Targeting Mitochondria in Tumor-Associated Macrophages using a Dendrimer-Conjugated TSPO Ligand that Stimulates Antitumor Signaling in Glioblastoma

Article abstract of DOI:10.1021/acs.biomac.0c01033

Mitochondria mediate critical cellular processes, including proliferation, apoptosis, and immune responses; as such, their dysfunction is pathogenic in many neurodegenerative disorders and cancers. In glioblastoma, targeted delivery of mitochondria-focuse

Full text of DOI:10.1021/acs.biomac.0c01033

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Article
Targeting Mitochondria in Tumor-Associated Macrophages using a Dendrimer
Conjugated TSPO Ligand Stimulates Anti-Tumor Signaling in Glioblastoma
Anjali Sharma, Kevin Liaw, Rishi Sharma, Ajit G. Thomas, Barbara
S. Slusher, Sujatha Kannan, and Rangaramanujam M. Kannan
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.0c01033 • Publication Date (Web): 06 Aug 2020
Downloaded from pubs.acs.org on August 13, 2020
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Targeting Mitochondria in Tumor-Associated
Macrophages using a Dendrimer Conjugated
TSPO Ligand Stimulates Anti-Tumor Signaling
in Glioblastoma
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Anjali Sharma,1§ Kevin Liaw,1,2§ Rishi Sharma,1 Ajit G. Thomas,3 Barbara S. Slusher,3-9 Sujatha
Kannan,1,10,11 and Rangaramanujam M. Kannan*1,2,11
1Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute
2Department of Chemical and Biomolecular Engineering
3Johns Hopkins Drug Discovery
4Department of Neurology
5Department of Neuroscience
6Department of Medicine
7Department of Oncology
8Department of Psychiatry and Behavioral Sciences
9Department of Pharmacology and Molecular Sciences
10Department of Anesthesiology and Critical Care Medicine,
11Hugo W. Moser Research Institute at Kennedy Krieger
§These authors contributed equally
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*Corresponding author:
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Rangaramanujam M. Kannan, Arnall Patz Distinguished Professor of Ophthalmology, Center for
Nanomedicine at the Wilmer Eye Institute, 400 North Broadway, Baltimore, Maryland 21231,
USA; Email: krangar1@jhmi.edu; Phone: 443-287-8634.
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Abstract
Mitochondria mediate critical cellular processes, including proliferation, apoptosis, and
immune responses; as such, their dysfunction is pathogenic in many neurodegenerative disorders
and cancers. In glioblastoma, targeted delivery of mitochondria-focused anti-cancer therapies
have failed to translate into clinical successes due to their nonspecific cellular localization,
heterogeneity of receptor expression across patients, poor transport across biological barriers to
reach the brain, tumor, and mitochondria, as well as systemic side effects. Strategies that can
overcome brain and solid tumor barriers and selectively target mitochondria within specific cell
types may yield improvements to glioblastoma treatment. Developments in dendrimer-mediated
nanomedicines have shown promise targeting tumor-associated macrophages (TAMs) in
glioblastoma following systemic administration. Here, we present a novel dendrimer conjugated
to the translocator protein 18KDa (TSPO) ligand 5,7-dimethylpyrazolo[1,5-α]pyrimidin-3-
ylacetamide (DPA). We developed a clickable DPA for conjugation on dendrimer surface and
demonstrate in vitro that the dendrimer-DPA conjugate (D-DPA) significantly increases
dendrimer co-localization with mitochondria. Compared to free TSPO ligand PK11195, D-DPA
stimulates greater anti-tumor immune signaling. In vivo, we show that D-DPA targets
mitochondria specifically within TAMs following systemic administration. Our results
demonstrate that dendrimers can achieve TAMs specific targeting in glioblastoma and can be
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further modified to target specific intracellular compartments for organelle-specific drug
delivery.
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Keywords
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Dendrimer, Glioblastoma, Tumor-associated macrophages, mitochondria, targeting
Introduction
The mitochondrion is widely known as the powerhouse of the cell and is chiefly
responsible for regulating cellular energy production and metabolism.1 In addition, mitochondria
play critical roles in regulating trafficking, proliferation, apoptosis, and immune responses.2 Due
to their position at the nexus of these vital physiological functions, mitochondrial dysfunction is
associated with diverse diseases ranging from neurodegenerative disorders to cancers to
autoimmune disorders.3, 4 Restoration of healthy mitochondrial function from pathological
activity is therefore necessary to address progression of these diseases.5
Defective mitochondria function in cell cycle regulation and apoptotic signaling is
characteristic of cancer progression.6 Therefore, intracellular targeting anti-cancer therapies
focused on mitochondria to slow cancer cell proliferation and induce apoptosis have been
explored.7 In addition, due to their roles in mediating inflammatory responses, mitochondria
have also been explored as targets in cancer immunotherapy approaches.8 For example,
mitochondrial damage in T cells have been found to mediate suppression of anti-cancer immune
response.9, 10 Tumor-associated macrophages (TAMs) have been found to mediate the cancer
immune response, and their activation state has been shown to be regulated by mitochondrial
metabolism.11, 12 Mitochondria mediate HIF-1α and HIF-2α in TAMs, and their localization to
mitochondria correlates with high grade and poor prognoses of cancers.11 In addition,
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dysfunctional mitochondria exhibit overproduction of reactive oxygen species, leading to high
oxidative stress in the tumor environment that promotes tumor growth.13, 14 As a therapeutic
target in TAMs, mitochondria can be leveraged to promote anti-tumor signaling by activating
pro-inflammatory immune signaling.15 However, TAMs-focused immunotherapies have suffered
from low response rates, poor brain and tumor penetration, and non-selective activity resulting in
systemic toxicities that have hampered their clinical translation.16, 17 In addition, the failure of
mitochondria-targeted therapies lies largely with their inability to preferentially accumulate in
mitochondria of specific cells of interest. Therefore, a nanotechnology-mediated strategy aimed
at cell-specific mitochondrial targeting may overcome these delivery challenges as an effective
treatment for glioblastoma and other cancers.8
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Translocator protein 18 kDa (TSPO) is a transport protein located on the outer
mitochondrial membrane that is responsible for transporting cholesterol into mitochondria for
synthesis of steroids.18 TSPO is minimally expressed in healthy tissues, but is highly
overexpressed in the context of neuroinflammation and cancer.19, 20 In the context of
glioblastoma, TSPO expression correlates with glioblastoma clinical outcomes.21 Targeting
TSPO has been explored to improve diagnostics through positron-emission tomography (PET)
imaging and therapies.22, 23 TSPO is highly upregulated in anti-inflammatory macrophages
consistent with the immunosuppressive TAMs phenotype.24 Targeting TSPO can leverage its
overexpression in TAMs to manipulate immune polarization. PK11195 is a first generation
TSPO ligand with nanomolar binding affinity that has been extensively explored as an inhibitor
of tumor cell proliferation and modulator of immune signaling.25, 26 5,7-Dimethylpyrazolo[1,5-
α]pyrimidin-3-ylacetamide (DPA) is a novel class of TSPO ligands which are selective, drug-like
ligands.27 While these TSPO targeting compounds have been explored extensively for
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diagnostics in PET imaging, their utility for targeted drug delivery in the brain have been limited
by poor brain penetration.28, 29 Therefore, nanomedicines that can penetrate into the brain and
brain tumors may enable effective TSPO targeting for improved drug delivery.
