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On-Demand Drug Releasing from Dual Targeting Small Nanoparticles Triggered by High Intensity Focused Ultrasound Enhanced Glioblastoma Targeting Therapy Zimiao Luo, Kai Jin, Qiang Pang, shun shen, Zhiqiang Yan, Ting Jiang, Xiaoyan Zhu, Lei Yu, Zhiqing Pang, and Xinguo Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10866 • Publication Date (Web): 01 Sep 2017 Downloaded from http://pubs.acs.org on September 1, 2017

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ACS Applied Materials & Interfaces

On-Demand Drug Releasing from Dual Targeting Small Nanoparticles Triggered by High Intensity Focused Ultrasound Enhanced Glioblastoma Targeting Therapy Zimiao Luo a,b, Kai Jin b, Qiang Pang b, Shun Shen b, Zhiqiang Yan a, Ting Jiang b, Xiaoyan Zhu b, Lei Yu a*, Zhiqing Pang b*, Xinguo Jiang b a

Biomedical Engineering and Technology Institute, Shanghai Engineering Research Center of

Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, 3663 N. Zhongshan Rd., Shanghai 200062, PR China; b

Department of Pharmaceutics, School of Pharmacy, Fudan University, Key Laboratory of Smart

Drug Delivery, Ministry of Education & PLA, 826 N. Zhangheng Rd., Shanghai 201203, PR China; * Corresponding author. Tel.: +86 21 62235411. E-mail: [email protected] (L. Yu). * Corresponding author. Tel.: +86 21 51980069. E-mail: [email protected] (Z. Pang).

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ABSTRACT Glioblastoma is one of the most challenging and intractable tumors with the difficult treatment and poor prognosis. Unsatisfactory traditional systemic chemotherapies for glioblastoma are mainly attributed to the insufficient and non-specific drug delivery into the brain tumors as well as the incomplete drug release at the tumor sites. Inspired by angiopep-2 peptide is an acknowledged dual targeting moiety for brain tumor targeting delivery and high-intensity focused ultrasound (HIFU) is an ideal trigger for drug release with the ultrahigh energy and millimeter-sized focus ability, in the present study, a novel HIFU-responsive angiopep-2-modified small poly(lactic-co-glycolic acid) (PLGA) hybrid nanoparticle drug delivery system holding doxorubicin/perfluorooctyl bromide (ANP-D/P) was designed to increase the intratumoral drug accumulation, further trigger on-demand drug release at the glioblastoma sites, and enhance glioblastoma therapy. It was shown ANP-D/P was stable and had a small size of 41 nm. The angiopep-2 modification endowed the ANP-D/P with improved BBB transportation and specific accumulation in glioblastoma tissues by 17 folds and 13.4 folds compared with unmodified nanoparticles, respectively. Under HIUF irradiation, ANP-D/P could release 47% of drug within 2 min and induced apoptosis of most tumor cells. HIUF-triggered instantaneous drug release at the glioblastoma sites eventually enabled ANP-D/P to achieve the strongest anti-glioblastoma efficacy with the longest median survival time (56 days) of glioblastoma-bearing mice and the minimum vestiges of tumor cells in the pathological slices among all the groups. In general, HIFU-responsive ANP-D/P in this study provided a new way for glioblastoma therapy with a great potential for clinical application.

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KEYWORDS on-demand drug release, small nanoparticles, dual targeting, high-intensity focused ultrasound (HIFU), glioblastoma

