Ultrasound-Enhanced Delivery of Doxorubicin-Loaded

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Applications of Polymer, Composite, and Coating Materials

Ultrasound-enhanced Delivery of Doxorubicin-loaded Nanodiamonds from Pullulan-all-trans-Retinal Nanoparticles for Effective Cancer Therapy Huanan Li, Ming Ma, Jingni Zhang, Wei Hou, Hangrong Chen, Deping Zeng, and Zhibiao Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03559 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 13, 2019

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ACS Applied Materials & Interfaces

Ultrasound-enhanced Delivery of Doxorubicin-loaded Nanodiamonds from Pullulan-all-trans-Retinal Nanoparticles for Effective Cancer Therapy

Huanan Lia, Ming Mab, Jingni Zhanga, Wei Houa, Hangrong Chenb*, Deping Zenga*, Zhibiao Wanga aState

Key Laboratory of Ultrasound Engineering in Medicine Co-Founded by

Chongqing and the Ministry of Science and Technology, College of Biomedical Engineering, Chongqing Medical University, 400016 Chongqing, P. R. China bState

Key Laboratory of High Performance Ceramics and Superfine Microstructure,

Shanghai Institute of Ceramics, Chinese Academy of Sciences, 200050 Shanghai, P. R. China * Correspondence: [email protected], [email protected].

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Abstract: Nanodiamond as drug carrier is of great significance in improving cancer therapy by overcoming chemoresistance. However, its clinical application is severely limited because of insufficient tumor vascular penetration. To address this limitation, pullulan-all-trans-Retinal (pullulan-ATR) self-assembled nanoparticles were prepared as nanocarriers, which encapsulated doxorubicin-loaded nanodiamonds (DOX-NDs), to construct core-shell structured co-loading nanosystem. The obtained composite nanoparticles show homogeneous size distribution with good dispersity and pH-sensitivity. Furthermore, ultrasound was utilized to promote the intratumoral penetration of these nanoparticles. As a result, the intracellular retention of DOX was efficiently enhanced, and DOX in the tumor tissue reached 17.3% of the injected dosage. The anti-tumor effect of this combined strategy was remarkably improved in both the DOX-sensitive HepG2 and DOX-resistant HepG2/ADR tumor models in vivo. This new strategy might serve as a powerful method to address the limitation of nanodiamond, and provide innovative ideas for the application of nanoparticles in clinical cancer therapy.

Keywords: nanodiamonds, good dispersity, ultrasound, nanoparticles, cancer therapy.


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1. Introduction The development of sophisticated nanoparticles for targeted delivery of anticancer drug into solid tumor is a promising strategy to improve therapeutic efficacy.1-3 Various innovative strategies to improve effectiveness of nanoparticles have been widely investigated and achieve meaningful results, such as by endowing nanoparticles with active-targeting activity4 and stimuli-response capacity,5 or by combined therapy.6 However, improving enhanced permeability and retention (EPR) effect of nanoparticles in the tumor is a currently well-known major challenge for a wide variety of nanoparticles therapy.7 Nanoparticles, once injected into the bloodstream, would face transport barriers on their journey to the targeting site. The first barrier in vivo is the clearance of the reticulo-endothelial system and rapid filtration in the kidney owing to the poor dispersal stability of nanoparticles.8-10 Second, passive transvascular delivery based on the EPR effect allows only 2-5% of the injected dose to enter into the tumor.7, 11 And the third barrier for drug delivery is transitory retention in the tumor cells.12-13 In general, the barriers mentioned above are the three inherent obstacles for nanoparticles to deliver sufficient amounts of drug into the tumor, thus the pivotal factors that might impede successful cancer therapy. To solve the predicament, nanodiamonds with the capacity to prevent drug efflux have drawn our attention as the potential drug delivery carriers to enhance drug retention in the tumor cells. Nanodiamonds have truncated octahedral carbon structure, and the surface potential variations allow nanodiamonds to load drugs or imaging agents by physical adsorption.14-15 Moreover, nanodiamonds bound with Doxorubicin/Epirubicin could prevent the efflux of drugs by ABC-transporters.16 Compared with small molecular ABC-transporter-inhibitors and other carbon nanocarriers, nanodiamonds are more biocompatible, leading to possible clinic application.16-19 With these merits, nanodiamonds might safely and effectively break through the limitation of transitory drug retention in the tumor cells. Recently, our previous work confirmed that the tumor vascular penetration and intracellular drug retention of drug-loaded nanodiamonds could be highly enhanced when the tumor was exposed to focused ultrasound, suggesting that ultrasound/nanomedicine combined strategy is an efficient way to improve their therapeutic efficacy.20 Herein, we aim to further address the problem of strong aggregation tendency and poor dispersal