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Many nanotechnology-based strategies are being developed to target drugs to
mitochondria.30, 31 However, platforms that enable mitochondria targeting within specific cell
types in the brain are still being developed. Dendrimers have shown significant promise for
specific gene and drug delivery to activated microglia, cancer cells and TAMs.31-37 In an
orthotopic brain cancer models, we have previously shown that systemic administration of
generation 4 hydroxyl-terminated polyamidoamine (PAMAM) dendrimers are able to fully
penetrate and distribute uniformly throughout the solid brain tumor selectively in TAMs, with
minimal accumulation in healthy brain and peripheral tissues.33, 38 This cell-type specific
targeting is achieved without the need for targeting ligands. This dendrimer platform has
potential for clinical applications, with favorable safety profile and scalability of production.39, 40
Here we present a novel dendrimer-DPA conjugate for specific targeting of mitochondria within
TAMs as a unique immunotherapy approach. We describe the design, synthesis, chemical
characterization, in vitro mitochondrial localization and immune repolarization, and in vivo
targeting properties of this dendrimer-DPA conjugate.
Materials and Methods
Synthesis
Materials and reagents: 2,4-Pentanedione, borontribromide, ethyleneglycol ditosylate (4),
sodium azide, potassium carbonate, 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide
methiodide (EDC), 4(dimethylamino)pyridine (DMAP), GABA_BOC_OH, N, N-diisopropyl
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ethyl amine (DIPEA), copper sulfate pentahydrate, sodium ascorbate, and 5-Hexynoic acid, were
purchased from Sigma Aldrich US. Cy5 NHS ester was purchased from GE healthcare and used
as received. All the anhydrous solvents were purchased from Sigma and were used as received.
Hydroxyl PAMAM dendrimer with ethylenediamine-core (generation 4, 64 hydroxyl terminal-
groups, Pharma grade, compound (7) was purchased from Dendritech as 13 wt% methanolic
solution. Methanol was evaporated using rotary evaporator prior to use. D-Cy5 was synthesized
using previously published protocols.41
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Characterization: Proton nuclear magnetic resonance spectra (NMR) were recorded using
Bruker spectrometer (500 MHz). The data is reported as chemical shift (δ ppm). The peaks are
relative to the residual protonated solvent resonance and are reported in terms of multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constants (J Hz) and
assignment. High resolution mass spectra (HRMS) was recorded using electrospray ionization
(ESI) recorded on a Bruker microTOF-II mass spectrometer in the positive mode. The samples
were introduced via direct flow using CH3CN/ H2O (9:1) solvent system. The empirical formula
confirmation is reported as protonated molecular ions [M + nH]n+ or adducts [M + nX]n+ (X =
Na). High pressure liquid chromatography (HPLC) was performed on a Waters system with 2998
photodiode array detector and a 2475 multi λ fluorescence detector, In-Line degasser, a 1525
binary pump and Waters Empower 2 Software. Separation was achieved on a Waters column
(C18 symmetry 300, 5µm, 4.6x250mm). Flow rate was maintained at 1.0 mL/min over a gradient
from 100:0 (A:B) gradually increasing to 50:50 (A:B) at 25 min, 10:90 (A:B) at 35 minutes, and
finally returning to 100:0 (A:B) at 40 minutes maintaining a flow rate of 1 mL/min. (A: 0.1%
TFA and 5% ACN in water and buffer B: 0.1% TFA in ACN) and eluent monitored at
wavelengths 210 and 650 nm. Flash chromatography was performed using Teledyne combiflash
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sytem. Size and zeta potential measurements were carried out via Dynamic light scattering using
Malvern Zetasizer Nano ZS as previously reported by our group.42
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Procedures for the synthesis of intermediates and dendrimer conjuagtes:
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Synthesis of compound 2: A solution of compound 1 (500 mg, 1.65 mmoles) and 2,4-
pentanedione (0.17 mL, 1.65 mmoles) in anhydrous ethanol (15mL) was heated at 60oC in a
microwave reactor for 20 hours. Upon completion, the solvent was evaporated and the residue
was dissolved in dichloromethane (DCM). The organic layer was washed with water and brine
and dried over sodium sulfate. The dried organic layer was then evaporated and the crude
product was purified using combiflash. The pure fractions were obtained in 3% methanol in
dichloromethane and were evaporated to afford an off-white solid product. Yield: 85%.
1H NMR (500 MHz, CDCl3) δ 7.86 – 7.66 (m, 2H), 7.08 – 6.87 (m, 2H), 6.51 (d, J = 0.7 Hz,
1H), 3.92 (s, 2H), 3.86 (s, 3H), 3.51 (q, J = 7.1 Hz, 2H), 3.42 (q, J = 7.1 Hz, 2H), 2.75 (s, 3H),
2.55 (s, 3H), 1.21 (t, J = 7.1 Hz, 3H), 1.12 (t, J = 7.1 Hz, 3H). Figure S1
13C NMR (126 MHz, CDCl3) δ 170.14, 159.81, 157.44, 155.05, 147.69, 144.70, 130.00, 126.39,
113.98, 108.15, 100.81, 77.31, 77.05, 76.80, 55.34, 42.33, 40.62, 28.21, 24.68, 16.94, 14.36,
13.11. Figure S2
ESI-MS: Theoretical for C21H26N4O2 is 366.21, observed is 367.21 [M+1]+ Figure S3
Synthesis of compound 3: To a stirring solution of compound 2 (450 mg, 1.23 mmoles) in
anhydrous DCM (5mL), 1M borontribromide in THF (6.13 mL, 5 equivalents) was added
dropwise at -60oC. The stirring was continued for 2 hours. Upon completion, the reaction
mixture was quenched by pouring onto ice. The reaction mixture was diluted with water and the
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product was extracted out in DCM. DCM layer was evaporated to afford compound 3 as yellow
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solid in quantitative yield.
