proliferation activity in vivo. Conclusions: The combination of cRGD Nano-MB and US is expected to be a promising strategy for peptide drug delivery and enhancing anti-proliferation and anti-angiogenesis.

. cRGD peptide (cyclic Arg-Gly-Asp) has high a nity with integrin a v b 3 , therefore it can exert a speci c inhibitory effect upon EDV (Pasqualini et al. 1997;Jin and Varner 2004). In particular, Wang et al found that cRGD also presented the inhibitory effect on VM formation in cancer cells (Tang et al. 2016). Thus, cRGD exhibits the combination suppressed ability upon EDV and VM formation, thereby possessing the effective anti-proliferative effect upon tumor cells. Over recent decades, nanoparticles (Nano) have presented great prospect for combining and delivering therapeutic agents simultaneously and speci cally to tumors due to their enhanced permeability and retention (EPR) effect, diverse surface chemistry and unique pharmacokinetics. Nevertheless, current peptide-based nanoparticle drug delivery platforms have not obtained the expected therapeutic e cacy due to rapid degradation of peptide and relative low tumor accumulation.
Finding effective ways to increase active pharmaceutical agent accumulation in tumor tissues is one of the major challenges for tumor therapy. Ultrasound-targeted microbubble destruction has been a prospective strategy for drug delivery to localize intratumoral drug release and enhance intracellular drug accumulation. Ultrasound (US) combined with microbubbles (MB) can enhance the intracellular drug delivery attributing to the sonoporation of US and inertial cavitation of MB (Zhang et al. 2018; Jing et al. 2019). However, the drug loading ability of MB is relatively limited thereby preventing its application in drug delivery due to the phospholipid MB lled with gas core (Lentacker et al. 2010). It was reported that the loading capacity of MB could be increased by attaching small nanoparticles to the surface of MB.
Accordingly, we attempt to design cRGD-based nanoparticles (cRGD Nano) attached to the surface of MB enabling to e ciently deliver cRGD Nano to ovarian cancer cells thereby enhancing its tumor accumulation and improving anti-proliferation and anti-angiogenesis assisted by US. Herein, heparin could react with cRGD to prepare cRGD-H conjugate and then conjugated with biotin to prepare cRGD Nano, and cRGD Nano was subsequently combined with MB to prepare cRGD Nano-MB via avidin-biotin bridge (Scheme 1). The prepared cRGD Nano-MB was further investigated the function of antiproliferation and anti-angiogenesis for ovarian cancer cells with or without US. To the best of our knowledge, no one has yet developed a cRGD Nano-MB combined with US to deliver cRGD thereby enhancing the cellular uptake ability and e ciently suppressing proliferation and the formation of EDV and VM. Preparation of cRGD Nano Succinylated-heparin (0.050 g) was dissolved by dry DMSO with gentle heating. cRGD peptide (0.006 g), and Biotin-NH2 (0.004 g), EDC (0.04 g, 0.2 mmol) and NHS (0.025 g, 0.2 mmol) were mixed and reacted at room temperature for 24 h. Biotinylated cRGD-H conjugate in solution was then dialyzed with distilled water for 48 h in a dialysis membrane (MWCO 3500) to fabricate cRGD Nano. After lyophilization, biotinylated cRGD-H conjugate (0.22 g) obtained as white powder. Oregon green488-labled biotinylated cRGD-H was prepared by a similar method as that of biotinylated cRGD-H. The amount of cRGD in nanoparticles was measured by bicinchoninic acid (BCA) assay according to the manufacturer's instruction. Folate content was measured by UV method at 366 nm. The loading e ciency of cRGD in cRGD Nano was calculated using the following equation: loading e ciency (%) = (weight of agent in nanoparticles/initial weight of agent) × 100%.
