Photodynamic therapy (PDT) is an established therapeutic modality for the treatment of a variety of premalignant and malignant diseases [1
]. It utilizes a photosensitizer activated by light to create irreversible photodamage to tumor tissues [3
]. Compared with traditional therapeutic methods, such as chemotherapy, radiotherapy, and surgery, PDT has the advantages of minimal invasiveness and extremely low systemic toxicity [5
]. Furthermore, PDT agents can act as fluorescent probes for in vivo cancer diagnoses and presentations, naturally combining fluorescence imaging and cancer therapy in a “see and treat” manner [6
]. Recently, it was also found that PDT can induce strong antitumor immune responses and thus contribute to the reduction of the residual tumor burden following surgical resection of tumors [8
]. For these reasons, PDT has emerged as an important therapeutic option for the management of cancers.
The enrichment of photosensitizers at tumor sites is very important for improving the treatment effect of PDT [10
]. It can increase the photosensitizer concentration in tumor sites, which is beneficial for improving the photodynamic killing ability and thus is very important for the treatment of large or deep-seated tumors [11
]. What is more, considerable damage occurs to the surrounding normal tissues if the photosensitizers that are used lack adequate tumor selectivity, because it is always difficult to precisely distinguish tumors from normal tissues by using precise light illumination because of the complex tumor infiltration in normal tissues [12
]. Because of this nonselectivity, the fluorescence detection capability is also detrimentally influenced. Based on this, much research has been focused on developing strategies to enhance the enrichment of photosensitizers at tumors sites.
The conjugation between photosensitizers and tumor homing ligands, such as monoclonal antibodies, epidermal growth factors, or peptide ligands, is a promising strategy for optimizing the tumor targeting of PDT agents [13
]. For example, Yamagaci et al. synthesized tumor-targeting photosensitizers by conjugating photosensitizer IR700 with antibody molecules, and the synthesized conjugates showed excellent tumor selectivity and enrichment towards receptor-positive tumors [15
]. The improved tumor enrichment using this strategy considerably improved the outcome of PDT, as demonstrated in the tumor treatment in a mouse model, and currently, they are undergoing advanced clinical trials, which represents a very promising and powerful method of cancer therapy. To overcome the difficulties in using large proteins and antibodies as targeting vehicles, conjugating with various tumor-specific small molecule ligands (e.g., short peptides or peptidomimetics) would provide another promising strategy because of the unique advantages of small molecule ligands [16
]. For example, unlike the obtained antibody conjugates, which are always complex mixtures because of random site conjugation [17
], small molecule ligands may easily afford conjugates with a single structure. Second, conjugates with small molecule ligands have a relatively short circulation half-life, which is beneficial for reducing the phototoxic side effects to the skin and eye that are otherwise due to the long-term presence of photosensitizers in the body. Furthermore, the stability and preparation costs of conjugates with small molecule ligands are also important issues for application. Thus, the development of photosensitizers conjugated with small molecule ligands has been extensively studied [18
However, compared with antibody molecules, small molecule ligands have a much weaker affinity to their receptors, which makes their tumor enrichment not always ideal. In our former work, we coupled a very promising photosensitizer, pyropheophorbide-a (Pyro), which has ideal photosensitizer properties, with a small molecule tumor homing ligand—cyclo(-RGDfK-)—to prepare a tumor-targeted conjugate [22
]. The cyclic peptide cyclo(-RGDfK-), which contains a conformationally restrained RGD sequence, has been often used for targeting tumor imaging and/or therapeutic agents as a high affinity ligand for the αv
integrin receptor [23
]. This conjugate showed improved tumor enrichment compared to free Pyro, thus greatly improving the PDT outcome against tumors in a mouse model and destroying implanted tumors with only one or two treatments of PDT. However, the tumor enrichment capability of this conjugate is still not very good because the cyclo(-RGDfK-) ligand only has a moderate affinity to the αv
integrin receptor compared with antibody molecules. Improving the affinity of small molecule ligands is very critical for improving the tumor enrichment of small molecule ligand-based conjugates and is thus very important for improving their clinical application potential.