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1H NMR (500 MHz, MeOD) δ 7.53 (d, J = 8.3 Hz, 2H), 6.95 – 6.75 (m, 3H), 3.98 (s, 2H), 3.54
(q, J = 7.1 Hz, 2H), 3.42 (q, J = 7.1 Hz, 2H), 2.83 (d, J = 7.6 Hz, 3H), 2.64 (s, 3H), 1.32 – 1.18
(m, 3H), 1.13 (t, J = 7.1 Hz, 3H). Figure S4
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13C NMR (126 MHz, MeOD) δ 172.5, 160.27, 156.99, 129.79, 115.12, 100.35 – 98.18, 42.24,
40.70, 27.43, 21.56, 15.86, 12.88, 11.85. Figure S5
ESI-MS: Theoretical for C20H24N4O2 is 352.19, observed is 353.19 [M+1]+ Figure S6
Synthesis of compound 5: To a stirring solution of compound 4 (3 g, 8.1 mmoles) in DMF,
sodium azide (790 mg, 12.15 mmoles) was added and the reaction mixture was heated at 40oC
for 24 hours. Upon cooling to ambient temperature, the reaction mixture was diluted with
ethylacetate and washed with water. The organic layer was dried over sodium sulfate and
evaporated. The crude product was purified via combiflash using 30% ethylacetate in hexanes.
The product was obtained as a transparent liquid. Yield: 60%.
1H NMR (500 MHz, CDCl3) δ 7.82 (d, J = 8.3 Hz, 2H), 7.37 (d, J = 8.1 Hz, 2H), 4.24 – 4.06 (m,
2H), 3.49 (t, J = 5.1 Hz, 2H), 2.46 (s, 3H). Figure S7
13C NMR (126 MHz, CDCl3) δ 145.26, 132.63, 130.00, 128.00, 68.04, 49.62, 21.71, 0.02.
Figure S8
Synthesis of compound 6 (DPA-azide): To a stirring solution of compound 3 (360 mg, 1.02
mmoles) in anhydrous anhydrous tetrahydrofuran (THF, 5mL), potassium carbonate (705 mg,
5.11 mmoles) was added and the stirring was continued for 30 minutes followed by the addition
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of compound 5 (344.5 mg, 1.42 mmoles) in DMF (2mL). The reaction mixture was stirred at
50oC for overnight. Upon completion, the solvent was evaporated. The residue was dissolved in
ethylacetate and washed with water. Column purification was performed to afford the pure
product. Yield: 82%
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1H NMR (500 MHz, CDCl3) δ 7.85 – 7.74 (m, 2H), 7.08 – 6.93 (m, 2H), 6.51 (d, J = 0.5 Hz,
1H), 4.26 – 4.15 (m, 2H), 3.93 (s, 2H), 3.62 (t, J = 5.0 Hz, 2H), 3.51 (q, J = 7.1 Hz, 2H), 3.41 (q,
J = 7.1 Hz, 2H), 2.75 (s, 3H), 2.55 (s, 3H), 1.21 (t, J = 7.1 Hz, 3H), 1.11 (t, J = 7.1 Hz, 3H).
Figure S9
13C NMR (126 MHz, CDCl3) δ 170.03, 158.46, 157.49, 130.11, 127.07, 114.68, 108.18, 100.91,
77.29, 77.04, 76.78, 67.00, 50.21, 42.34, 40.63, 28.19, 24.55, 16.97, 14.38, 13.12, 0.02. Figure
S10
ESI-MS: Theoretical for C22H27N7O2 is 421.22, observed is 422.23 [M+1]+ Figure S11
Synthesis of compound 8: To a stirring solution of compound 7 (800mg, 0.056 mmoles) in
anhydrous DMF (10mL), pentynoic acid (82.44 mg, 0.840 mmoles) was added followed by the
addition of DMAP (108.12mg, 0.885 mmoles) and EDC (169.65mg, 0.885mmoles). The reaction
mixture was stirred at room temperature for 24 hours. This was followed by the dialysis against
DMF and then water. The aqueous solution was lyophilized to yield compound 8 as a
hygroscopic white solid. Yield: 82%
1H NMR (500 MHz, DMSO) δ 8.10-7.62 (D-NH-CO), 4.71 (s, D-OH), 4.03 (t, D-CH2-COO),
3.49-3.22 (m, D-CH2), 3.18-2.94(m, D-CH2), 2.78 (s, linker -C≡CH), 2.82-2.55 (m, D-CH2),
2.45-2.30 (m, D-CH2), 2.29-2.18 (m, D-CH2). Figure S12
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Synthesis of compound 9: To a stirring solution of compound 8 (500 mg, 0.033 mmoles) in
anhydrous DMF (10mL), BOC-GABA-OH (33 mg, 0.166 mmoles) was added followed by the
addition of DMAP (24.1 mg, 0.198 mmoles) and EDC (37.8 mg, 0.198 mmoles). The reaction
mixture was stirred at room temperature for 24 hours. This was followed by the dialysis against
DMF and then water. The aqueous solution was lyophilized to yield compound 9 as a
hygroscopic white solid. Yield: 82%
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1H NMR (500 MHz, DMSO) δ 8.19-7.68 (D-NH-CO), 4.72 (s, D-OH), 4.09-4.34 (m, D-CH2-
COO), 3.53-3.00 (m, D-CH2), 2.80-2.57 (m, D-CH2), 2.47-1.92 (m, D-CH2), 1.62 (t, linker –
CH2), and 1.37 (BOC H). Figure S13
Synthesis of compound 10: A solution of compound 9 (400mg) in 30% trifluoroacetic acid
(TFA) in DCM was stirred vigorously for 12 hours. The solution was co-evaporated with
methanol to remove TFA to afford compound 10 as hygroscopic solid in quantitative yield which
was used directly for next step.