Preparation of cRGD Nano loaded microbubbles (cRGD Nano-MB) and characterization cRGD Nano-MB was prepared by the described method with minor modi cations. (Yin et al. 2013) The biotinylated MB reacted with 500 mL avidin (10 mg/mL) for 10 min at room temperature with gentle shaking. Free avidin were separated from the avidin-bound biotinylated MB by washing for 2 times. And then biotinylated cRGD Nano (50 mL) mixed with avidinylated MB (1 mL) for 15 min with gentle shaking. Free biotinylated cRGD Nano were removed by washing for 2 times. Oregon green488 labled cRGD Nano loaded MB was using similar method. DiI was mixed with MB for 20 min to prepare DiI labeled MB (MB-DiI). To investigate the binding e ciency and construction of NPs-loaded MB, Oregon green488 labled cRGD Nano loaded MB-DiI was visualized using a confocal laser scanning microscope (Olympus FV1000, Japan) with the excitation wavelength of 405 nm for blue channel, 488 nm for green channel and 559 nm for red channel. The 1 H NMR spectra of cRGD-H were recorded on a Bruker-400 MHz NMR in D 2 O. Size distributions of cRGD Nano and cRGD Nano-MB were detected by dynamic lighting scattering (DLS) using a Zetasizer Nano-Zs (Malvern Instruments, UK) and Coulter Multisizer IIe (Beckman Coulter, USA), respectively. 1 H NMR spectra of Biotinlyated cRGD-F-H in D2O were detected for structural characterization (Fig. S1).

Cell Culture
Human umbilical cord vein endothelial cells (HUVEC), human ovarian carcinoma SKOV3 cells and human lung carcinoma A549 cells were come from ATCC. All cell lines were cultured in DMEM (Corning, USA) containing 10% fetal bovine serum (Bioind, Israel) and 100 units/mL penicillin/streptomycin. All cell lines were maintained in humid environment containing 5% CO 2 at 37 ºC.

Ultrasound Setup
Ultrasound-waves were produced from an unfocused single element transducer (Vermon SA, Tours, France) with a center frequency of 1 MHz, driven by a waveform generator (Agilent, City, CA, USA). A power ampli er (ADECE, Artannes sur Indre, France) was generated a pulsed US at acoustic pressure amplitude determined by a calibrated PDVF needle hydrophone (0.2 mm diameter, Precision Acoustics, Dorchester, UK). cRGD Nano-MB were scanned under the transducer (30 s, a center frequency of 1 MHz, 50% duty cycle) and negative peak pressure amplitude for HUVEC cells, SKOV3 cells and A549 cells was Cellular uptake of cRGD Nano and cRGD Nano-MB with and without US Oregon green488-labeled cRGD Nano and cRGD Nano-MB were synthesized by the similar procedure.
HUVEC, SKOV3 and A549 cells were respectively cultured in 6-well plates (3´ 10 5 cell/well) and incubated for 24 h at 37 o C. The culture medium was replaced by fresh medium with Oregon green488 labeled cRGD Nano and cRGD Nano-MB and then followed by US exposure. And the bottom of the plate was drowned in water for acoustic coupling without contaminating the sample in the well of the plate. Meanwhile, the US transducer was submerged in water upward aiming at the plates. HUVEC SKOV3 and Confocal laser scanning microscope was used to visualize the cellular uptake of Oregon green488 labeled cRGD Nano and cRGD Nano-MB in HUVEC and SKOV3 cells. The cells were incubated in 6-well plate for 24 h and then the culture medium was replaced with medium containing Oregon green488labeled cRGD Nano and cRGD Nano-MB with or without US. The ultrasound parameters were the same as the above procedure. The plates were incubated for 4 h at 37 o C after US treatment, and cells were washed by PBS and xed by 4% (w/v) para-formaldehyde solution. The uorescent images were observed by confocal microscope.
In vitro anti-angiogenesis (anti-EDV and anti-VM) assays Endothelial tube formation in vitro was conducted by the described method. (Kheirolomoom et al. 2007) Matrigel (50 mL) was added to a 24-well plate and allowed to polymerize. HUVEC cells were suspended in medium with 3´ 10 5 cells/mL, and the cell suspension (0.1 mL) was added to each well coated with Matrigel. And then Cells were treated with cRGD Nano or cRGD Nano-MB (each at an equivalent cRGD dose of 600 mg/mL) with or without US and then incubated for 12 h at 37 o C. After that, the cells were photographed using an inverted light microscope (NiKon ECLPSE 80i system, Japan) and branch points from 4 to 6 high-power elds (200) were counted and averaged.