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INTRODUCTION Glioblastoma multiforme (GBM), with the difficult treatment and poor prognosis, is one of the most challenging and intractable diseases in unmet medical needs, which greatly threat the human health.1-3 In systemic chemotherapy, the blood-brain barrier (BBB), as the primary barrier, restricts most of the administered chemotherapeutic agents to transport into the brain.1, 4, 5 In addition, the chemotherapeutic agents in clinic such as Temodal® and Doxil® suffer from the limit of poor targeting.2, 6, 7 The seriously insufficient and non-specific delivery of the chemotherapeutic agents eventually results in the unsatisfactory therapeutic effects on glioblastomas.6, 8 In recent decades, with the advantages of biodegradability, biocompatibility, non-toxicity, and prolonged circulation, nanoparticle drug delivery systems have received increasing attention and achieved exciting therapeutic effects in solid tumors benefited from enhanced permeability and retention (EPR) effect.9, 10 However, the EPR effect in glioblastomas (U87 MG cell line) is relatively weak with the cut-off size only about 7~100 nm.1, 11 Moreover, it has been shown brain delivery of nanoparticles was size-dependent and smaller nanoparticle favored to transport across the BBB.12 Therefore, the smaller-sized targeted nanoparticle drug delivery might be a promising strategy to effectively overcome these barriers of BBB and weak EPR effect. The non-specific distribution of drugs in the brain is considered as another barrier for brain tumor drug delivery. Thus, dual-targeting delivery strategies in which nanoparticle drug delivery system can target both the BBB and brain tumor cells are proposed for brain tumor treatment.13, 14 Receptor-mediated transcytosis is one of the most important transport mechanisms for endogenous molecules into the brain with high specificity, selectivity and affinity.15, 16 The low density 4


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lipoprotein receptor-related protein (LRP) overexpressed on the BBB has been reported to mediate multiple ligands to transport across the BBB.16, 17 What’s more, the LRP is also highly expressed on the glioblastoma cells.18, 19 Hence, as the acknowledged specific ligand of LRP, angiopep-2 is an ideal dual-targeting moiety to transport small nanoparticle across the BBB and further target to glioblastoma cells, achieving sufficient drug delivery to brain tumors.20-22 The incomplete drug release at glioblastoma sites is another formidable obstacle to kill tumor cells effectively, with residual tumor cells eventually leading to the recurrence.23 In consequence, nowadays, the chemotherapeutic drugs in clinic require more than a sustained drug release with prolonged circulation time, but an on-demand delivery strategy in which drugs are delivered to the exact foci and the drug release is actuated by some specific stimulus.24-27 As one kind of external mechanical force stimulus,27 ultrasound induced thermal effect, mechanical effect or radiation force and could make the on-demand drug release come true by turning on/off the stimulus in a non-invasive, remote and spatiotemporally controlled manner.23, 28, 29 Especially, the high intensity focused ultrasound (HIFU), widely applied in clinic, depending on the ultrahigh energy and millimeter-sized focus ability, has been proposed as an ideal trigger for controlled drug release in recent years.29-31 Compared with common liposome bubbles, poly (lactic-co-glycolic acid) (PLGA) nanoparticles are considered as the preferable HIFU-responsive carriers because of their excellent mechanical strength and thermostability.30 Although the depolymerization of PLGA was observed under a high frequency ultrasound of 5~10 MHz and the temperature of the focused spot (60~100 °C) of HIFU 28, 29, 31 was higher than the glass transition temperature of the PLGA (50:50) (43~48 °C),32 the rapid energy attenuation of HIFU during penetrating soft tissues still could not be ignored, which greatly hindered the activating of the drug release from PLGA nanoparticles.33 5



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Therefore, a series of fluorocarbon-like perfluorooctyl bromide (PFOB) were selected as the reinforcing agent to intensify the cavitation effect of HIFU.30, 33, 34 In consideration of these above, HIFU was introduced into the present study as an ideal trigger to greatly increase the local release of the targeted chemotherapeutic drugs from PLGA nanoparticles at the glioblastoma sites. Given these above, in the present study, we developed a novel HIFU-responsive angiopep-2-modified small hybrid PLGA nanoparticle drug delivery system holding doxorubicin/perfluorooctyl bromide (ANP-D/P) to enhance glioblastoma targeting therapy (Fig. 1 A). Doxorubicin, one of the most effective broad-spectrum anticancer agents, was used as a model drug. The small hybrid nanoparticles (NP) were prepared through a nanoprecipitation method and the angiopep-2 was conjugated onto the NP though a maleimide-thiol reaction. The cellular uptake, transport across the BBB monolayers , in vivo fluorescence imaging, nanoparticle co-localization with tumor cells, and bio-distribution studies were performed to evaluate the dual targeting property of the ANP-D/P. Meanwhile, the fluorescence dyes, DiD and DiR, were utilized to track the nanoparticles in vitro and in vivo. The HIFU-responsive performances of ANP-D/P were characterized through the structure changes and the drug release behaviors of the ANP-D/P. The tumor inhibition effects of the ANP-D/P on U87 MG cells were assessed by the IC50 and the apoptosis assay. The in vivo anti-glioblastoma effects of the ANP-D/P under HIFU irradiation were evaluated by the median survival of tumor-bearing mice as well as the H&E staining and TUNEL assay of tumor slices.