1H NMR (500 MHz, DMSO) δ 8.19-7.64 (D-NH-CO), 4.71 (s, D-OH), 4.09 (m, D-CH2-COO),
4.02 (m, D-CH2-COO), 3.53-3.00 (m, D-CH2), 2.78 (s, linker -C≡CH), 2.85-2.53 (m, D-CH2),
2.46-2.07 (m, D-CH2), 1.72 (t, linker –CH2). Figure S14
Synthesis of compound 11: To a stirring solution of compound 10 (403 mg, 0.026 mmoles) and
compound 6 (165.45 mg, 0.392 mmoles) in DMF/THF mixture, a solution of CuSO4.5H2O
(1mg, 0.004mmoles) in water (1mL) was added. This was followed by the addition of sodium
ascorbate (1.5mg, 0.008 mmoles) in water (1mL). The reaction mixture was left to stir overnight
at 40oC. The aqueous solution was lyophilized to afford compound 11 as white solid. Yield:
83%.
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1H NMR (500 MHz, DMSO) δ 8.05-7.67 (D-NH-CO), 7.65-7.56 (m, DPA ArH), 7.00-6.90 (m,
DPA ArH), 6.77-6.71 (m, DPA ArH), 4.66 (DPA H), 4.35 (DPA H), 4.06-3.94 (m, D-CH2-
COO), 3.79-3.72 (m, linker-CH2), 3.50-3.11 (m, D-CH2), 3.11-2.89 (m, D-CH2), 2.38-2.25 (m,
D-CH2), 2.22-1.99 (m, D-CH2), 1.10 (t, DPA –CH3), 0.93 (t, DPA –CH3). Figure S15
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Synthesis of compound 12 (DPA-D-Cy5): To a stirring solution of compound 11 (193 mg, 0.009
mmoles) in DMF, Cy5-NHS ester (12.13 mg, 0.019 mmoles) was added at pH 7.5. The reaction
mixture was left to stir overnight at room temperature. The solution was then dialyzed against
DMF followed by water. The aqueous solution was lyophilized to afford compound 12 as a blue
solid. Yield: 72%.
1H NMR (500 MHz, DMSO) δ 8.39-8.29 (m, Cy5H), 8.13-7.74 (D-NH-CO), 7.72-7.63 (m, DPA
ArH), 7.34-7.27 (m, Cy5H), 7.04-6.96 (m, DPA ArH), 6.82-6.75 (m, DPA ArH), 6.62-6.52 (m,
Cy5H), 6.32-6.21(m, Cy5H), 4.9-4.56 (m, DPA H), 4.48-4.28 (m, DPA H), 4.05-3.94 (m, D-
CH2-COO), 3.87-3.75 (m, linker-CH2), 3.50-3.19 (m, D-CH2), 3.18-3.00 (m, D-CH2), 2.72-2.57
(m, D-CH2), 2.31-2.011 (m, D-CH2).76-1.62 (Cy5H), 1.44-0.89 (DPA –CH3, linker H and
Cy5H). Figure S16
HPLC: Retention time: 23.1 minutes, Purity: 99.8% Figure S17
Synthesis of compound 13 (D-DPA): To a stirring solution of compound 8 (500 mg, 0.033
mmoles) and compound 6 (165.45 mg, 0.392 mmoles) in DMF/THF mixture, a solution of
CuSO4.5H2O (1mg, 0.004mmoles) in water (1mL) was added. This was followed by the addition
of sodium ascorbate (1.5mg, 0.008 mmoles) in water (1mL). The reaction mixture was left to stir
overnight at 40oC. The aqueous solution was lyophilized to afford compound 13 as white solid.
Yield: 87%.
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1H NMR (500 MHz, DMSO) δ 8.09-7.73 (D-NH-CO), 7.72-7.62 (m, DPA ArH), 7.04-6.98 (m,
DPA ArH), 6.85-6.77 (m, DPA ArH), 4.73 (t, DPA H), 4.43 (DPA H), 4.00 (m, D-CH2-COO),
4.82 (t, DPA H), 3.50 (m, DPA H), 3.48-2.97 (m, linker-CH2), 2.73-2.57 (m, D-CH2), 2.28-2.07
(m, D-CH2), 1.84 (t, linker H), 1.17 (t, DPA –CH3), 1.01 (t, DPA –CH3). Figure S18
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HPLC: Retention time: 24.2 minutes, Purity: 99% Figure S19
Synthesis of D-DPA: The synthetic protocol for the synthesis of DPA-triazole is described in
supplementary section. The 1H and 13C NMR spectra for DPA-triazole are presented in figures
S20 and S21.
Biology studies
Materials
DMEM media, RPMI media, fetal bovine serum (FBS), penicillin-streptomycin (P/S), 0.25%
trypsin-EDTA, NucBlue fixed cell stained, goat anti-rabbit Alexafluor 488, TRIzol, and MTT
reagent were purchased from Invitrogen (Carlsbad, CA, USA). Lysine-coated glass bottom
dishes were purchased from MatTek Inc. (Ashland, MA, USA). Anti-AIF antibody was
purchased from Abcam (Cambridge, UK). Methanol, 4% formalin solution, Triton-X, bovine
serum albumin (BSA), and lipopolysaccharide (LPS) were purchased from Sigma Aldrich (St.
Louis, MO, USA). Mitochondria isolation for mammalian cells kit and normal goat serum (NGS)
were purchased from ThermoFisher (Waltham, MA, USA). Lectin Dylight 594 was purchased
from Vector Laboratories Inc. (Burlingame, CA, USA). Phosphate buffered saline (PBS) and tris
buffered saline (TBS) were obtained from Corning Inc. (Corning, NY, USA).
Mitochondrial isolation
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To assess comparative binding affinities, mitochondria were isolated from kidneys and
adrenal glands harvested from neonatal rabbits. These tissues were chosen because TSPO has
been shown to have high expression in steroid producing tissues such as adrenal glands.68
Mitochondrial isolation was performed using a mitochondria extraction from mammalian tissues
kit as per manufacturer’s procedure (ThermoFisher).
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TSPO binding affinity of D-DPA
Binding of compounds to the TSPO receptor were evaluated using a radiolabeled
membrane binding assay in tandem with size exclusion chromatography. [3H]-PK11195 (Cat#
NET885250UC, Perkin Elmer, Boston, MA) was used as the radioligand and mitochondria, as
isolated above, used as the receptor source. Briefly, mitochondria were incubated with a range of
concentrations of the tested compounds (1 pM – 100 µM) and [3H]-PK11195 (1.2 nM) in 50 mM
Tris.HCl buffer at pH 7.4 and in a total volume of 100 µl for 30 min and at room temperature
(RT). Total binding was determined in the presence of [3H]-PK11195 alone and non-specific
binding was determined in the presence of both [3H]-PK11195 and 100 nM unlabeled PK11195.