Matrigel (150 mL) was placed on 24-well plates and allowed to polymerize for 1 h at 37 o C. 1.5´ 10 4 number of MDA-MB-231 were seeded on the well to observe the ability of capillary-like structure formation. The mild dosage was selected to guarantee its cell viability around 80%. cRGD Nano or cRGD Nano-MB (each at an equivalent cRGD dose of 600 mg/mL) with or without US were added to the cell suspensions. After that, images were photographed using an inverted light microscope after 96 h.
In vivo assessment of angiogenesis using chicken chorioallantoic membrane (CAM) White Leghorn chicken eggs (South China Agricultural University, Guangzhou) were incubated under routine conditions (constant humidity and 37 o C) in CAM assay, and a square window was opened in the egg shell at day 3 incubation which exposed the CAM. Gelatin sponges were cut to 1 cm 3 size and placed on the top of the CAM under sterile conditions at day 8. The sponges were then absorbed with cRGD Nano or cRGD Nano-MB (each at an equivalent cRGD dose of 600 mg/mL) with or without US and then they were tested. Sponges with PBS were used as negative controls and each group consisted of three eggs. The blood vessel inhibition ratio was identi ed as the percentage of treated group vessels area (VA) occupied by the whole area of CAM under the microscopic eld. CAMs were observed daily and photographed in vitro at 72 h. Image-Pro Plus 7.0 software was used to automatically assess and quantify the blood vessel areas.
Cell viability detected by MTT assay HUVEC and SKOV3 cells were separately seeded in 6-well plates (3´ 10 5 cell per well) and incubated for 24 h at 37 o C. After incubation of 24 h, cRGD Nano or cRGD Nano-MB were added to the medium with drug concentration of 0.06, 0.12, 0.3 and 0.6 mg/mL. The bottom surface of the plate was drowned in water for irradiation and the US transducer was then submerged in water upward aiming at the plates. US condition were set to 0.7 MPa-1 MPa for 30 s and then cells were incubated for 4 h. After that, the cells were washed and trypsinized to transfer 96-well plates with fresh medium for 20 h incubation. The culture medium was replaced with MTT solution and then the cells were further incubated for another 4 h. The medium was replaced by DMSO and the plates were slightly shaken for 10 min. The plates were measured by a microplate reader (Labsystem, Multiskan, Ascent, Finland) at optical absorbance of 570 nm. Cells with PBS were used as control.

In vivo anti-tumor experiments
Athymic nude female nu/nu mice (4-5 weeks old) were injected with 100 mL of SKOV3 cells (1´ 10 6 ) on the right ank. The tumors were allowed to grow until their size reached between 9 and 25 mm3. Mice were randomly divided into three groups (PBS, cRGD Nano-MB and cRGD Nano-MB+US, n = 5) and injected tail intravenously. The cRGD Nano-MB and cRGD Nano-MB+US (each at an equivalent dose of 150 ug cRGD per mice) were injected at one day intervals. For the cRGD Nano-MB +US group, tumors were irradiated with US after every injection under the following settings: 1 MHz, 2 % duty cycle, duration of 1 min and intensity of 2 W/cm 2 .T umor length (a), width (b) measurements, and body weight were recorded at one day intervals. Tumor volume was calculated according to the formula: V= (a´b 2 )/2

Statistical analysis
Statistical analysis was conducted to determine the differences of the experiments. One-way analysis of variance was used to analyze by a statistical program (Statistical Package for the Social Sciences, Version 19.0, SPSS Inc., USA). All the data were performed in triplicate and showed as a mean value with its standard deviation indicated (mean ± SD). Differences were considered the signi cance at * P < 0.05, * *P < 0.01, * * * P < 0.001.

Results And Discussion
Although nanotechnology-based drug delivery platforms presented promising prospect for delivering therapeutic agents simultaneously and speci cally to tumors, the current peptide-based nanoparticles still could not achieve the expected therapeutic e cacy due to the rapid degradation of peptide and their low tumor accumulation. The combination US with MB could enhance the intracellular drug delivery owing to the sonoporation of US and inertial cavitation of MB. Herein, we designed NPs-MB complex using nanoparticles attached to the surface of MB thereby increasing the drug loading capacity of MB and enhancing the intracellular uptake ability of cRGD Nano assisted by US effect. In the cRGD NPs-MB complex, the carboxyl group of succinlyated-heparin was rst conjugated with the amine of cRGD to synthesize cRGD-Heparin and then introduced the small amount of biotin to facilitate combination with MB. The cRGD-Biotin-Heparin could be dialyzed to prepare cRGD Nano. And then, cRGD Nano was incorporated with MB by avidin-biotin bridge to create a new drug delivery system cRGD Nano-MB. The design could increase loading capacity of MB, simultaneously enhance cRGD tumor accumulation and improve the bioavailability of cRGD Nano with the aid of US (Scheme 1).