MATERIALS AND METHODS Materials 6


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Angiopep-2 (CTFFYGGSRGKRNNFKTEEY) was synthesized by CHINESE PEPTIDE (Hangzhou, China). Carboxylic acid-terminated poly(lactic-co-glycolic acid) (PLGA-COOH; 0.67 dL/g, 50 : 50 ratio) was purchased from LACTEL (USA). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-3400] (DSPE-PEG3400-MAL) and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were purchased from Laysan Bio.Co (USA). The perfluorooctyl bromide (PFOB), perfluoro-15-crown-5-ether, and deuterochloroform (CDCl3, 99.96 atom % D) were purchased from Sigma (USA). The Doxil® (doxorubicin hydrochloride liposome injection) (Lip-D) was purchased from Janssen (USA). 1,1’-Dioctadecyl-3,3,3,‘3’-tetramethylindodicarbocyanine perchlorate (DiD) and 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) were obtained from AAT Bioquest (USA). Doxorubicin hydrochloride (Dox) and Tris-HCl buffer (pH: 8.0) were ordered from Meilun Biotechnology Co., Ltd. (Dalian, China). The ultracentrifuge filter device with 30 k Da molecular weight cutoff (MWCO) membrane was purchased from Millipore (USA). Thiazoyl Blue Tetrazolium Bromide-MTT and PE Annexin V Apoptosis Detection Kit were from AMRESCO (USA) and Dojindo Molecular Technologies (Japan), respectively. The CD31 rabbit polyclonal primary antibody was obtained from Abcam (USA). The Alexa Fluor 647-conjugated goat-anti-rabbit secondary antibody was purchased from Yeasen Biotechnology Co., Ltd. (Shanghai, China). The 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Beyotime (Nantong, China). Plastic cell culture dishes and plates were purchased from Corning Incorporation (USA). Laser confocal cell culture dishes and 24-well transwell filters were purchased from NEST (Suzhou, China) and from Corning (USA), respectively. Dulbecco's 7



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modified eagle's medium (high glucose) (DMEM), fetal bovine serum (FBS), trypsine-EDTA (0.25%), and penicillin-streptomycin were purchased from Gibco (CA). All of the other chemicals with analytical reagent were provided by Sinopharm Chemical Reagent (Shanghai, China). Cells and animals The U87 MG cell line and brain capillary endothelial cells (BCEC) were purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China). The U87 MG cells and BCEC cells were cultured in DMEM medium with 10% FBS, 100 IU/mL penicillin and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. BALB/c nude mice (male, 4-5 weeks, 18-20 g) were purchased from the Shanghai Slac Laboratory Animal Ltd. (China) and all animal experiments were performed in the context of the protocols approved by the Animal Ethics Committee of School of Pharmacy, Fudan University. The intracranial glioblastoma animal model was established based on a previous report.35 In brief, U87 MG cells with the density of 5 × 105 were inoculated into the right striatum guided by a stereotactic fixation device (Stoleting, USA) according to the coordinates as below: 2 mm right lateral to the bregma and 4 mm depth from the dura.11 Preparation of ANP-D/P Unmodified small lipid–polymer hybrid nanoparticles (NP) were prepared through a modified nanoprecipitation method.36-38 In brief, 16 mg of PLGA-COOH, 3.6 mg of DSPE-PEG2000 and 0.4 mg of DSPE-PEG3400-MAL were dissolved in 1mL of acetone containing 0.15 mg of triethylamine. The polymer solution was then injected into 2 mL of water containing 10 mM of Tris-HCl at pH 8. The acetone was then removed under a ZX-98 rotary evaporator (Shanghai Institute of Organic Chemistry, China). Dox-loaded, Dox/PFOB-loaded, DiD or 8