stability of nanodiamonds in vivo, and achieve the traverse of the first barrier by drug-loaded nanodiamonds. Several strategies have been studied to improve the dispersity and long-term stability of nanodiamonds by modifying their surface with polymers or other biomolecules. It was reported that polymer-diamonds hybrid materials could enhance drug delivery into breast tumor cells and suppress the tumor cell viability.21 And various studies illustrated that nanoparticles constructed by modified polysaccharide showed considerable stability in vivo and kept their original size without aggregation, which contributed to traversing the first barrier.22-24 In our previous work, modified pullulan polysaccharide nanocarriers were used to encapsulate anticancer drugs, and demonstrated to exhibit excellent stability, great drug loading capability and precise hepatic targeting efficacy.25-28 In this study, a pullulan encapsulation strategy was proposed to concurrently enhance the dispersity of DOX-NDs and improve loading capability of nanocarriers as well as modulate the drug release rate. Herein, ATR linked on pullulan via hydrazone bond exhibits on-demand release capacity and is converted to anti-tumor-active ATRA in acidic lysosomal compartments, helping to realize combined therapy with DOX. Furthermore, we employed ultrasound to induce the preferential accumulation of nanoparticles in tumor in the way that ultrasound irradiation opened up tight junctions of tumor vascular endothelium cells (Figure 1). Systematic studies on the in vivo drug biodistribution and anti-tumor effect were performed on nude mice bearing HepG2 and HepG2/ADR-induced tumors. Our findings suggest that this combined strategy could significantly enhance accumulation of drugs in the tumor, and show great clinical potential in cancer therapy.

2. Experimental Section Preparation of doxorubicin-nanodiamonds loaded pullulan-ATR nanoparticles. Doxorubicin-nanodiamonds loaded pullulan-ATR (DOX-NDs/PR) nanoparticles were prepared in three steps as follows. (1) Preparation of PR copolymers: Pullulan-hydrazine hydrate (Pu-NH-NH2) was synthesized as described in our previous report.27 Briefly, ATR was added to aqueous solutions of Pu-NH-NH2 at