To improve the affinity of RGD ligand-coupled Pyro conjugates for αv
integrin targeting, we decided to synthesize multimeric conjugates with a polyvalent RGD sequence that can bind simultaneously to several integrins. Multimeric ligands have a higher receptor binding affinity and better tumor retention because the polyvalence results in enhanced binding and steric stabilization [32
]. Multivalent interactions are thought to be a very practical strategy for amplifying weak ligand–receptor interactions that are more biologically relevant, thus affording more effective ligands with better targeting capability. For example, Chen [34
] and Ryppa [35
] et al. constructed conjugates of paclitaxel with multivalent RGD peptides, and the conjugates showed enhanced selective killing of cancer cells, excellent integrin specific accumulation and very good tumor/background contrast in vivo. However, until now, not much attention has been paid to preparing the conjugates of photosensitizers with multimeric ligands. To this end, we hope to adopt a multivalence strategy to improve the tumor enrichment capability of the RGD-coupled Pyro photosensitizer to enhance its clinical application potential.
3.1. Instruments and Materials
Fmoc-Asp-OAll, Fmoc-Dphe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, N-hydroxybenzotriazole (HOBt), and 2-(1H-benzotriazol-1-YL)-1.1.3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from GL Biochem Ltd. (Shanghai, China). Phenylsilane, triisopropylsilane (Tis), tetratriphenylphosphine palladium (Pd(PPh3)4), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA), and N,N-dimethylformamide (DMF) were purchased from Heowns (Tianjin, China). 1H and 13C NMR spectra were recorded in CDCl3 or in D2O solutions on a Bruker AV400 or AV300 spectrometer (Bruker Bioscience, Billerica, MA, USA.) The Cary 5000 spectrophotometer (Varian Co., Palo Alto, CA, USA) was used for UV–Vis spectra. Fluorescence spectra were recorded using a Hitachi model F-4500 FL spectrophotometer (Tokyo, Japan). Mass data were obtained using a Varian 7.0 T FTMS. High-performance liquid chromatography (HPLC) (Shimadzu, Japan) was used for the analysis and purification of the conjugates. In vivo imaging analysis was performed using an IVIS Lumina imaging system (IVIS Lumina II, Xenogen, Alameda, CA, USA). Human glioblastoma U87-MG cells were purchased from a typical culture preservation commission cell bank of the Chinese Academy of Sciences (Shanghai, China) and were grown in DMEM culture medium, which contained L-glutamine, 10% fetal bovine serum (FBS), and 1% Pen/Strep (10 000 U penicillin, 10 mg streptomycin). BALB/c nude mice (male and female, 7−8 weeks old) were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. Animal studies were conducted in accordance with animal ethic guidelines and ethic approval (Number 20180002) by ethic committee on animals of Nankai University (Tianjin, China) which was abtained prior the study.
3.2. Solid-Phase Synthesis of Side Chain-Protected Cyclic RGD Pentapeptide Cyclo(-Arg[Pbf]-Gly-Asp[tBu]-D-Phe-Asp-)
The syntheses were manually performed in a sealed tube using a solid-phase strategy. 2-chlorotrityl chloride resins (8.0 g) were swollen in dichloromethane for 1 h prior to synthesis. Fmoc-Asp-OAll (949 mg, 2.4 mmol, 0.3 mmol/g loading) and DIPEA (2.0 mL, 5.0 mmol) were dissolved in 30 mL of dichloromethane and added to the resin. The mixture was agitated for 5 h. Afterwards, a sealing agent (CH2Cl2:MeOH:DIPEA = 17:1:2, 20 mL) was used to seal the unreacted chlorine group for 30 min. The Fmoc protecting group was removed by treatment with 20% piperidine in DMF (20 mL) for 30 min. Coupling reactions of the other four amino acids (Fmoc-D-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, and Fmoc-Arg(Pbf)-OH) were performed using 2 equiv of a Fmoc-protected derivative and activated in situ with HBTU (2.0 equiv), HOBt (1.5 equiv), and DIPEA (5 equiv) in DMF for 12 h. Excess reagents were washed five times with DMF for each coupling step.