We characterized 1 H NMR spectrum of cRGD-Heparin conjugate in D2O. It was observed chemical shift of 6.79 ppm and 7.05 ppm corresponding to cRGD fragment, indicating we prepared successfully cRGD-H conjugate ( Figure S1). Due to the small of amount of biotin, it was hard to detect the signal of biotin using 1H NMR. We prepared cRGD-biotin-Heparin conjugate which could present amphiphilic property facilitating to self-assemble into cRGD Nano. Their size distribution and morphology were determined by DLS and TEM. TEM detected they presented approximately spherical shape (Fig. 1a). Also, DLS results displayed the mean diameter of cRGD Nanos was approximately 51 ± 2 nm (Fig. 1b). The content of cRGD was about 14.4% as determined by BCA assay. The amount of cRGD Nano bound to MB was detected by removing the unbound cRGD Nano from MB via centrifugation. We screened the optimal ratio of cRGD Nano and MB by detecting cRGD amount. The loading concentration of cRGD was about 85.2 mg/10 8 MB when 1 mg/mL of cRGD Nano was added to the MB. With the increased cRGD Nano concentration, the drug loading e ciency of cRGD Nano-MB was increased. When the optimal procedure was the mixture of 2 mg/mL of cRGD Nano and 10 8 mg /mL of MB, it could obtain around 178.7 mg cRGD per 10 8 MB. The size and concentration of cRGD Nano-MB were determined by Coulter Multisizer, and the drug loading e ciency of cRGD Nano-MB was investigated (Tab. 1). In comparison with the method of MB directly carrying free cRGD, our designed method presented higher drug loading e ciency (Kheirolomoom et al. 2007;Lentacker et al. 2010;Wang et al. 2019). To further con rm the successful attachment of cRGD Nano to the surface of MB shells, we prepared Oregon green488 labeled cRGD Nano and DiI encapsulated to MB (Oregon green488-cRGD Nano-MB-DiI). Oregon green488-cRGD Nano was attached to the surface of MB-DiI to prepare Oregon green488-cRGD Nano-MB-DiI via avidin-biotin bridge. Confocal laser scanning microscopy was used to detect two uorescent signals of Oregon green488 and DiI and we could observed MB morphology in bright eld. It clearly showed that the surface of MB was homogeneously covered by green and red uorescent signals (Fig.  1c), indicating cRGD Nano had ability to patch on the surface of the MB shells.
Recent studies showed that cavitation and sonoporation induced by US could assist drug delivery system to enhance cell membrane permeability thereby facilitating drug accumulation at the speci c region (Yan et al. 2013;Li et al. 2018). In this study, we compared the cellular uptake e ciency of cRGD Nano and cRGD Nano-MB with an without US (Fig. 2). Their cellular uptake ability could be affected by US irradiation. It was found that HUVEC and SKOV3 cells treated with cRGD Nano-MB with US displayed the highest cellular uptake e ciency compared to cRGD Nano and cRGD Nano-MB without US. The results demonstrated US irradiation could improve the cellular uptake e ciency of cRGD Nano. cRGD Nano presented the second higher cellular uptake in these cells, while without US exposure cRGD Nano-MB group could not e ciently enter into these cells, which possibly attributed to the its large size. To further con rm the cellular uptake e ciency, these cells treated with cRGD Nano and cRGD Nano-MB with or without US were visualized with a confocal microscope. As shown in Fig. 3, Oregon green488-cRGD Nano were observed in cytoplasm, while the uorescence of cRGD Nano-MB without US was not stronger than cRGD Nano. It was speculated the increased size of cRGD Nano-MB limited its cellular uptake ability in the absence of US. In comparison, the uorescence was signi cantly enhanced when cRGD Nano-MB exposed to US, suggesting the cavitation effect induced by US could improve cellular uptake e ciency of cRGD Nano-MB. The results of confocal images were in accordance with that of ow cytometry. Collectively, the synergistic effect from US irradiation were bene cial to the enhancement of peptide drug delivery.