At the end of the incubation period, 90 µl of the reaction mixture was transferred to pre-
equilibrated, 7K molecular weight cut off, 96-well, Zeba Spin Desalting plates (Cat# 89807,
Thermo Scientific, Rockford, IL). [3H]-PK11195-bound mitochondria was eluted upon
centrifugation at 1000 x g for 2 min (Beckman GS-6R centrifuge with PTS-200 swinging bucket
rotor) and the radioactivity in 80 µl of the flow through detected using solid scintillator-coated
96-well LumaPlates (Perkin Elmer, Cat# 6005630) in conjunction with Perkin Elmer’s TopCount
instrument. Finally, IC50 values were determined from CPM results of the specific binding using
Microsoft Office Excel, IDBS’ XLfit and GraphPad Prism programs.
Cell culture
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HMC3 human macrophages were acquired from ATCC (Manassas, VA, USA). BV2
murine microglia were obtained from the Children’s Hospital of Michigan’s cell culture facility.
GL261 murine glioblastoma cells were obtained from the DTP/DCTD/NCI Tumor Repository
(Frederick, MA, USA). HMC3 and BV2 cells were maintained in DMEM supplemented with
10% FBS and 1% P/S. GL261 cells were maintained in low glutamine RPMI supplemented with
10% FBS, 1% P/S, and 1% L-glutamine. Cells were kept at 37ºC in 5% CO2 atmosphere.
Treatments were performed in half serum media (5% FBS).
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Mitochondrial colocalization and quantification
Imaging and colocalization of mitochondria with dendrimers were performed according
to previously published protocols.41 Briefly, HMC3 macrophages were seeded on glass bottom
culture dishes. HMC3 cells were used to image mitochondrial targeting due to their large cell
bodies. Cells were treated with fluorescently labeled dendrimer (D-Cy5) and DPA-conjugated
dendrimer (Cy5-D-DPA) for 48 hours at 50 µg/mL. Cells were then fixed in 4% formalin
solution. Cells were stained with rabbit anti-mouse AIF primary antibody (1:200) and anti-rabbit
Alexafluor 488 (1:200) to label mitochondria, Lectin Dylight 594 to label cell membranes, and
DAPI to label cell nuclei.
For quantification of dendrimer content in isolated mitochondria, BV2 murine microglia
were used due to greater yield of isolated mitochondria. Dendrimer quantification from cell
extracts were performed as described previously with modifications for mitochondrial
isolation.69 Cells were treated with 50 µg/mL D-Cy5 or Cy5-D-DPA for 24 hours. Cells were
freshly fractionated to yield isolated mitochondria and cytosolic fractions. Isolated mitochondria
were then resuspended in isolation buffer, and dendrimers were extracted via three freeze/thaw
cycles with liquid nitrogen. Dendrimer fluorescence in each fraction was measured using a
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Shimadzu RF-3501PC spectrofluorophotometer (Shimadzu Corporation, Columbia, MD, USA).
Fluorescence intensities were converted into masses using calibration curves of known
dendrimer concentrations.
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Analysis of inflammatory expression
BV2 murine microglia were stimulated with LPS at 300 EU/mL for 3 hours, followed by
cotreatment with PK11195 or DPA-conjugated dendrimer (D-DPA) with LPS for 24 hours.
Then, cells were exposed to fresh media for 24 hours and collected for analysis of cellular and
extracellular signals. LPS was chosen as a stimulant despite induction of pro-inflammatory
immune response due to its role in mediating cancer cell metastasis, proliferation, and immune
recruitment.41, 42, 70 Cells were collected in TRIzol and mRNA extracted according to
manufacturer’s procedure. cDNA conversion was then performed using a cDNA conversion kit
(ThermoFisher) and thermocycler (Bio-Rad Laboratories, Hercules, CA, USA). Cells were then
analyzed for immune expression using rt-qPCR StepOne Plus system (Applied Biosystems).
Primer sequences used were as follows: TNFα (F: CCA GTG TGG GAA GCT GTC TT; R:
GTG TAA TTA AGC CTC CGA CTT G), IL1β (F: AGC TTC AAA TCT CGA AGC AG; R:
TGT CCT CAT CCT GGA AGG TC), arginase-1 (F: TCA TGG AAG TGA ACC CAA CTC
TTG; R: TCA GTC CCT GGC TTA TGG TTA CC), and GAPDH (F: TGT CGT GGA GTC
TAC TGG TGT CTT C; R: CGT GGT TCA CAC CCA TCA CAA). Cell supernatants were
assessed for TNFα and nitrite secretion using mouse TNFα Quantikine ELISA kit (R&D
Systems, Minneapolis, MN, USA) and Griess reagent assay kit (Promega, Madison, WI, USA)
according to manufacturer’s procedure, respectively. Samples were read on a Synergy Mx
microplate reader (BioTek, Winooski, VT, USA). Cytotoxicity of D-DPA compared to PK11195
was assessed using MTT assay.
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Tumor inoculations
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All animals were housed at the Johns Hopkins University animal facilities and were
given free access to food and water. All animal experiments were conducted in accordance with
protocols approved by the Johns Hopkins University institutional animal care and use committee.
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To establish the GL261 orthotopic immunocompetent model of glioblastoma, male and
female C57BL/6 (Jackson Laboratory, Bar Harbor, ME, USA) mice 6-8 weeks of age were
intracranially implanted with GL261 cells. Cells were brought to a concentration of 100,000 cells
per 2 µL. Mice were anesthetized using a ketamine/xylazine for survival surgeries. A midline
scalp incision was created, and a burr hole was drilled 1 mm posterior to the bregma and 2 mm
lateral to the midline to inject cells into the striatum. A 2 µL Hamilton syringe (Hamilton
Company, Reno, NV, USA) was lowered to a depth of 2.5 and injected 2 µL of cell solution over
10 minutes with an automated syringe pump (Stoelting Co., Wood Dale, IL, USA). The syringe
was withdrawn slowly. The incision was sutured together, and antibiotic cream was applied to
the wound.