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DiR-labeled NP was prepared with the same procedure except that 0.4 mg of Dox, 0.6 mg of the mixture of Dox and PFOB (the weight ratio 2:1), 2 µg of DiD or 2 µg of DiR was dissolved in 1 mL of polymer solution in advance. ANP was prepared via a maleimide-thiol coupling reaction between angiopep-2 and NP.39, 40 The angiopep-2 was added in the NP solution at the molar ratio of peptide to DSPE-PEG3400-MAL 1:2.5 and stirred at room temperature for 2 h. Dox-loaded, Dox/PFOB-loaded, DiD or DiR-labeled ANP was prepared with the same procedure except that Dox-loaded, Dox/PFOB-loaded, DiD or DiR-labeled NP was reacted with angiopep-2. Characterization of ANP-D/P The surface morphology of the ANP-D/P and the surface morphology changes of the ANP-D/P after the HIFU irradiation at 8.5 W for 5 min were observed under transmission electron microscopy (TEM) (Hitachi, Japan) after negative staining with 2% sodium phosphotungstate solution. The particle size and zeta potential of the NP, NP-D/P, ANP-D and ANP-D/P were determined by dynamic light scattering (DLS) using a Malvern Nano ZS (Malvern Instruments, UK). The stability of the ANP-D/P in vitro was assessed by monitoring the particle size and zeta potential every day during one-week at 4 °C in PBS. Peptide conjugation efficiency and peptide density on the surface of ANP-D/P The unconjugated angiopep-2 was collected by ultrafiltration (30 k Da MWCO Membrane, Millipore, USA) and analyzed by a high performance liquid chromatography (HPLC) system (Agilent 1200, USA). The mobile phase was the mixture of solvent A and solvent B (80/20, v/v). Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 80% acetonitrile solution containing 0.09% trifluoroacetic acid. The peptide conjugation efficiency was calculated with the 9



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formula: =

(

!"#$ )

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×100%.

The peptide density on the nanoparticle surface (n) was calculated with the formula: =

&' ×* (' +×&, -$.

, where NA was Avogadro’s constant, m1 was the weight of conjugated angiopep-2

peptide on ANP, M1 was the molar mass of angiopep-2 peptide, m2 was the weight of ANP, d was the mean diameter of nanoparticles and ρ was the density of nanoparticles, estimated to be 1.2 g/cm3.41 Drug loading capacity and encapsulation efficiency of ANP-D/P The free Dox was separated from nanoparticles by ultracentrifugation and quantified at 485 nm by an UV spectrophotometer (Shimadzu, Japan). The encapsulation efficiency (EEDOX) and drug loading capacity (DLCDOX) of the Dox in nanoparticles were calculated as follows: //012 =

03 0345## 03

03#

;? @>=A>?B × 100%.

The ANP-D/P was lyophilized and then dissolved into CDCl13 containing perfluoro-15-crown-5-ether as an internal standard to determine the amount of PFOB in ANP-D/P by 19F NMR.42 The encapsulation efficiency (EEPFOB) and drug loading capacity (DLCPFOB) of the PFOB in nanoparticles were calculated as follows: //CD1E =

CD1E#

;=A>?B

× 100%.