Anti-EDV effect in vitro and in vivo of cRGD Nano, cRGD Nano-MB with or without US was conducted by using microtube formation experiments of HUVECs and the CAM assay. Regard to the microtube formation experiments of HUVECs, cRGD Nano-MB with US could present best inhibitory ability of microtubule formation (70.3 ± 2% inhibition) than cRGD Nano (48 ± 4% inhibition) and cRGD Nano-MB without US (30.6 ± 7% inhibition) in HUVECs (P < 0.001) (Fig. 4a). Regard to the CAM assay, the untreated CAM displayed a normal pattern of vascularization (29.8 ± 1%, vessel area/CAM area) and cRGD Nano-MB without US could produce slight inhibition (23.5 ± 2%, P < 0.05). The blood vessel formation within the allantoic membrane showed that cRGD Nano group presented a signi cant anti-angiogenic response (18.5 ± 2%, vessel area/CAM area, P < 0.01), in comparison spontaneous angiogenesis was signi cantly inhibited under cRGD Nano-MB with US (13.8 ± 1.7%, vessel area/CAM area, P < 0.001) (Fig. 4b). It was shown the boundaries and capillaries of the CAM disappeared, suggesting that US could help cRGD Nano-MB to present a strong anti-angiogenic response of EDV.
Anti-VM effect in vitro of cRGD Nano and cRGD Nano-MB with or without US was performed by using microtube formation experiments of MDA-MB-231 cells (Fig. 4c). It was shown cRGD Nano-MB with US exhibited preferable destructive effect upon the vessel-like channels (56.9 ± 3% inhibition), while cRGD Nano exhibited better inhibitory effect than cRGD Nano-MB without US. The order of anti-VM effect: cRGD Nano-MB with US> cRGD Nano> cRGD Nano-MB without US. The results indicated US could further assist cRGD Nano-MB to improve the inhibitory ability to VM formation. Collectively, anti-EDV and anti-VM effect were attributed to the ability of cRGD Nano and US could enhance the inhibitory ability. In addition, the cRGD Nano-MB combined with US presented the highest cytotoxicity than that of cRGD Nano and cRGD Nano-MB without US. Meanwhile, cRGD Nano exhibited better the cell death ability than cRGD Nano-MB without US. The results suggested that ultrasound-mediated cavitation contributed to enhance anti-proliferation ability of cRGD Nano.
We then compared their anti-tumor effect of cRGD Nano, cRGD Nano-MB with or without US in the SKOV3 xenograft mouse model. Each treatment group was intravenously injected into mice model using PBS as the control. After the mice were treated with cRGD Nano, cRGD Nano-MB with or without US, tumor growth, neovascularization and the inhibition of proliferation were monitored as shown in Figure 6, US irradiation could signi cantly enhance the antitumor activity of cRGD Nano-MB; and cRGD Nano presented slightly better anti-tumor activity than cRGD Nano-MB, which was attributed to its larger size. In addition, the body weight of mice in treatment groups was no obvious change, indicating antiangiogenesis therapy almost had no cytotoxicity in vivo ( Figure S). We then detected tumor proliferation and VM expression in tumor tissues using Ki67 and CD34/PAS dual staining (Fig. 6B). The immunohistochemistry results showed cRGD Nano-MB with US exhibited lowest the expression level of Ki67; and the order of Ki67 expression was as followed: PBS> cRGD Nano-MB> cRGD Nano> cRGD Nano-MB+US. CD34/PAS double staining was used to verify VM. VM vessels were negative for CD34 immunostaining, but the walls of these VM vessels were PAS-positive. It was observed the number of VM channels was the fewest in the SKOV3 xenograft treated with cRGD Nano-MB in the presence of US, while cRGD Nano-MB had no signi cant difference with cRGD Nano. Collectively, the immunohistochemistry results were consistent with that of anti-tumor experiments.

Conclusions
In summary, the combined strategy of cRGD Nano-MB with US could increase peptide bioavailability, enhance drug uptake e ciency and exhibited great anti-tumor effect. The enhancement of antiproliferation, anti-EDV and anti-VM for cRGD Nano induced from cRGD effect and cavitation triggered by US. It provides a promising strategy for improving bioavailability of peptide in anticancer therapy.