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ratios of 1:10, 2:10 or 3:10 (w/w), respectively. Subsequently, isopropanol was added until the solution was transparent. After stirring overnight, isopropanol was volatilized and the reactant was centrifuged to remove unconjugated ATR, followed by lyophilizing supernatant to obtain PR copolymers. (2) Preparation of DOX-NDs: Briefly, DOX·HCl solution (2.5 mg/mL) was added dropwise into nanodiamonds solution (5 mg/mL) at 1:1 (w/w) under mild mixing. Subsequently, NaOH solution was added into the above mixture until its final concentration was 2.5 mM. After stirring overnight, the reactant was centrifuged to collect the obtained precipitate.16-17 (3) Preparation of DOX-NDs/PR nanoparticles: DOX-NDs solution (2.5 mg/mL) was added into PR solution (20 mg/mL). After 30 min of stirring, the mixture was added dropwise into distilled water under ultrasound exposure. The obtained nanoparticles were then dialyzed, filtrated, and concentrated. To measure the drug loading capacity (LC), the freeze-dried DOX-NDs/PR was dissolved in acidic DMSO. DOX and ATRA were measured by ultraviolet spectroscopy (UV, PerkinElmer, UK) with absorbing wavelength of 488 nm and 432 nm. LC (wt %) = [weight of loaded drug/weight of drug loaded nanoparticles] × 100% Fluorescein isothiocyanate (FITC)-NDs were prepared as follows. (1) The aminated nanodiamonds (NDs-NH2) were produced as previously reported.29 (2) FITC-NDs were prepared by covalently linking FITC to the NDs-NH2 (1:1, w/w) in aqueous solution (2.5 mg/mL). The remaining detailed experimental procedures could be found in supporting information. 3. Results and Discussion 3.1 Preparation of DOX-NDs/PR nanoparticles. In this work, DOX-NDs/PR nanoparticles were prepared according to Figure 1. ATR reacted with Pu-NH-NH2 through pH-sensitive cleavable hydrazone bond to generate PR conjugate. A series of PR with different degrees-of-substitution (DS, w/w) of ATR/PR was successfully synthesized by mixing ATR and Pu-NH-NH2 at different weight ratios (Table S1 in Supporting Information). PR (DS, 8.9%) was



chosen to reach better loading capacity and smaller particle size. Furthermore, DOX-NDs (DOX, 48.3% w/w) were prepared by mixing nanodiamonds and DOX (5:5, w/w) in the NaOH environment. During this process, doxorubicin hydrochloride was converted to hydrophobic DOX, which was later absorbed on the hydrophobic surfaces of nanodiamonds. The obtained DOX-NDs were hydrophobic and easy to be loaded into the hydrophobic core of PR. To prepare DOX-NDs/PR nanoparticles with desired ratio of DOX and ATRA, a series of DOX-NDs/PR nanoparticles were fabricated by regulating the ratio of DOX-NDs to PR. DOX-NDs/PR nanoparticles with weight ratio (~8:2) of DOX/ATRA were chosen for further study according to our previous report on the synergistic effect of DOX/ATRA.20 The LC of ATR and DOX in PR nanoparticles were 6.56% and 26.15% respectively, indicating its high loading capacity for DOX-NDs. The high loading capacity might be related to the modified pullulan, whose superior hydrophilic property enhances the balance of system, and to the ATR, whose hydrophobic property enhances the loading of DOX-NDs. 3.2 Characterization of DOX-NDs/PR nanoparticles. The chemical structure of PR was verified by 1H-NMR and FITR spectra by comparing the peaks of Pu-NH-NH2, ATR and PR (Figure S1 in Supporting Information). PR 1H-NMR spectrum allowed the identification of the protons: 1.42 ppm (2H, t, a-H2), 3.00-4.00 ppm (4H, glucose C2, C3, C4, and C5), 4.60-5.40 ppm (glucose, -OH), 4.67 ppm (1H, s, 1-glueose a-1,6), 5.03 ppm (1H, s, 2-glueose a-1,4). The 1H-NMR results show the characteristic peak of ATR at 1.42 ppm, indicating the conjugating of ATR to Pu-NH-NH2 (Figure S1a). As shown in the FITR spectra (Figure S1b), Pu-ATR exhibits the characteristic band at 1704 cm-1 due to the carbonyl group, suggesting the conjugating of ATR to Pu-NH-NH2, whereas Pu-NH-NH2 shows no vibration bands at these indicated regions. DLS and zeta-potential analysis were performed to measure the particle size and surface charge of DOX-NDs/PR. Compared with DOX-NDs, DOX-NDs/PR nanoparticles possess increased hydrodynamic size of 109.0 nm (Figure 2a) with an appropriate polydispersity index and decreased zeta potential of 0.8 mV (Table S2), which indicates the DOX-NDs have been successfully encapsulated into PR nanoparticles. Furthermore, the decreased surface charge of DOX-NDs/PR nanoparticles could potentially lay the foundation for their cellular uptake and in vivo biocompatibility.