After the final Fmoc deprotection, the resin was treated with PhSiH3 (24 equiv) and Pd(PPh3)4 (0.3 equivalents) in dichloromethane for 1 h under N2 protection for selective alpha-carboxyl deprotection of the allyl groups. Cyclization between α-NH2 of Arg and α-CO2H of Asp was conducted overnight in DMF using PyBOP (2 equiv)/HOBt (1.5 equiv)/DIPEA (5 equiv). The resin was washed with DMF and dichloromethane before it was dried under vacuum to afford the resin-bound cyclic RGD pentapeptide.
The peptide was cleaved from the resin by treating the dried resin with 5 mL of 1% TFA at room temperature 5 times, and excess TFA was neutralized with DIPEA. The mixture was concentrated under reduced pressure, and the solid was washed with dry diethyl ether (1.5 mL) three times. Then, the mixture was purified by column chromatography (silica gel, CH2Cl2-MeOH, 10:1) to afford pure peptapeptide 1 (57 mg, 42%). HRMS-ESI: m/z = 899.3956, calcd for C42H59N8O12S m/z = 899.3973 [M + H]+.
3.3. Synthesis of Amino-Modified Cyclic Pentapeptide Cyclo(-Arg[Pbf]-Gly-Asp[tBu]-D-Phe-Asp[PEG-amine]-)
Peptapeptide 1 (44 mg, 0.050 mmol, 1.0 eq), HOBt (10 mg, 0.075 mmol, 1.5 eq), HBTU (38 mg, 0.10 mmol, 2.0 eq), and DIPEA (41 µL, 0.25 mmol, 5.0 eq) were dissolved in 500 µL of DMF, and then 4,7,10-trioxa-1,13-tridecanediamine (220 µL, 1.0 mmol, 20 eq) was added. The mixture was stirred overnight at r.t. The reaction mixture was precipitated with diethyl ether, and the solid was dissolved in dichloromethane. The organic layer was washed with water and dried over Na2SO4. After concentration, the mixture was purified by column chromatography (silica gel:CH2Cl2-MeOH, 10:1) to produce amino-modified cyclic pentapeptide 2 as a light yellow solid in an 81% yield (44 mg, 0.041 mmol). ESI-HRMS: m/z = 1101.5633, calcd for C52H81N10O14S m/z = 1101.5654 [M + H]+.
3.4. Synthesis of Monomeric RGD Conjugate Pyro-MonoRGD
Pyro (4.9 mg, 0.009 mmol, 1.0 eq), HOBt (1.8 mg, 0.014 mmol, 1.5 eq) and EDC (3.5 mg, 0.018 mmol, 2.0 eq) were dissolved in 150 µL of DMF. DIPEA (7.4 µL, 0.045 mmol, 5.0 eq) and amino-modified pentapeptide 2 (10 mg, 0.009 mmol, 1.0 eq) were added. The mixture was reacted overnight in the dark. The reaction mixture was precipitated with diethyl ether. The solid was dissolved in dichloromethane and washed with water. The organic layer was dried (MgSO4), concentrated under reduced pressure, and purified via a silica gel column (CH2Cl2/MeOH = 15:1) to afford condensed compound 3 in an 80% yield (12.0 mg, 7.2 µmol). Deprotection was performed with 100 µL of the cleavage solution (TFA/Tis/water = 95:2.5:2.5) for 40 min at room temperature. The product was precipitated, washed three times with ethyl ether (0.5 mL), and purified with HPLC to produce Pyro-MonoRGD in a 71% yield (6.7 mg, 5.1 µmol). ESI-HRMS: m/z = 1309.6694, calcd for C68H89N14O13 m/z = 1309.6734 [M + H]+.