To determine TSPO overexpression in this model of glioblastoma, tumors and
contralateral hemisphere tissues were dissected from brains collected 14 days after tumor
inoculation. Tissues were processed for mRNA extraction and rt-qPCR as described above, with
additional agitation initially to dissociate tissues. Premade TSPO primers were obtained from
Bio-Rad.
To assess systemic biodistribution of Cy5-D-DPA compared to D-Cy5, glioblastoma
brain tumor bearing mice were intravenously injected with dendrimers at 55 mg/kg on day 14
post inoculation. Clearance organs (kidney, liver, spleen) were collected 24 hours later after
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perfusion. Organs were dissected into equivalent mass samples and homogenized with stainless
steel beads in a bullet blender homogenizer (Next Advance Inc., Troy, NY, USA) to extract
dendrimers into methanolic solution. Fluorescence was measured using the
spectrofluorophotometer and converted to quantities using calibration curves of known
dendrimer concentration.
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Tissue processing and immunohistochemistry
Glioblastoma brain tumor bearing mice were injected intravenously with D-Cy5 or Cy5-
D-DPA on day 14 post-inoculation. Mice were perfused and brain collected 24 hours after
injection. Brains were fixed in 4% formalin solution, followed by sucrose gradient to remove
residual formalin (10%, 20%, then 30% sucrose in PBS overnight each). Brains were sectioned
axially into 30 µm using a Leica CM 1905 cryostat (Wetzlar, Germany). Brains were stained
with DAPI to label nuclei, Iba1 (1:200) to label TAMs, and AIF (1:200) to label mitochondria.
Slices were blocked in 1x TBS + 0.1% Triton-X + 1% BSA + 5% NGS for 4 hours at room
temperature. Slices were then incubated with primary antibodies diluted in 1x TBS + 0.1%
Triton-X + 1% BSA overnight at 4ºC. Then slices were washed and incubated with secondary
antibodies (goat anti-rabbit 488) for 2 hours at room temperature. Finally, slices were incubated
with DAPI for 15 minutes, mounted, and sealed.
Confocal imaging
Images were acquired using a Zeiss LSM710 confocal microscope (Hertfordshire, UK).
Background fluorescence in cells and tissues were set with untreated samples. Microscope
settings such as laser intensity, gain, and offset were kept constant across compared images.
Zenlite 2011 software was used to process the obtained images, and any adjustments to
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brightness and contrast were kept constant across all compared images. Colocalization and
fluorescence profiles were obtained with the Zenlight 2011 software.
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Statistical analyses
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Graphs and statistical analyses were performed using GraphPad Prism v8.0 software (San
Diego, CA, USA). Error bars presented represent mean ± standard errors. Statistical significance
between colocalization coefficients was determined with Student’s t-test. Significances in
comparisons between PK11195 and D-DPA were performed with two-way ANOVAs.
Results
Synthesis of the dendrimer-DPA conjugate and intermediates
To enable mitochondria targeting within TAMs in glioblastoma, DPA was conjugated to
generation 4 hydroxyl-terminated polyamidoamine dendrimers via non-cleavable linkages.
Chemical conjugation of DPA on the surface of dendrimer is challenging due to the absence of
functional groups that can be easily modified to attach a linker. To address this issue, we first
synthesized a conjugable form of DPA (Figure 1A) with azide terminal group separated by a
two carbon linker. This served two purposes, i) the presence of an azide terminal group
participated in copper (I) catalyzed alkyne-azide click (CuAAC) reaction on dendrimer surface,
and ii) the resulting aromatic triazole ring with the two carbon spacer for enhanced TSPO
binding affinity, as modifications with phenethyl ether side-chains have resulted in DPA analogs
with highest TSPO binding affinities.27 The synthesis of DPA-azide began with the condensation
of compound 1 with 2,4 pentanedione to generate pyrazolopyrimidine derivative 2, which on
subsequent cleavage of methoxy group with borontribromide afforded phenolic derivative 3. The
two carbon orthogonal linker was synthesized by mono-azidation of ethyleneglycol ditosylate 4
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to afford linker 5. The phenolic intermediate 3 was subsequently reacted with 5 using potassium
carbonate and mild heating to afford clickable version of DPA as DPA-azide (6). To enable
fluorescent imaging of DPA-conjugated dendrimers, the dendrimer surface was functionalized
with two orthogonal functional groups to obtain a trifunctional dendrimer for the simultaneous
conjugation of a cyanine 5 (Cy5) fluorescent probe and DPA (Figure 1B). Using simple
esterification reactions, dendrimers (7) were first modified with pentynoic acid to bring alkyne
groups to enable click reaction with azide-terminated DPA (6). The alkyne-terminating
dendrimer 8 was then modified in two steps by first reacting with GABA-BOC-OH followed by
BOC deprotection in mild acidic conditions, to afford amine groups to enable conjugation to
Cy5-NHS ester, resulting in trifunctional dendrimer with two orthogonal functional groups for
further attachment (10). The alkyne groups in trifunctional dendrimer 10 were further reacted
with DPA-azide (6) via CuAAC click reaction to afford dendrimer 11, which was subsequently
reacted with Cy5-NHS via activated acid-amine coupling reaction to obtain the final
fluorescently labeled dendrimer-DPA conjugate (12). For binding and efficacy studies, a non-
fluorescently labeled dendrimer-DPA conjugate was synthesized by simply reacting alkyne-
terminating dendrimer 8 with DPA-azide (6) to afford D-DPA (13, Figure 1C). We conjugated
only ~9 molecules of DPA (19%w/w) on the surface of hydroxyl dendrimer to keep the inherent
biodistribution and targeting potential of the parent dendrimer intact. In addition, for comparison
purpose, we also synthesized a DPA-triazole analogue by reacting DPA-azide with propargyl
alcohol (Scheme S1).
