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Optical Modulation of the Blood-Brain Barrier for Glioblastoma Treatment

血脑屏障的光学调节在胶质母细胞瘤治疗中的应用

QC Qi Cai
HF Hanwen Fan
XL Xiaoqing Li
MG Monica Giannotta
RB Robert Bachoo
ZQ Zhenpeng Qin

发布: 2024年01月20日第14卷第2期 DOI: 10.21769/BioProtoc.4920 浏览次数: 2637

评审: Mary Luz UribeAnonymous reviewer(s)

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Aug 2023

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Abstract

The blood–brain barrier (BBB) is a major obstacle to the diagnostics and treatment of many central nervous system (CNS) diseases. A prime example of this challenge is seen in glioblastoma (GBM), the most aggressive and malignant primary brain tumor. The BBB in brain tumors, or the blood–brain–tumor barrier (BBTB), prevents the efficient delivery of most therapeutics to brain tumors. Current strategies to overcome the BBB for therapeutic delivery, such as using hyperosmotic agents (mannitol), have impeded progress in clinical translation limited by the lack of spatial resolution, high incidences of complications, and potential for toxicity. Focused ultrasound combined with intravenously administered microbubbles enables the transient disruption of the BBB and has progressed to early-phase clinical trials. However, the poor survival with currently approved treatments for GBM highlights the compelling need to develop and validate treatment strategies as well as the screening for more potent anticancer drugs. In this protocol, we introduce an optical method to open the BBTB (OptoBBTB) for therapeutic delivery via ultrashort pulse laser stimulation of vascular targeting plasmonic gold nanoparticles (AuNPs). Specifically, the protocol includes the synthesis and characterization of vascular-targeting AuNPs and a detailed procedure of optoBBTB. We also report the downstream characterization of the drug delivery and tumor treatment efficacy after BBB modulation. Compared with other barrier modulation methods, our optical approach has advantages in high spatial resolution and minimally invasive access to tissues. Overall, optoBBTB allows for the delivery of a variety of therapeutics into the brain and will accelerate drug delivery and screening for CNS disease treatment.


Key features

• Pulsed laser excitation of vascular-targeting gold nanoparticles non-invasively and reversibly modulates the blood–brain barrier permeability.

• OptoBBTB enhances drug delivery in clinically relevant glioblastoma models.

• OptoBBTB has the potential for drug screening and evaluation for superficial brain tumor treatment.


Graphical overview


Keywords: Glioblastoma (胶质母细胞瘤) search Blood–brain barrier (血脑屏障) search Blood–brain–tumor barrier (血脑肿瘤屏障) search Nanoparticles (纳米颗粒) search Pulsed laser (脉冲激光) search Drug delivery (药物输送) search Chemotherapy (化疗) search

Background

High-grade gliomas represent the most malignant primary brain tumors in adults. Among these, grade IV astrocytomas, also referred to as glioblastoma multiforme (GBM), constitute over 50% of all primary brain tumors (Straehla et al., 2022). Despite standard-of-care treatment that includes maximal safe resection, fractionated radiation to 60 Gy with concurrent daily temozolomide (TMZ) followed by up to six months of adjuvant TMZ, as well as the progress made in developing new treatment approaches, GBM continues to remain predominantly incurable (Stupp et al., 2005). In addition to the heterogeneous and immunosuppressive tumor microenvironment, a significant challenge for GBM therapy lies in the difficulty of delivering sufficient drug concentrations in the tumor due to the protective blood–brain barrier (BBB), which is also known as blood–brain–tumor barrier (BBTB) in brain tumors. The BBB governs the exchange of ions, molecules, and cells between the bloodstream and the brain, ensures optimal neuronal functioning, and shields brain tissue from harmful toxins and pathogens. Nevertheless, the BBB also prevents more than 98% of small-molecule drugs and all macromolecular therapeutics from entering the brain, resulting in a challenge in drug delivery for brain disease treatment. Although a variety of pathologic conditions, including brain tumors, can disrupt the integrity of the BBB (Sarkaria et al., 2018), the disruption is heterogeneous or inadequate to facilitate clinically meaningful drug penetration. Additionally, evidence suggests that GBM tumor cells infiltrate neighboring tissues without disrupting the BBB, leading to inevitable recurrence (Belykh et al., 2020). To date, numerous strategies have been developed to overcome the BBB for drug delivery (van Tellingen et al., 2015; Belykh et al., 2020; Rosenblum et al., 2018; Brunner and Borchard, 2021). Nonetheless, GBM is still considered incurable, and many promising results from preclinical studies do not translate successfully to clinical trials. Thus, it is of utmost importance to create and validate treatment strategies using clinically relevant GBM models to achieve a more efficient transition from preclinical testing to clinical implementation.