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TEM image shows that DOX-NDs/PR nanoparticles are well-dispersed with regular spherical core-shell structure (Figure 2b) comparing with DOX-NDs (Figure S2). Both the DLS and TEM results indicate that the constructed pullulan-ATR nanocarrier could prevent aggregation of DOX-NDs and improve system dispersity. The dispersal stability of DOX-NDs/PR nanoparticles was evaluated in the PBS and DMEM containing 10% FBS, respectively. The DLS sizes of DOX-NDs/PR nanoparticles (the values of PDI is less than 0.3) exhibit negligible change within 8 h, however, obvious increase of hydrodynamic size is found for DOX-NDs in the PBS and the DMEM containing 10% FBS (Figure 2c). Severe aggregation of nanodiamonds in aqueous solutions could be explained by rich surface chemistry and small size, which is probably caused by the formation of covalent bonds among the nanoparticles, hydrogen bonds, van der Waals forces and π-π stacking.30 In addition, the size of DOX-NDs changed in the DMEM containing 10% FBS, might be related to interaction between charge of protein in FBS and surface charge of DOX-NDs. Compared with DOX-NDs, DOX-NDs/PR shows remarkably enhanced dispersal stability mainly due to the pullulan shell of nanoparticles, which can generate sufficient steric hindrance to reduce aggregation among the nanodiamonds.31 3.3 In vitro drug release from DOX-NDs/PR nanoparticles. ATR reacted with pullulan through pH-sensitive cleavable hydrazone bond to generate PR conjugate. Thus, in acidic endosomes/lysosomes, ATR could be released and convert into ATRA. The release behaviors of ATRA and DOX were evaluated at different pH values. At pH 7.4, about 15% ATRA was released from DOX-NDs/PR nanoparticles at 24 h. At pH 5.0, about 70% ATRA was released, indicating the cleavage of hydrazone bond (Figure 2d). And the released ATRA contributed to the cytotoxicity of DOX.6 Moreover, the pH-dependent releasing curve of DOX is similar to that of ATRA, which further guarantees the combined effect of ATRA and DOX in the cells. 3.4 Ultrasound-enhanced penetration of DOX-NDs/PR nanoparticles through vascular barrier in vitro and biosafety evaluation of ultrasound. To evaluate the effect of PR nanocarrier material on nanoparticles dispersity and the effect of ultrasound on penetration of nanoparticles through vascular barrier, the



simulated tumor vessel wall was prepared by culturing a monolayer of CRL1730 on Transwell insert, and it was treated with ultrasound (0.6 W/cm2, 1 min) before exposing it to nanoparticles (Figure 3a). The representative images of CRL1730 intercellular space with and without ultrasound treatment are shown in Figure 3b. It is clear that after ultrasound treatment, cells on the insert surface get thinner and intercellular gaps are widened due to the disruption of desmosomes connecting the cells, even if many cells are not adherent to the insert surface under ultrasonic radiation force. Nevertheless, cell floating is temporary and reversible. Then, cells adhere to the wall again and continue to grow. After 24 h, the survival rate of CRL1730 cells with ultrasound treatment is still above 95% (Figure S3), which indicates that ultrasound could not inhibit the growth of endothelial cells. Moreover, only a small amount of DOX-NDs/PR and DOX-NDs nanoparticles diffused through the untreated vessel wall (Figure 3c), whereas the diffusion rate of DOX-NDs/PR and DOX-NDs nanoparticles through the pretreated vessel wall significantly increased. The number of intracellular DOX-NDs/PR nanoparticles was approximately 3.14 folds higher than that of DOX-NDs. This result indicates that ultrasound could open the tight junctions of tumor vascular endothelium cells in vitro, and good dispersal stability of DOX-NDs/PR nanoparticles contributes to penetrating the tumor vessel wall. 3.5 Cellular uptake, intracellular distribution and retention of DOX-NDs/PR nanoparticles. HepG2/ADR cells were cultured with DOX-NDs/PR for 4 h, washed and allowed for drug efflux for 12 h. Compared with free DOX·HCl-treated cells, higher amount of intracellular DOX fluorescence could be observed and quantitatively analyzed in cells treated with DOX-NDs/PR (Figure 4a and 4b). FITC was used to label the nanodiamonds. The fluorescence signal of FITC indicated that a portion of nanodiamonds was distributed in the cytoplasm, and the rest was distributed in the nuclei (Figure 4a). The phenomenon observed in the nuclei might imply that small-sized nanodiamonds could be well loaded by PR nanocarrier, be released from PR nanocarrier and enter the cell nuclei. It also provides evidence for improving the dispersity of nanodiamonds by utilizing PR nanocarrier material. Moreover, the intracellular distribution of DOX is consistent with that of nanodiamonds, which