Size and zeta changes of ANP-D/P under HIFU irradiation 5 mg/mL of the ANP-D/P samples were added into five glass tubes to receive different HIFU treatments. The CZF-200 HIFU therapy system (Chongqing Haifu Technology, China) was employed to irradiate these samples at 8.5 W or 10.5 W for 5 min or 10 min, respectively. After HIFU irradiation, the particle size and zeta potential of different samples were determined by dynamic light scattering (DLS) using a Malvern Nano ZS (Malvern Instruments, UK). 10


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HIFU-triggered drug release from ANP-D/P 5 mg/mL of the ANP-D/P samples were added into several glass tubes to receive different HIFU treatments. Different irradiation powers (8.5 W or 10.5 W) and exposure times (5 min, 10 min or 20 min) were selected to investigate their influences on HIFU-triggered drug release. After HIFU irradiation, the UV spectra of these samples were detected by an UV spectrophotometer (Shimadzu, Japan). To further demonstrate the drug release behavior of the ANP-D/P before and after HIFU irradiation, the UV absorbance of these samples at 485 nm was detected at pre-set time points and the accumulated amount of Dox released from ANP-D/P was plotted with time. In vitro cellular uptake U87 MG cells (2 × 104 cells/well) in the logarithmic phase were seeded into 24-well plates. When the cells reached 80% confluence, they were incubated with 600 µL of different Dox formulations (Lip-D, NP-D, ANP-D, ANP-D/P and ANP-D/P +A) at a Dox concentration of 50 µg/mL at 37 °C for 0.5 h, respectively. The ANP-D/P +A group indicated ANP-D/P with the presence of excessive free angiopep-2 (100 µg/mL). For qualitative assay, U87 MG cells were washed with PBS for 3 times, fixed with 4% paraformaldehyde for 15 min, stained with DAPI for 5 min, and then observed by a laser scanning confocal microscope (ZEISS, 710, LSM, Germany). To quantitatively determine the cellular uptake, U87MG cells were collected by trypsin digestion, suspended in PBS, and analyzed by a FACS Aria cell sorter (BD, USA) equipped with a 488-nm Arion laser. Transport of ANP-D/P across the BBB monolayers BBB monolayers were established according to previous works.43-45 Briefly, BCEC cells were seeded at a density of 5×104 cells per cm2 into 24-well transwell filters with 3.0-µm pore size 11



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and 0.3-cm2 filter membranes (Falcon Cell Culture Insert, Becton Dickinson Labware, NJ, USA). The confluency of the BCEC cells was examined under the microscope. The monolayer integrity was monitored by an epithelial voltohmmeter (Millicell ERS®, Millipore, MA, USA) and the trans-endothelial electrical resistance (TEER) was recorded every day. When the TEER was sustained over 200 Ω•cm2, the BBB monolayers could be used for further transport studies. When the BBB monolayers were established, 400 µL of DiD-labelled NP-D/P and ANP-D/P were added into the donor chambers, and then transferred to a 24-well plate containing 500 µL of 10 % FBS in DMEM. At 1, 2, 3, 4 and 6 h, 100 µL of sample was removed from each acceptor chamber and replaced with the same volume of fresh medium, respectively. The concentration of nanoparticles in samples was determined by a Tecan Infinite M200 Pro Multiplate Reader (Switzerland) with the excitation wavelength of 640 nm and emission wavelength of 670 nm. The amount of the nanoparticles across the BBB model were calculated according to their DiD loading capacity. The permeation rate (Pr) of the nanoparticles across the BBB model was calculated with @

the formula: Pr = = ∙ . , where m3 was the amount of the nanoparticles across the BBB model, t was the cumulative time, and A was the surface area of the filter membrane. In vitro cytotoxicity assay The cytotoxicity of different Dox formulations (Lip-D, NP-D, ANP-D and ANP-D/P) against U87 MG cells were evaluated by MTT assay.46-48 Briefly, U87MG cells in the logarithmic growth phase were seeded into 96-well plates (2000 cells per well). After 24 h, cells were incubated with different Dox formulations at the Dox concentration ranging from 0.25 ng to 250 µg for 24 h.49, 50 The cytotoxicity was determined by the MTT assay according to the routine protocol and the absorbance was measured by a microplate reader (Bio-TEK, USA) at the wavelength of 570 nm. 12