Here, we describe an optical method to open the BBTB (optoBBTB) to modulate the BBB for drug delivery in two clinically relevant genetically engineered mouse models (GEMMs) that recapitulate key GBM characteristics (e.g., PS5A1 GEMM for infiltrative tumor margin with an intact BBTB and 73C GEMM for angiogenic tumor core with a gradually disrupted BBTB) (Cai et al., 2023). We show that optoBBTB facilitates the delivery of paclitaxel (Taxol) to both GEMMs, which further reduces tumor growth and prolongs the mice’s survival. These results demonstrate that optoBBTB is effective for drug delivery and GBM treatment in these two clinically relevant GEMMs.

Materials and reagents

Biological materials

  1. Immunodeficient nude mice Foxn1nu, 7 weeks old, female, 20–25 g (Jackson Laboratory, catalog number: Nu/J 002019)

  2. PS5A1 cell line, generated from Dr. Robert Bachoo’s laboratory at the University of Texas Southwestern Medical Center (Singh et al., 2017; Gao et al., 2020)

  3. 73C cell line, generated from Dr. Robert Bachoo’s laboratory at the University of Texas Southwestern Medical Center (Singh et al., 2017; Gao et al., 2020)

  4. Anti-JAM-A antibodies BV11, provided by Dr. Monica Giannotta at FIRC Institute of Molecular Oncology Foundation


Reagents


Reagents for glassware cleaning

  1. Hydrochloric acid (HCl) 37% w/w (Fisher Scientific, catalog number: 7647-01-0)

  2. Nitric acid (HNO3) 70% w/w (Fisher Scientific, catalog number: 7697-37-2)


Reagents for gold nanoparticle (AuNP) synthesis and antibody conjugation

  1. Gold (III) chloride trihydrate (HAuCl4·3H2O) (Sigma-Aldrich, catalog number: 520918-1G)

  2. Sodium citrate tribasic dihydrate (Sigma-Aldrich, catalog number: S4641-500G)

  3. Hydroquinone (Sigma-Aldrich, catalog number: H9003-100G)

  4. OPSS-PEG-SVA 3400 Da (Laysan Bio, Inc., catalog number: OPSS-SVA-3400)

  5. mPEG-thiol 1,000 Da (Laysan Bio, Inc., catalog number: M-SH-1K)

  6. Cytiva HyCloneTM phosphate-buffered saline (PBS) (Fisher Scientific, catalog number: SH3025801)

  7. PierceTM borate buffer (20×) (Fisher Scientific, catalog number: PI28341)

  8. Endotoxin-free ultrapure water (Sigma-Aldrich, catalog number: TMS-011-A)


Reagent for cell culture

  1. GibcoTM DMEM high glucose GlutaMAXTM supplement (Fisher Scientific, catalog number: 10-566-016)

  2. GibcoTM DMEM/F-12, no glutamine (Fisher Scientific, catalog number: 21-331-020)

  3. GibcoTM Hanks' Balanced Salt Solution, no calcium, no magnesium, and no phenol red (HBSS) (Fisher Scientific, catalog number: 14-175-095)