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indicates that the enhanced drug retention of DOX-NDs/PR is mediated by nanodiamonds. 3.6 Cytotoxicity of PR and DOX-NDs/PR nanoparticles in vitro. The biocompatibility of nanodiamonds and Pu-NH-NH2 was evaluated by co-incubation with HepG2 and L929 cells. CCK8 assay results (Figure 4c) show that the nanocarriers have commendable biocompatibility after 24 h of co-incubation. Table 1 IC50 of different formulations. Samples

DOX·HCl

DOX-NDs

DOX-NDs/PR

IC50(HepG2, mg/L)

0.55 ± 0.03

0.25 ± 0.02

0.013 ± 0.004

IC50(HepG2/ADR, mg/L)

-

0.20 ± 0.03

0.035 ± 0.006

Compared with free DOX·HCl and DOX-NDs, significant decreased cell viability is observed when HepG2 or HepG2/ADR cells are treated with DOX-NDs/PR (Figure 4d), suggesting the superior anti-tumor activity of ATRA and DOX co-loaded nanoparticles. Afterwards, the half maximal inhibitory concentration (IC50) values were determined in our study (Table 1). However, the mortality of HepG2/ADR cells in the DOX·HCl group was very low, so that IC50 could not be obtained. It is found that the IC50 for DOX-NDs/PR in the HepG2 or HepG2/ADR cells is the lowest. The main reason might be that DOX-NDs/PR enhances the cellular uptake and retention of drug, and the combination of ATRA and DOX achieves synergistic anti-tumor effect as our previous result suggested.20 In this study, the combination index (CI) was calculated by Chou-Talalay method.20,

32-33

As illustrated in Table 2, all CI values

were lower than 1, thus indicating the synergistic anti-tumor effect of DOX and ATRA.