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A. Synthetic route to clickable DPA-azide
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OH
CH
3C(O)CH2C(O)CH3
O
N
N
N
NH
N
BBr
3, DCM
EtOH, Microwave,
N
O
H2N
N
N
-60 oC to RT
O
O
2
3
O
NEt2
1
NEt2
NEt2
O
S
O
NaN
3, DMF
O
, THF, 50
O
K2CO3
oC
S
O
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N3
O
S
O
O
0 oC to RT
5
O
4
O
N
N
N3
N3
DPA
N
6
DPA-N3
O
NEt2
B. Synthetic route to Cy5-D-DPA
OH
GABA-BOC-OH
EDC, DMAP
OH
O
O
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OH
55
O
O
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D
OH 64
O
D
D
O
O
EDC, DMAP
7
DMF, RT, 24 hr
8
9
DMF, RT, 24 hr
NHBOC
9
RT, 12 hr
TFA
Click: CuSO4.5H2O
NaAscorbate
N
N
OH
O
Et2N
O
OH
O
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N3
O
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O
N3
DPA
9
N
D
D
O
O
6
O
O
N
DMF/THF/H2O
N
NH2
N
NH2
40 o
9
11
C, 18 hr
Trifunctional dendrimer
10
Cy5 NHS ester
DMF, 24 hr, RT
Et2N
N
N
O
OH
54
O
N
N
N
D
O
O
OH
O
O
54
N
O
O
N
O
D
DPA
H
O
O
N
N
N
9
9
NH
Cy5
N
O
SO3
DPA-D-Cy5 (12)
N
1
12
O3S
C. Synthetic route to D-DPA
N
Et2N
O
O
55
OH
N
N
OH
N3
DPA
6
55
O
D
D
O
O
Click: CuSO4.5H2O
NaAscorbate
DMF/THF/H2O
O
N
9
8
N
N
9
40 o
C, 8 hr
D-DPA (13)
Figure 1. Synthesis of DPA derivative and dendrimer conjugates. Schematic representation of
the synthesis pathways for A) conjugable azide-terminated DPA derivative (6, DPA-N3), B) Cy5
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fluorescently labelled dendrimer-DPA conjugate (12, Cy5-D-DPA), and C) dendrimer-DPA (13,
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D-DPA).
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The structures of intermediates, DPA-triazole analogue, and the final dendrimer-DPA
conjugates were characterized using 1H NMR, mass spectroscopy and HPLC (Figures S1-S21).
The 1H NMR spectra of trifunctional dendrimer 10 (Figure 2 A, green spectrum) exhibited
characteristic ester methylene protons from two linkers at δ 4.90 and 4.02 ppm. The comparative
integration of protons from dendrimer internal amides at δ 8.19-7.64 ppm with ester methylene
protons suggested the attachment of on an average ~9 alkyne linkers and ~2 amine terminating
linkers. The success of the click reaction between DPA-azide and trifunctional dendrimer was
analyzed via 1H NMR by comparing the spectra of the resulting product (blue spectrum) with the
starting materials (red and green spectra), showing the characteristic dendrimer peaks along with
the DPA aromatic peaks in between δ 7.65-7.56, 7.00-6.90, and 6.77-6.71 ppm and aliphatic
methyl protons at δ 1.10 and 0.93 ppm. Finally, the success of cy5 conjugation was evident from
the Cy5 protons in the aromatic region (magenta spectrum). The purities of the final conjugates
(D-DPA and Cy5-D-DPA) were >99% as analyzed by HPLC (Figure 2B, Figures S17 and
S19). Conjugation of DPA to the dendrimer surface resulted in a shift in retention time from 19.9
minutes for trifunctional dendrimer to 23.1 minutes for Cy5-D-DPA (Figure 2B). DLS and zeta
potential measurements revealed a hydrodynamic radius of 4.8 nm (Figure 2C and D) and ζ-
potential of +3.6 mV (Figure 2D and E).
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Figure 2. Chemical characterization of intermediate products and final conjugates. A)
Comparative 1H NMR spectra of fluorescently labelled dendrimer-DPA conjugate (Cy5-D-
DPA), dendrimer-DPA (D-DPA), DPA-azide, and trifunctional dendrimer. Characteristic proton
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signals are labelled. B) Comparative HPLC chromatogram of intermediates and final conjugate.
C) Size distribution of D-DPA measured using dynamic light scattering (DLS). D) A table
showing physicochemical characterization of D-DPA. E) Zeta potential distribution of D-DPA
measured by DLS.
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TSPO binding affinity of dendrimer-DPA conjugates
To evaluate whether D-DPA was active as a TSPO ligand, TSPO binding affinity in
mitochondria was measured for D-DPA and was compared to free PK11195, DPA-triazole, and
empty dendrimer (D-OH) (Table 1). The half-maximal inhibitory concentration (IC50) of D-
DPA was 70nM +/- 4, approximately 14-fold less potent relative to free PK11195 (5nM +/- 0.5)
and ~1.4-fold more potent than the DPA-triazole analogue (100nM+/-20). The negative control
empty dendrimer (D-OH) displayed no binding affinity.
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Table 1. Comparative half-maximal inhibitory concentration (IC50) of PK11195, DPA-triazole
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monomer, D-DPA and PAMAM-D-OH affinity to TSPO receptor.
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DPA conjugation to dendrimer enables mitochondrial targeting
Before proceeding with in vitro experiments, we first assessed the potential cytotoxicity
of PK11195 and D-DPA via cell viability measurements. BV2 murine microglia were exposed to
PK11195 or D-DPA over a concentration range of 1-1000 µg/mL for 24 hours. Consistent with
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previous in vitro studies,43, 44 PK11195 demonstrated significant dose-dependent toxicity, with
the 1000 µg/mL dose reducing cell viability to ~5% of controls (Figure S22). In contrast, D-
DPA exhibited significantly less cytotoxicity (p < 0.0001 PK11195 vs. D-DPA), with the 1000
µg/mL dose exhibiting ~60% cell viability. PK11195 has been shown to induce apoptosis via
interference with mitochondrial permeability pores, so the slightly weaker binding affinity by D-
DPA may contribute to the significantly reduced cytotoxicity.45 This indicates that dendrimer
delivery of a TSPO ligand may ameliorate cytotoxic effects for more effective and safer
therapies.
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Previous in vivo dendrimer work has shown that dendrimers selectively target TAMs in
gliomas.33 To explore the mitochondria targeting capabilities of DPA-conjugated dendrimers,
HMC3 human macrophages were exposed to Cy5-D-DPA for 48 hours at 50 µg/mL. Cells were
then fixed and stained to label nuclei, cell membranes, and mitochondria. Cy5-D-DPA exhibited
highly punctated signal corresponding to the mitochondria, as shown by the yellow signal
indicating overlap between dendrimer (red) and mitochondria (green) signals (Figure 3A). In
contrast, unmodified dendrimer (D-Cy5) exhibits diffuse perinuclear signal consistent with what
we have observed previously in vitro and in vivo.46 While some overlap between dendrimer and
mitochondrial signal is observed with D-Cy5, this appears to arise from the broad, cytosolic
signal pattern of D-Cy5 rather than specific interactions with mitochondria. Representative
fluorescence line profiles through cells show that dendrimer and mitochondria signal correlate
closely with Cy5-D-DPA, whereas D-Cy5 exhibits regions of mitochondrial signal without
corresponding dendrimer signal and vice versa (Figure 3B). Semi-quantitative analysis shows
that conjugation of DPA to the dendrimer increases its colocalization coefficient by ~2-fold
(Figure 3C, p < 0.0001).