  4. Epidermal growth factor (EGF) (Sigma-Aldrich, catalog number: E4127)

  5. Recombinant human fibroblast growth factor basic (FGF2) (Sigma-Aldrich, catalog number: F0291)

  6. Progesterone (Sigma-Aldrich, catalog number: P6149)

  7. GibcoTM B-27TM supplement (50×), serum-free (Fisher Scientific, catalog number: 17-504-044)

  8. GibcoTM insulin-transferrin-selenium-ethanolamine 100× (Fisher Scientific, catalog number: 51-500-056)

  9. GibcoTM fetal bovine serum (FBS) (Fisher Scientific, catalog number: A3160502)

  10. GibcoTM penicillin-streptomycin (Fisher Scientific, catalog number: 15-140-122)

  11. GibcoTM StemProTM AccutaseTM cell dissociation reagent (Fisher Scientific, catalog number: A1110501)

  12. GibcoTM Trypsin-EDTA (0.25%), phenol red (Fisher Scientific, catalog number: 25-200-056)


Reagents for optoBBTB and drug delivery

  1. 0.9% saline solution, sterile (Fisher Scientific, catalog number: S5815)

  2. Isoflurane USP (Covetrus, catalog number: NDC 66794-017-25)

  3. Cremophor® EL (Millipore Sigma, catalog number: 61791-12-6)

  4. Ethanol absolute (Fisher Scientific, catalog number: BP2818100)

  5. Taxol Janelia Fluor® 646 (Tocris Bioscience, catalog number: 6266)

  6. Paclitaxel (Sigma-Aldrich, catalog number: T7191)

  7. Buprenorphine SR-LAB 5 mL (1 mg/mL) (ZooPharm, catalog number: BSRLAB1)


Reagents for tissue collection and ex vivo drug delivery analysis

  1. Paraformaldehyde, 4% in PBS (Fisher Scientific, catalog number: AAJ61899AP)

  2. Sucrose (Millipore Sigma, catalog number: 57-50-1)

  3. Hoechst 33342 solution (Fisher Scientific, catalog number: PI62249)

  4. Scigen Tissue-PlusTM O.C.T. compound (Fisher Scientific, catalog number: 23-730-571)

  5. InvitrogenTM Fluoromount-GTM mounting medium (Fisher Scientific, catalog number: 50-187-88)


Solutions

  1. HAuCl4 solution, 1 M (see Recipes)

  2. HAuCl4 solution, 25 mM (see Recipes)

  3. Sodium citrate solution, 112.2 mM (see Recipes)

  4. Sodium citrate solution, 15 mM (see Recipes)

  5. Hydroquinone solution, 25 mM (see Recipes)

  6. 2 mM borate buffer stock solution, pH = 8.5 (see Recipes)

  7. 10% sucrose stock solution (see Recipes)

  8. 20% sucrose stock solution (see Recipes)

  9. 30% sucrose stock solution (see Recipes)


Recipes

  1. HAuCl4 solution, 1 M

    *Note: Upon unsealing the bottle, rapidly transfer the HAuCl4·3H2O into a tube to measure the weight. Aliquot the stock solution into 0.5 mL Eppendorfs and snap-freeze the solution with liquid nitrogen for future utilization. The stocks can be stored at -80 °C.

    ReagentFinal concentrationQuantity or Volume
    HAuCl4·3H2O1 M1 g (see note*)
    Ultrapure H2On/a2.539 mL
    Total (optional)n/an/a


  2. HAuCl4 solution, 25 mM

    Note: This solution should be prepared freshly.

    ReagentFinal concentrationQuantity or Volume
    HAuCl4 (1 M)25 mM0.05 mL
    Ultrapure H2On/a1.95 mL
    Total (optional)n/a2 mL


  3. Sodium citrate solution, 112.2 mM

    Note: This solution should be prepared freshly.

    ReagentFinal concentrationQuantity or Volume
    Sodium citrate112.2 mM0.165 g
    Ultrapure H2On/a5 mL
    Total (optional)n/an/a


  4. Sodium citrate solution, 15 mM

    Note: This solution should be prepared freshly.

    ReagentFinal concentrationQuantity or Volume
    Sodium citrate15 mM0.022 g
    Ultrapure H2On/a5 mL
    Total (optional)n/an/a


  5. Hydroquinone solution, 25 mM

    Note: This solution should be prepared freshly.