Table 2 CI at different weight ratios of DOX/ATRA. Weight ratios DOX/ATRA CI

of

8:2

6:4

5:5

4:6

2:8

0.12±0.01

0.57±0.1

0.16±0.07

0.56±0.08

0.49±0.03



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3.7 In vivo pharmacokinetic study, drug accumulation and intratumoral penetration. The in vivo pharmacokinetics of the DOX-NDs/PR and that of free DOX·HCl have been conducted for comparison. Blood samples were extracted from the orbit at different intervals after injection of DOX-NDs/PR. As illustrated in Figure S4, the DOX-NDs/PR protected by pullulan displayed longer blood circulation time than free DOX·HCl. At 24 h, 5.4% of the injected DOX-NDs/PR was in the plasma compared with 0.3% of free DOX·HCl. The biodistribution of DOX-NDs/PR was investigated by in vivo imaging and tissue distribution method. We treated the HepG2 or HepG2/ADR-induced tumor with ultrasound after the injection of DOX-NDs/PR (DOX dose of 10 mg/kg) to obtain clear imaging effect within safe limit. The in vivo imaging of mice was performed at 2 and 8 h postinjection by in vivo imaging system at excitation wavelength of 488 nm. As displayed in Figure 5a, DOX fluorescence in the ultrasound+DOX-NDs/PR group was observed to accumulate preferentially in the HepG2 or HepG2/ADR-induced tumor tissue at 2 h post injection. Moreover, the nanoparticles exhibited lasting florescence in the tumor for 8 h after the injection, which indicated that the prolonged blood circulation was closely related to drug biodistribution. It is worth noting that free DOX·HCl exhibits no obvious accumulations in the HepG2/ADR-induced tumor (yellow circle), but ultrasound+DOX-NDs/PR exhibits strong accumulation in the HepG2/ADR-induced tumor, which provides a powerful evidence for the enhanced drug retention and overcoming of chemoresistance mediated by nanodiamonds. After live imaging, tumor tissues were harvested, and comparative quantification of DOX distribution in the tumors was also conducted. The tissue distribution result (Figure

5b)

shows

that,

DOX-NDs/PR,

ultrasound+DOX-NDs

and

ultrasound+DOX-NDs/PR significantly elevate the fluorescence intensity of DOX-NDs in the HepG2-induced tumors by ~2.8, 4.1, 6.9 folds, and in the HepG2/ADR-induced tumor by ~3.1, 3.7, 6.3 folds, respectively. These results indicate that ultrasound can enhance the penetration of DOX-NDs/PR into the tumor site. This is consistent with the Wu’s and our previous report.20,

34

Additionally,

comparing with ultrasound+DOX-NDs, ultrasound+DOX-NDs/PR significantly


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improved the fluorescence intensity of DOX by 1.9 (HepG2) or 1.7 (HepG2/ADR) folds,

which

further

verify

the

improved

DOX-NDs

dispersity

by

PR

nanocarrier. After 24 h of injection, the DOX distributions in tumor are still as high as 15.6 (HepG2) or 17.3 (HepG2/ADR) percent-of-injected-dose (%ID). To further evaluate the intratumoral penetration, HepG2 or HepG2/ADR-induced tumor-bearing mice were administered with DOX-NDs/PR at 10 mg/kg before exposing ultrasound irradiation (low intensity focused ultrasound, 1 MHz, 0.6 W/cm2, 5 min). Comparative quantification of DOX distribution in the central interstitial regions of tumors was also conducted. The intratumoral penetration result (Figure

S5)

indicated

that,

DOX-NDs/PR,

ultrasound+DOX-NDs

and

ultrasound+DOX-NDs/PR enhanced the fluorescence intensity of DOX-NDs in the HepG2-induced tumors by ~1.9, 3.3, 4.8 folds, and in the HepG2/ADR-induced tumor by ~2.1, 2.9, 5.8 folds, respectively. This result intuitively demonstrates that ultrasound can enhance delivery of DOX-NDs/PR into the tumor site. To clearly observe the drug accumulation and release of drug in tumor cell, tumors were collected at 8 h for frozen section and a precise location of fluorescence signal of DOX was displayed by CLSM imaging (Figure 5c). The images showed that DOX signals of HepG2/ADR-induced tumor were as strong as that of HepG2-induced tumor. Obvious signal of DOX was localized in the nucleus. This result revealed that drug could be effectively accumulated and released from DOX-NDs/PR in tumor cells, which was in accordance with the in vitro CLSM study. The above results provide powerful evidences for the effect of ultrasound, PR nanocarrier material and nanodiamonds on enhancing the penetration, release and retention of DOX in the tumor. 3.8 In vivo anti-tumor effect. After ascertained preferential accumulation of DOX-NDs/PR in tumor under exposure to ultrasound, we evaluated the therapeutic efficacy of this strategy. When their tumor volume reached 100 mm3, nude mice were treated with DOX formulas for 5 dosages every three days (DOX dose: 5 mg/kg). No significant difference in tumor growth was found between nude mice that were treated with and without ultrasound exposure (Figure S6), which indicates that ultrasound could not inhibit the growth of