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The half maximal inhibitory concentration (IC50) of different Dox formulations (Lip-D, NP-D, ANP-D and ANP-D/P) against U87 MG cells were analyzed by GraphPad Prism v6.02. In vitro cell apoptosis assay Cell apoptosis was measured by the PE Annexin V apoptosis detection method.51 Briefly, U87 MG cells (1 × 105/mL) were seeded into 12-well plates and cultured for 24 h. Afterwards, U87 MG cells were incubated with different Dox formulations (Lip-D, NP-D, ANP-D, ANP-D/P and ANP-D/P H+) at a Dox concentration of 1 µg/mL for 24 h. For the ANP-D/P H+ group, U87 MG cells were incubated with ANP-D/P for 2 h, irradiated by HIFU at 10.5 W for 5 min, and then cultured for 22 h. Finally, cells in all groups were treated according to the PE Annexin V Apoptosis Detection Kit. The percent of early apoptosis and late apoptosis were analyzed by the flow cytometry (BD, USA). In vivo fluorescence imaging The accumulation of different Dox formulations (NP-D, ANP-D and ANP-D/P) in the intracranial glioblastoma-bearing nude mice was investigated by in vivo fluorescence imaging.46, 52 In brief, fifteen days after the U87 MG cell inoculation, the glioblastoma-bearing mice were injected with 200 µL of different DiR-labelled Dox formulations (NP-D, ANP-D and ANP-D/P) at the DiR dose of 2 µg via the tail vein, respectively, and then subjected to in vivo fluorescence imaging by an IVIS spectrum imaging system (PerkinElmer, USA) equipped with a DiR filter sets (excitation/emission, 745/780 nm) at various time points (6, 12, 24 and 48 h). Forty-eight hours post injection, the mice were euthanized followed by heart perfusion with saline and the major organs were sampled for the ex vivo fluorescence imaging by the IVIS spectrum imaging system (PerkinElmer, USA). 13



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Pharmacokinetics experiments In pharmacokinetics experiments, the nude mice were injected with 200 µL of different DiR-labelled Dox formulations (NP-D, ANP-D and ANP-D/P) at the DiR dose of 2 µg via the tail vein, respectively. Blood samples were collected from the orbital plexus at 0.25, 0.5, 1, 3, 8, 24 and 48 h following administration. The nanoparticle concentration in blood samples were quantified by a Tecan Infinite M200 Pro Multiplate Reader (Switzerland) with the excitation wavelength of 745 nm and emission wavelength of 780 nm 53 and expressed as percentage of injected dose per milliliter (% ID/mL). The pharmacokinetics parameters including the area under the concentration-time curve (AUC0-t), the mean residence time (MRT0-t), the maximum concentration (Cmax), and the half-life (t1/2) were calculated by DAS 3.0 software. In vivo tissue bio-distribution The tissue bio-distribution of different Dox formulations was performed on intracranial glioblastoma-bearing nude mice.54 Fifteen days after tumor implantation, mice were injected with 200 µL of different DiR-labelled Dox formulations (NP-D, ANP-D and ANP-D/P) at the DiR dose of 2 µg via the tail vein, respectively. At pre-set time points (24 and 48 h) after administration, the mice were euthanized followed by heart perfusion with saline and the major organs including hearts, livers, spleens, lungs, kidneys and brain tumors were collected. After tissue homogenization, the nanoparticle concentration in tissue samples was quantified by a Tecan Infinite M200 Pro Multiplate Reader (Switzerland) with the excitation wavelength of 745 nm and emission wavelength of 780 nm. Localization of nanoparticles in glioblastoma tissues The distribution of different Dox formulations (Lip-D, NP-D, ANP-D and ANP-D/P) in 14