    ReagentFinal concentrationQuantity or Volume
    Hydroquinone25 mM0.0138 g
    Ultrapure H2On/a5 mL
    Total (optional)n/an/a


  6. 2 mM borate buffer stock solution, pH = 8.5

    ReagentFinal concentrationQuantity or Volume
    Borate buffer (20×)2 mM0.2 mL
    Ultrapure H2On/a99.8 mL
    Total (optional)n/a100 mL


  7. 10% sucrose stock solution

    *Note: PBS should be added in increments of 10 mL. Allow the solution to mix completely before adding any more PBS.

    ReagentFinal concentrationQuantity or Volume
    Sucrose10% (w/v)10 g
    PBS (1×)n/asee note*
    Total (optional)n/a100 mL


  8. 20% sucrose stock solution

    *Note: PBS should be added in increments of 10 mL. Allow the solution to mix completely before adding any more PBS.

    ReagentFinal concentrationQuantity or Volume
    Sucrose20% (w/v)20 g
    PBS (1×)n/asee note*
    Total (optional)n/a100 mL


  9. 30% sucrose stock solution (100 mL)

    *Note: PBS should be added in increments of 10 mL. Allow the solution to mix completely before adding any more PBS.

    ReagentFinal concentrationQuantity or Volume
    Sucrose30% (w/v)30 g
    PBS (1×)n/asee note*
    Totaln/a100 mL


Laboratory supplies


Supplies for AuNP synthesis and antibody conjugation

  1. FisherbrandTM polygon stir bars (Fisher Scientific, catalog number: 14-512-127)

  2. FisherbrandTM IsotempTM stirrer (Fisher Scientific, catalog number: S88850200)

  3. PYREXTM short-neck heavy-wall round-bottom boiling flasks, standard taper joints (Fisher Scientific, catalog number: 10-068-1C)

  4. PYREXTM narrow-mouth heavy-duty glass Erlenmeyer flask 250 mL (Fisher Scientific, catalog number: 10-040F)

  5. DWK Life Sciences KimbleTM KIMAXTM west condensers (Fisher Scientific, catalog number: K452000-2430)

  6. PYREXTM crystallizing dishes (Fisher Scientific, catalog number: 08-741-D)

  7. FisherbrandTM sterile polystyrene disposable serological pipettes with magnifier stripe 10 mL (Fisher Scientific, catalog number: 13-678-11E)

  8. FisherbrandTM sterile polystyrene disposable serological pipettes with magnifier stripe 50 mL (Fisher Scientific, catalog number: 13-678-11F)

  9. FisherbrandTM premium microcentrifuge tubes 2.0 mL (Fisher Scientific, catalog number: 05-408-138)

  10. FisherbrandTM premium microcentrifuge tubes 1.5 mL (Fisher Scientific, catalog number: 05-408-129)

  11. Thermo ScientificTM low protein binding microcentrifuge tubes 1.5 mL (Fisher Scientific, catalog number: PI90410)

  12. Thermo ScientificTM NuncTM 15 mL conical sterile polypropylene centrifuge tubes (Fisher Scientific, catalog number: 12-565-269)

  13. Thermo ScientificTM NuncTM 50 mL conical sterile polypropylene centrifuge tubes (Fisher Scientific, catalog number: 12-565-271)

  14. 0.22 μm syringe filters (VWR, catalog number: 76479-030)

  15. SpectrumTM trial size kits for biotech-grade CE dialysis membrane tubing (1 m), MWCO: 20 kd (Fisher Scientific, catalog number: 08-801-251)


Supplies for AuNP characterization

  1. FisherbrandTM 384-well polystyrene plates (Fisher Scientific, catalog number: 12-566-625)

  2. FisherbrandTM disposable cuvettes (Fisher Scientific, catalog number: 14-955-127)

  3. Electron microscopy sciences carbon support film 5–6 nm thick on square 200 mesh copper grid (Fisher Scientific, catalog number: NC9044609)