3.5. Synthesis of Tert-butyl (1,3-dihydroxypropan-2-yl)carbamate
2-Amino-1,3-propanediol (1.62 g, 17.8 mmol, 1.0 eq) was dissolved in 60 mL of tetrahydrofuran/H2
O (1/1). Di-tert-butyl dicarbonate (Boc2
O) (5.82 g, 26.7 mmol, 1.5 eq) and K2
(6.15 g, 44.5 mmol, 2.5 eq) were added. The reaction was stirred at room temperature overnight. The reaction system was allowed to stand for stratification, and the aqueous phase was extracted three times with tetrahydrofuran. The organic phase was combined, dried over anhydrous Na2
, evaporated in vacuo, and purified by column chromatography (silica gel:petroleum ether/ethyl acetate, 1:1) to produce compound 4
as a white solid in a 62% yield (2.1 g, 11.0 mmol). 1
H-NMR (400 MHz, CDCl3
) δ 5.24 (s, 1H), 3.81 (qd, J
= 11.1, 4.3 Hz, 4H), 3.69 (s, 1H), 2.50 (dd, J
= 22.1, 14.7 Hz, 2H), 1.46 (s, 9H), which agrees with published data [39
3.6. Synthesis of Di-tert-butyl-3,3′-((2-((tert-butoxycarbonyl)amino)propane-1,3-diyl)bis(oxy))dipropionate
Compound 4 (200 mg, 1.05 mmol, 1.0 eq) was dissolved in 500 µL of DMSO and treated with 5.0 M NaOH (21 µL, 0.105 mmol, 0.1 eq). Afterwards, tert-butyl acrylate (440 µL, 3.03 mmol, 2.9 eq) was added, and the solution was stirred at room temperature overnight. The reaction solution was diluted with ethyl ester, washed with H2O and then with saturated brine, and dried over anhydrous Na2SO4. After evaporation, the residue was purified by column chromatography (silica gel, petroleum ether/ethyl ester, 8:1), and compound 5 was obtained as a colorless oil in a 58% yield (273 mg, 0.61 mmol). 1H-NMR (400 MHz, CDCl3) δ 4.98 (d, J = 7.2 Hz, 1H), 3.81 (s, 1H), 3.71 – 3.60 (m, 4H), 3.52 (dd, J = 9.0, 3.5 Hz, 2H), 3.42 (dd, J = 9.3, 6.2 Hz, 2H), 2.45 (t, J = 6.3 Hz, 4H), 1.44 (s, 18H), 1.42 (s, 1H). 13C NMR (100.6 MHz, CDCl3): δ 170.89, 155.46, 80.53, 79.19, 77.38, 77.07, 76.75, 69.21, 66.81, 49.46, 36.27, 28.36, 28.08. LC-MS: m/z = 448.5, calcd for C22H43NO8 m/z = 448.3 [M + H]+.
3.7. Synthesis of Dimeric Linker 3,3′-((2-Aminopropane-1,3-diyl)bis(oxy))dipropionic acid hydrochloride
Compound 5 (134 mg, 0.30 mmol, 1.0 eq) was dissolved in 0.5 mL of dichloromethane, and hydrochloride in ethyl ether (2.8 M, 3 mL) was added. The reaction was allowed to proceed for 20 h at room temperature and then concentrated under reduced pressure. Dimeric linker 6 was obtained as a white solid in a 93% yield (75 mg, 0.28 mmol). 1H-NMR (300 MHz, D2O) δ 3.93–3.76 (m, 6H), 3.73 (dd, J = 9.4, 6.1 Hz, 3H), 2.73 (t, J = 5.8 Hz, 4H). 13C NMR (100.6 MHz, D2O): δ 176.33, 67.21, 66.50, 50.54, 34.23. LC-MS: m/z = 236.4, calcd for C9H18NO6 m/z = 236.1 [M + H]+.