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glioblastoma slices was investigated to evaluate the targeting property of the ANP-D/P in vivo. Fifteen days after tumor implantation, mouse models were injected with 100 µL of different Dox formulations (Lip-D, NP-D, ANP-D and ANP-D/P) at the Dox dose of 10 µg via the tail vein, respectively. Twenty-four hours later, the mice were euthanized followed by heart perfusion with saline. Then, the brains were harvested, dehydrated in 15% and 30% sucrose successively, embedded in OTC, and cut into 10-µm slices. Neo-vascular cells were stained with rabbit polyclone CD 31 (1:100) at 4 °C overnight and further stained with Alexa fluor®647-conjugated goat anti-rabbit IgG secondary antibody (1:100) for 1 h at room temperature. Cell nuclei were stained by DAPI (1 µg/mL) at room temperature for 5 min, and eventually, the slices were observed under a laser scanning confocal microscope (ZEISS, 710, LSM, Germany). Ex vivo mouse liver irradiation by HIFU To further demonstrate the effectiveness of the PFOB encapsulated in ANP-D/P under HIFU irradiation, the degassed ex vivo mouse liver models were prepared in degassed water and received an injection of 100 µL of different formulations (NP-D, ANP-D, ANP-D/P or saline) at the Dox dose of 10 µg, respectively.30, 55 Then, the injection sites of the excised mouse livers were exposed to HIUF irradiation at 10.5 W for 12 s (every 1s with an interval of 4 s). After HIFU irradiation, the digital photo of treated mouse livers were recorded and the radiation volume of HIFU in mouse livers were measured using the following formula: V = π/6×L×W×D (L: length, W: width, D: depth).30 The optimal parameters of HIFU applied in vivo were investigated in Supporting information and Supplementary figure 2 and figure 3. Anti-glioblastoma efficacy in vivo The intracranial glioblastoma-bearing nude mice were established as described above. The 15



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mice were randomly divided into six groups to receive different treatments including Lip-D, NP-D, ANP-D, ANP-D/P, ANP-D/P H+ (2 mg/kg of Dox) and saline via i.v. injection at 2, 5, 8 and 11 days after U87 MG cell inoculation, respectively. Twenty-four hours after the last administration, the ANP-D/P H+ group further received HIFU irradiation at 10.5 W for 12 s (every 1s with an interval of 4 s), respectively. Meanwhile, the craniectomy was performed before the HIFU irradiation to reduce the energy absorption of skull bone.56, 57 The body weight of the tumor-bearing mice was monitored every two days. The survival data were analyzed with log-rank test in a Kaplan-Meier nonparametric analysis by GraphPad Prism v6.02. To investigate tumor cell apoptosis induced by different treatments, three mice from each group were euthanized fifteen days after implantation. The brains were harvested, fixed with 4% paraformalehyde for 48 h, embedded in paraffin and cut into 10-µm slices. Then, the slices were subjected to the histological treatments including haematoxylin and eosin (H&E) staining using routine protocols and Tdt-mediated dUTP nick-end labeling (TUNEL) staining according to the protocol of in situ cell death detection kit. Finally, the slices were visualized under a fluorescence microscope (Leica, Germany). Statistical analysis Unpaired student’s t-test was used for assessment of statistically significant differences between two groups and one-way ANOVA comparison followed by Bonferroni analysis was used for multiple-group analysis. Data were expressed as mean ± standard deviation (SD), and P value < 0.05 was considered statistically different.