Supplies for cell culture

  1. Thermo ScientificTM NuncTM EasYFlaskTM cell culture flasks 75 cm2 (Fisher Scientific, catalog number: 12-565-349)

  2. Thermo ScientificTM NunclonTM SpheraTM cell culture flasks 75 cm2 (Fisher Scientific, catalog number: 12-566-440)


Supplies for tumor cell transplantation in mouse and optoBBTB

  1. Micropipette puller (Sutter Instrument, catalog number: Co. P-1000)

  2. Instech PE-20 tubing (Fisher Scientific, catalog number: 50-890-048)

  3. BD Micro-FineTM IV insulin syringes (Fisher Scientific, catalog number: 14-829-1D)

  4. Med Vet International Exel butterfly infusion set, with 27 G × ¾ in. needle, 12 in. tubing (Fisher Scientific, catalog number: 50-209-2293)

  5. PuritanTM cotton-tipped applicators (Fisher Scientific, catalog number: 22-029-553)

  6. FisherbrandTM sterile alcohol prep pads (Fisher Scientific, catalog number: 22-363-750)

  7. World Precision Instrument mouse dissecting kit (Fisher Scientific, catalog number: 50-822-920)

  8. Henkel Loctite 4014 medical device instant adhesive clear (Henkel Adhesives, catalog number: 202152)

  9. Body double standard set (Reynolds Advanced Materials)


Supplies for tissue processing and ex vivo drug delivery analysis

  1. BD VacutainerTM eccentric tip syringe (Fisher Scientific, catalog number: B300613)

  2. FisherbrandTM SuperfrostTM plus microscope slides (Fisher Scientific, catalog number: 12-550-15)

  3. FisherbrandTM cover glasses: rectangles (Fisher Scientific, catalog number: 12-544-18P)

Equipment

  1. New Era Pump Systems Inc single channel syringe pump (Fisher Scientific, catalog number: NC1072839)

  2. FisherbrandTM accuSpinTM Micro 17 microcentrifuge (Fisher Scientific, catalog number: 13-100-675)

  3. BioTek Synergy 2 plate reader multi-mode (The LabWorld Group, catalog number: 18531)

  4. Dynamic light scattering (DLS) zetasizer nano zs (Malvern Panalytical)

  5. Electron microscope (JEOL Ltd., model: JEM-1400 plus)

  6. Diode-pumped high energy picosecond Nd: YAG lasers (EKSPLA, catalog number: PL2230)

  7. Coherent® LabMax touch power and energy meter (Coherent®, catalog number: 2256258)

  8. Coherent® EnergyMax laser energy sensors (Coherent®, catalog number: 1110746)

  9. Isoflurane vaporizer (Kent Scientific, catalog number: VetFlo-1231)

  10. DC temperature controller system (FHC, catalog number: 40-90-8D)

  11. Rectal thermistor probe (FHC, catalog number: 40-90-5D-02)

  12. Heating pad (FHC, catalog number: 40-90-2-07)

  13. Syringe pump controller (World Precision Instruments, catalog number: micro-2T)

  14. Brushless micromotor system (Osada, catalog number: EXL-M40)

  15. Dual small animal stereotaxic instrument (KOPF, catalog number: 902)

  16. Zeiss Stemi 305 microscope (Zeiss, catalog number: STEMI305-T-B)

  17. HM525 NX cryostat (Epredia, catalog number: HM525 NX)

  18. Virtual slide microscope (Olympus, model: VS120)

  19. Spinning disk super-resolution microscope (Olympus, model: SD-OSR)

  20. IVIS® Lumina Series III (PerkinElmer, catalog number: CLS136334)

Software and datasets

  1. ImageJ (version 1.53c, 6/27/2020)

  2. GraphPad Prism (version 9.5.0, 12/6/2022)

Procedure

English
中文翻译

文章信息

版权信息

© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).

如何引用

Cai, Q., Fan, H., Li, X., Giannotta, M., Bachoo, R. and Qin, Z. (2024). Optical Modulation of the Blood-Brain Barrier for Glioblastoma Treatment. Bio-protocol 14(2): e4920. DOI: 10.21769/BioProtoc.4920.

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分类

神经科学 > 基础技术 > 光遗传学

癌症生物学 > 通用技术 > 癌症治疗

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