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Figure 3. Conjugation of DPA to dendrimers enables targeting of mitochondria. HMC3
human macrophages were treated with fluorescently labelled dendrimer (red) with (Cy5-D-DPA)
or without (D-Cy5) DPA conjugation. Following 48 hours of exposure cells were fixed and
stained with DAPI to label cell nuclei (blue) and AIF to label mitochondria (green) for confocal
imaging. A) Conjugation of DPA to dendrimers improves mitochondrial localization, as
indicated by the yellow regions signifying colocalization of the green mitochondria and red
dendrimer signal. D-Cy5 exhibits diffuse perinuclear signal. B) Representative fluorescence line
profiles through cells demonstrates the close association between Cy5-D-DPA and mitochondrial
signal, whereas D-Cy5 signal does not exhibit such correspondence. C) Cy5-D-DPA exhibits
significantly greater colocalization coefficient with mitochondrial signal than D-Cy5. *** p <
0.001.
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To further explore the mitochondria targeting properties of D-DPA, BV2 murine
microglia were treated with unmodified or DPA-conjugated dendrimers for 24 hours. BV2
microglia were chosen due to their higher yield of isolated mitochondria compared to HMC3
macrophages. Cells were then fractionated to yield mitochondrial and cytosolic fractions. Cy5-
D-DPA exhibited significantly greater partitioning to the isolated mitochondrial fraction
compared to D-Cy5 (Figure S23A, p = 0.0021). However, total cellular internalization was not
changed (Figure S23B, p = 0.859), indicating that dendrimer uptake properties were preserved
with DPA conjugation, consistent with previous studies where up to 20 wt% loading of
conjugated therapies did not interfere with dendrimer interactions with cells.47
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Dendrimer-DPA promotes anti-tumor immune signaling
To evaluate the impact of PK11195 or D-DPA treatment on modulating TAMs-like
phenotype, markers of immune activation were assessed after treatment in LPS stimulated BV2
murine microglia. LPS was chosen due to its role in promoting tumor immune recruitment,
metastasis, and proliferation.48 D-DPA treatment (100 μg/mL on a DPA loading basis)
significantly increased secretions of TNFα by activated microglia ~2-fold compared to PK11195
treatment (100 μg/mL) (Figure 4A). D-DPA treatment similarly upregulated expression of anti-
tumor, pro-inflammatory cytokines TNFα and IL1β ~3-fold compared to PK11195 treatment
(Figure 4C and D). Notably, both PK11195 and D-DPA treatment did not upregulate anti-
inflammatory signal Arg-1 (Figure 4E). Both PK11195 and D-DPA did decrease secretion of
reactive oxygen species (Figure 4B), which are implicated in tumor recruitment of TAMs,
tumorigenesis via inducing DNA damage, and tumor proliferation.49, 50 These results indicate
that in the context of TAMs-like immune activation, D-DPA exerts TSPO agonism activity to
promote pro-inflammatory, anti-tumor immune signaling while reducing oxidative stress.
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Figure 4. D-DPA treatment increases anti-tumor inflammatory signals. BV2 murine
microglia were stimulated with LPS at 300 EU/mL for 3 hours, followed by cotreatment with
PK11195 or DPA-conjugated dendrimer (D-DPA) for 24 hours. Then, cells were exposed to
fresh media for 24 hours and collected for analyses of cellular and extracellular signals. A)
Treatment with D-DPA significantly increases the secretion of TNFα, a tumor-killing signal,
from stimulated BV2 microglia compared to PK11195 treatment. ** p < 0.01. B) Both
treatments reduced secretion of nitrite, a reactive oxygen species indicative of oxidative stress.
Analyses of mRNA expression via rt-qPCR demonstrates that D-DPA significantly increases
expression of anti-tumor signals C) TNFα and D) IL1β while limiting expression of E) pro-
tumor cytokine arginase-1 (Arg1). *** p < 0.001.
Systemically administered dendrimer-DPA targets mitochondria specifically within TAMs in vivo
Based on these promising in vitro results, we proceeded to evaluate in vivo targeting
properties of the dendrimer-DPA conjugate in an orthotopic, immunocompetent model of
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glioblastoma. First, to validate the rationale for TSPO targeting in this model of glioblastoma, we
compared expression of TSPO within and outside the brain tumor. TSPO was significantly
upregulated in the tumor ~5-fold compared to the contralateral hemisphere (Figure S24, p <
0.0001). In addition, we also examined the systemic biodistribution of Cy5-D-DPA compared to
unmodified D-Cy5. TSPO has been shown to exhibit high expression in kidneys, livers, and
spleens,20, 51 creating the potential for off-target Cy5-D-DPA accumulation. Organs were
collected 24 hours after systemic injection, and dendrimers were extracted and quantified using
fluorescence spectrometry. Cy5-D-DPA exhibited similar levels in kidney, liver, and spleen
compared to D-Cy5 (Figure S25). This indicates that Cy5-D-DPA is not interacting with TSPO
expressed in peripheral organs for increased accumulation. Therefore, the dendrimer transport
properties dominate, and DPA interactions with TSPO arise only once dendrimers have carried
DPA into the intracellular space.
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Cy5-D-DPA was intravenously injected into mice with orthotopic glioblastoma brain
tumors 14 days after tumor inoculation. Brains were collected 24 hours after injection, fixed, and
stained for confocal imaging. Imaging in the tumor core indicated that Cy5-D-DPA fully
penetrated the solid brain tumor upon systemic administration and targeted mitochondria within
the tumor (Figure 5, white arrows). This signal contrasts with our previously observed diffuse,
cytosolic signal pattern in vivo in TAMs of unmodified dendrimers, indicating that the addition
of TSPO ligand DPA confers mitochondrial targeting.38 Unlike in the in vitro images, nuclear
targeting of Cy5-D-DPA was not observed. Notably, green signal corresponding to mitochondria
were observed throughout the tumor, but Cy5-D-DPA only exhibited cellular signal in specific
cells.
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