RESULTS 16


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Characterization of ANP-D/P The surface morphology of the ANP-D/P observed by TEM was generally spherical with a regular shape (Fig. 1 B). The mean particle size of the ANP-D/P was 41 nm (PDI = 0.16) and the zeta potential of the ANP-D/P was around -20.7 mV (Fig. 1 C). The angiopep-2 modification might be the main factor contributing to the changes of particle size (30±1.24 nm) and zeta potential (-48±1.27 mV) of small NP (Fig. 1 C). The ANP-D/P showed an excellent stability in PBS at 4 °C during one week, without significant changes in both size and zeta potential (Fig. 1 D). The conjugation efficiency of the angiopep-2 for ANP-D/P was 89.0±1.32 %. The EEDOX and DLCDOX of the Dox for ANP-D/P were 91±1.21 % and 1.8±1.28 %, respectively. The density of the angiopep-2 on the ANP-D/P surface was 179±2.3 per nanoparticle. The EEPFOB and DLCPFOB of the PFOB for ANP-D/P were 89±1.7 % and 0.9±2.6 %, respectively. Structure changes of ANP-D/P under HIFU irradiation The effect of HIFU irradiation on the structure of ANP-D/P was assessed by TEM and DLS. As shown in Fig. 1 B, an apparent morphology change and size increase of the ANP-D/P could be observed after HIFU irradiation at 8.5 W for 5 min. In addition, some PLGA segments were found expelled from the nanoparticles after HIFU irradiation (Fig. 1 B and Supplementary Figure 1), which intuitively demonstrated the structural collapse of the ANP-D/P induced by HIFU irradiation. The size of the ANP-D/P under HIFU irradiation significantly enlarged with the increase of irradiation power or exposure time (Fig. 2 A and B). The decreased zeta potential of the ANP-D/P after HIFU irradiation might attribute to the exposure of the carboxyl groups in PLGA after HIFU irradiation (Fig. 2 C). All the structural changes of the ANP-D/P above could deduce that the instantaneous vaporization of the encapsulated PFOB mainly contributed to the 17



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structural collapse of the ANP-D/P under HIFU irradiation. HIFU-triggered drug release of ANP-D/P The influences of irradiation power and exposure time on the HIFU-triggered drug release behavior were investigated. As shown in Fig. 2 D, an obviously improved Dox release after HIFU irradiation could be observed in the UV-vis spectra, and elevating the irradiation power (10.5 W) could significantly enhance the drug release with the same exposure time (5 min). The samples collected after different exposure times (5 min, 10 min and 20 min) at the same irradiation power (10.5 W) provided the evidences that prolonged exposure time was favorable for the HIFU-responsive drug release as well (Fig. 2 E). Additionally, as shown in Fig. 2 F, before or without HIFU irradiation, almost no Dox released from the ANP-D/P. However, a dramatically increased drug release was observed in the ANP-D/P after HIFU irradiation. The accumulative Dox release reached 47%, 65% and 76% at 2, 5 and 10 min after HIFU irradiation, respectively. This distinctive rapid drug release might ascribe to the PFOB-induced structural collapse of the ANP-D/P 30 as well as the depolymerization or glass-transition of the PLGA under HIFU irradiation.32 In vitro cellular uptake As shown in Fig. 3 A, the fluorescence intensity of U87 MG cells in the ANP-D and ANP-D/P groups were much higher than that in other groups, and the process could be inhibited by the excessive free angiopep-2. The flow cytometry results further exhibited that the angiopep-2 modification on small nanoparticles increased the cellular uptake of the ANP-D and ANP-D/P by 3.6 and 3.8 folds compared with that of the NP-D, respectively (Fig. 3 B and C). There was no significant statistical difference in the fluorescence intensity between ANP-D/P +A and NP-D. 18


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These results suggested that the angiopep-2 modification significantly enhanced the uptake of the ANP-D and ANP-D/P by U87 MG cells through the LRP-mediated endocytosis. Furthermore, the significant differences in fluorescence intensity between the angiopep-2-modified nanoparticle groups (ANP-D and ANP-D/P) and the Lip-D group demonstrated that the ANP-D and ANP-D/P owned the superior targeting properties to the Lip-D, which might be due to their optimal size (40~50 nm) for efficient nanoparticle uptake into cells,58 negative charge-facilitated cell uptake,59, 60

and LRP-mediated endocytosis.

Transport of ANP-D/P across the BBB monolayers As shown in Figure 3 D and E, the amount of ANP-D/P across the in vitro BBB model were significantly higher than that of NP-D/P at each time point (P

On-Demand Drug Release from Dual-Targeting ... - ACS Publications

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