3.8. Synthesis of Pyro-Conjugated Dimeric Linker 7
A mixture of Pyro (32 mg, 0.06 mmol, 1.0 eq), NHS (70 mg, 0.6 mmol, 10 Equation), and EDC (115 mg, 0.6 mmol, 10 eq) was stirred in DMF (5 mL) at r.t. for 20 h without light. The reaction mixture was diluted with ethyl acetate, washed with water and saturated salt water, and dried with Na2SO4. After evaporation, the activated ester was obtained. The activated ester (10 mg, 0.016 mmol, 1.0 eq) was dissolved in 300 µL of DMF, and compound 6 (5 mg, 0.019 mmol, 1.2 eq) and DIPEA (13 µL, 0.08 mmol, 5.0 eq) were added. The reaction solution was diluted with water, the pH was adjusted with 1.0 M HCl, and then it was extracted with EtOAc. The organic layer was combined and evaporated, and the residue was purified via silica gel (DCM/MeOH = 10:1) to produce Pyro-conjugated dimeric linker 7 as a black solid in a 92% yield (11.0 mg, 0.055 mmol). 1H-NMR (300 MHz, CDCl3) δ 9.23 (s, 1H), 9.15 (s, 1H), 8.43 (s, 1H), 8.00 (s, 3H), 7.86 (dd, J = 17.8, 11.5 Hz, 1H), 6.64 (d, J = 8.1 Hz, 1H), 6.26–6.07 (m, 2H), 5.47 (d, J = 20.3 Hz, 1H), 5.05 (d, J = 20.2 Hz, 1H), 3.47 (s, 3H), 3.33 (s, 3H), 3.14 (s, 3H), 2.96 (s, 8H), 2.89 (d, J = 0.6 Hz, 9H), 2.65 (s, 1H), 2.07 (s, 1H), 1.84 (d, J = 7.2 Hz, 3H), 1.63 (t, J = 7.6 Hz, 3H), 1.27 (s, 3H). HRMS-ESI: m/z = 752.3651, calcd for C42H50N5O8 m/z = 752.3659 [M + H]+.
3.9. Synthesis of Conjugate Pyro-DiRGD
Pyro-conjugated dimeric linker 7 (2.3 mg, 0.003 mmol, 1.0 eq), HOBt (1.8 mg, 0.013 mmol, 4.4 eq), and HBTU (5.0 mg, 0.013 mmol, 4.4 eq) were dissolved in 50 µL of DMF. Then, DIPEA (5.5 µL, 0.033 mmol, 11 eq) and amino-modified RGD peptide 2 (8.0 mg, 0.0073 mmol, 2.4 eq) were added. The mixture was reacted overnight in the dark. The reaction mixture was precipitated with diethyl ether. The solid was dissolved in dichloromethane and washed with brine. The organic layer was dried (MgSO4), concentrated under reduced pressure, and purified via silica gel (DCM/MeOH = 10:1) to afford protected conjugate 8 in a 72% yield (6.5 mg, 0.002 mmol). Deprotection was performed with 100 µL of the cleavage solution (TFA/Tis/water = 95:2.5:2.5) for 40 min at room temperature. The product was precipitated, washed three times with anhydrous ether (0.5 mL) and purified with HPLC to produce Pyro-DiRGD in a 67% yield (3.2 mg, 1.4 µmol). HRMS-ESI: m/z = 1151.0880, calcd for C112H159N25O28 m/z = 1151.0893 [M + 2H]2+/2.
3.10. Synthesis of Tris((2-(tert-butoxycarbonyl)ethoxyl)methyl)methylamine
Tris(hydroxymethyl)aminomethane (2.4 g, 0.02 mol, 1.0 eq) was dissolved in DMSO (4 mL) and treated with NaOH (0.4 mL, 5 M). Afterwards, tert-butylacrylate (9.0 g, 0.07 mol, 3.5 eq) was added. The opaque solution was stirred at room temperature for 20 h. Water was added to the reaction mixture, and it was extracted with dichloromethane three times and then washed with brine. The organic layer was dried (Na2
), concentrated, and purified by silica gel column chromatography (petroleum ether/ethyl ester, 1:2) to afford compound 9
as a pale yellow oil in a 38% yield (3.86 g, 7.63 mmol). 1