tumor. Compared with saline and free DOX·HCl, DOX-NDs show more favorable inhibitory effect on tumor growth, which could be attributed to the retention effect of nanodiamonds. Ultrasound+DOX-NDs group exhibits much stronger anti-tumor efficacy, confirming the ultrasound effect on enhancing delivery of nanoparticles. Among all the formulations administrated, ultrasound+DOX-NDs/PR shows the strongest anti-tumor efficacy. The total volume of detectable tumors treated with ultrasound+DOX-NDs/PR is significantly lower than that without pullulan nanocarrier (Figure 6a), indicating that dispersal stability of DOX-NDs enhanced by pullulan nanocarrier could improve therapeutic efficacy. Furthermore, we evaluated the therapeutic efficacy against mice bearing HepG2/ADR-induced tumor. The administration of free DOX·HCl shows no inhibiting effects on HepG2/ADR tumors growth. Interestingly, DOX-NDs, ultrasound+DOX-NDs, DOX-NDs/PR, and ultrasound+DOX-NDs/PR nanoparticles could suppress the tumor growth without exception, which further confirm the effect of nanodiamonds on overcoming chemoresistance. Histological observation show that ultrasound+DOX-NDs/PR causes much more remarkable apoptosis in tumor tissues, which is consistent with its inhibiting effect on tumor growth (Figure 6b). The important reason is that the combination of DOX and ATRA synergistically inhibits tumor cell growth by inducing cellular senescence, which relies on retinoic acid receptors.6 There is no significant damage of heart and kidney in ultrasound+DOX-NDs/PR treated mice. However, glomerular pyknosis and myocardial fiber fracture emerge in the DOX·HCl treated groups (Figure S7). These results show that ultrasound+DOX-NDs/PR could reduce drug toxicity. Next, we also periodically monitored the change of mice weight and survival time (Figure 6c and 6d). After 5 dosages, the mice of ultrasound+DOX-NDs/PR group exhibited a stable increase in weight. Until 90th day, most mice in this group still survived. Although the tumor growth was suppressed on the 25th day, the recurrence and metastasis caused by individual differences or other uncontrollable factors could not be excluded yet after 25 days. Overall, ultrasound+DOX-NDs/PR significantly prolongs the survival time of mice. These results provide strong evidence that this combined strategy could reduce the toxic side effect of DOX and enhance therapeutic efficacy in vivo.


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The above results verify the in vivo effectiveness of DOX-NDs/PR. The safety of nanodiamonds is reported to be related to the dose and kinetic size. With regard to the safety of nanodiamonds, kinetic size of 450 nm and dose of 20 mg/kg in mice is relatively safe based on the reported results.19,

35-37

In non-human primates, no

significant differences were found in serum composition or organ function between treatment groups (nanodiamonds dose: 25 mg/kg) and control group.38 In this study, the safety of nanodiamonds was further evaluated by periodically monitoring the change of nude mice weight, which is an important indicator of biomaterials safety in vivo. The nude mice were intravenously injected with nanodiamonds (nanodiamonds dose: 20 mg/kg), however, no significant differences in body weight were found between nude mice that were treated with the control group and nanodiamonds (Figure S8). After DOX-NDs were encapsulated in the PR nanocarriers, the size and dosage of nanodiamonds in vivo were kept within the safe limit. The effectiveness and safety of DOX-NDs/PR might lead us to further researches for clinical tumor therapy. 4. Conclusions In summary, a novel combined strategy of ultrasound-enhanced delivery of DOX-NDs based on pH-sensitive pullulan-ATR nanoparticles has been developed. The strategy presented here combines the advantages of: (1) enhanced dispersal stability of DOX-NDs and improved tumor accumulation via EPR effect by introducing pullulan nanocarrier; (2) improved loading capability of nanocarrier material for DOX-NDs; (3) promoted tumor vascular penetration of nanoparticles through ultrasound; (4) increased drug intracellular retention via nanodiamonds; (5) improved therapeutic efficacy with synergistic pathway. Yet still, more preclinical work is needed before clinical translation. This research demonstrates the benefits of a combined strategy in biomedical applications and provides a new insight to relieve the nanocarrier burden and maximize the therapeutic efficacy of nanoparticles. Therefore, the superiorities of this method will facilitate its future clinical applications.

Acknowledgements This project was financially supported by Yuzhong District Research Program of Basic Research and Frontier Technology of Chongqing (No. 20180110 to Huanan Li),



Natural Science Foundation of Shanghai (No. 18ZR1444800 to Ming Ma) and National Natural Science Foundation of China (No. 51772316 to Hangrong Chen).

Supporting Information Experimental procedure, FTIR and 1H-NMR characterizations of Pu-ATR, particle size of PR with different ATR/PR degrees of substitution, particle size and surface charge of DOX-NDs/PR, TEM measurement of DOX-NDs nanoparticles, CRL1730 cells viability treated with focused ultrasound, in vivo pharmacokinetics, DOX content in the central interstitial regions of tumor, body weight change of nude mice administrated nanodiamonds.

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Figures

Figure 1. Schematic illustrations of ultrasound-enhanced delivery of doxorubicin-nanodiamonds

loaded

pullulan-ATR

(DOX-NDs/PR)

nanoparticles into the tumors.



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Figure 2. Characterizations of DOX-NDs/PR. (a) The size distribution of DOX-NDs/PR nanoparticles by DLS measurement. (b) The morphology distribution of DOX-NDs/PR nanoparticles by TEM measurement. (c) The stability of DOX-NDs/PR nanoparticles. (d) In vitro drug releases from DOX-NDs/PR nanoparticles.


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Figure 3. Ultrasound-enhanced penetrating of DOX-NDs/PR nanoparticles through

vascular

barrier

in

vitro.

(a)

Schematic

illustration

of

ultrasound-enhanced penetrating of DOX-NDs/PR across the vascular barrier. (b) Representative images of intercellular space of CRL1730 cells with ultrasound exposure. (c) Flow cytometry results of HepG2 cells treated with DOX-NDs/PR on Transwell assay with ultrasound exposure.



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Figure 4. Cellular uptake, intracellular drug distribution, drug retention and cytotoxicity of DOX-NDs/PR. (a) CLSM images of HepG2/ADR cells treated with DOX-NDs/PR/FITC-DOX-NDs/PR for 4 h before efflux and after 12 h efflux. (b) Flow cytometry results of HepG2/ADR cells treated with DOX-NDs/PR/fluorescein isothiocyanate-DOX-NDs/PR (FITC-DOX-NDs/PR) for 4 h before efflux and after 12 h efflux. The nucleus of the cell was stained with DAPI (blue). Red fluorescence represented DOX. Green fluorescence represented

FITC-NDs.

(c,d)

Cytotoxicity

of

carrier

materials

and

DOX-NDs/PR for 24 h.


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Figure 5. In vivo drug accumulation. (a) In vivo fluorescence imaging. (b) DOX content in the tumor tissue for 8 h. (c) CLSM images of the tumor cryosections of DOX-NDs/PR for 8 h.



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Figure 6. In vivo anti-tumor effect. (a) Tumor growth curves. (b) Histological observation. (c) Body weight change. (d) survival rates of nude mice bearing HepG2 or HepG2/ADR tumors treated with (1) Saline, (2) free DOX·HCl, (3) DOX-NDs,

(4)

ultrasound+DOX-NDs,

(5)

DOX-NDs/PR

and

(6)

ultrasound+DOX-NDs/PR.


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142x70mm (300 x 300 DPI)


Ultrasound-Enhanced Delivery of Doxorubicin-Loaded

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