H-NMR (400 MHz, CDCl3
): δ 3.60 (t, J
= 6.4 Hz, 6H), 3.27 (s, 6H), 2.41 (t, J
= 6.4 Hz, 6H), 1.40 (s, 27H), which agrees with the published data [40
3.11. Synthesis of Pyro-Conjugated Compound
Compound 9 (100 mg, 0.197 mmol, 1.0 eq) was dissolved in DMF (5 mL). Afterwards, EDC (77 mg, 0.4 mmol, 2.0 eq) and DIPEA (165 µL, 1 mmol, 5.0 eq) were added to the solution followed by the addition of Pyro (105 mg, 0.197 mmol, 1.0 eq). The solution was stirred overnight at RT. The reaction mixture was diluted with dichloromethane, washed with water and brine and dried with Na2SO4. After concentration, the residue was purified by silica gel column chromatography (CH2Cl2/MeOH = 50:1) to afford compound 10 as a blue-purple solid in a 90% yield (180 mg, 0.176 mmol). 1H-NMR (400 MHz, CDCl3) δ 9.51 (s, 1H), 9.40 (s, 1H), 8.56 (s, 1H), 8.02 (dd, J = 17.8, 11.5 Hz, 1H), 6.23 (dd, J = 47.3, 14.6 Hz, 3H), 6.04 (s, 1H), 5.21 (dd, J = 88.1, 19.9 Hz, 3H), 4.55 (d, J = 6.7 Hz, 1H), 4.33 (d, J = 8.7 Hz, 1H), 3.58 (t, J = 6.3 Hz, 6H), 3.41 (s, 3H), 3.25 (s, 3H), 2.36 (t, J = 6.2 Hz, 6H), 1.82 (d, J = 7.2 Hz, 3H), 1.70 (t, J = 7.6 Hz, 3H), 1.57 (s, 8H), 1.27 (s, 27H), 0.45 (s, 1H), −1.69 (s, 2H). 13C NMR (CDCl3, 100.6 MHz): δ 196.35, 172.41, 171.72, 170.91, 161.02, 154.96, 150.58, 148.96, 144.81, 141.34, 137.75, 136.01, 135.90, 135.63, 131.40, 130.50, 129.21, 128.16, 122.39, 106.12, 103.85, 96.95, 93.11, 80.30, 77.32, 77.20, 77.00, 76.68, 69.08, 66.95, 59.68, 51.70, 49.83, 48.06, 36.01, 33.63, 30.35, 28.07, 27.89, 23.19, 19.41, 17.43, 12.09, 12.00, 11.19. ESI-HRMS: m/z = 1022.5861, calcd for C58H80N5O11 m/z = 1022.5854 [M + H]+.
3.12. Synthesis of Deprotected Pyro-Conjugated Trimeric Linker
Trifluoroacetic acid (TFA) (2.0 mL) was added to the dichloromethane (2.0 mL) solution of compound 10 (100 mg, 0.098 mmol, 1.0 eq). The mixture was stirred overnight at room temperature and concentrated under reduced pressure. Then, the residues were dissolved in dichloromethane, washed with water, and dried with MgSO4. Concentration under reduced pressure afforded deprotected Pyro-conjugated trimeric linker 11 as a purple solid in an 85% yield (75 mg, 0.088 mmol). 1H-NMR (400 MHz, CDCl3) δ 9.95 (s, 1H), 9.76 (s, 1H), 8.87 (s, 1H), 7.87 (dd, J = 17.6, 11.8 Hz, 1H), 6.59 (s, 1H), 6.25 (dd, J = 14.5, 9.7 Hz, 2H), 5.41 (d, J = 19.9 Hz, 1H), 5.16 (d, J = 19.7 Hz, 1H), 3.74–3.59 (m, 8H), 3.58–3.44 (m, 10H), 3.32 (s, 6H), 2.32 (d, J = 4.9 Hz, 7H), 1.85 (d, J = 4.9 Hz, 3H), 1.74 (t, J = 7.3 Hz, 3H), 1.25 (s, 5H), 0.84 (d, J = 4.0 Hz, 4H). ESI-HRMS: m/z = 854.3974, calcd for C46H55N5O11 m/z = 854.3976 [M + H]+.
3.13. Synthesis of the Trimeric Conjugate Pyro-TriRGD
Pyro-conjugated trimeric linker 11 (1.2 mg, 0.0014 mmol, 1.0 eq), HOBt (1.2 mg, 0.009 mmol, 6.6 eq), and HBTU (3.5 mg, 0.009 mmol, 6.6 eq) were dissolved in 50 µL of DMF. DIPEA (3.8 µL, 0.023 mmol, 16.5 eq) and amino-modified RGD peptide 2 (5.0 mg, 0.0046 mmol, 3.3 eq) were added. The mixture was reacted overnight in the dark. The reaction mixture was precipitated with diethyl ether, and the solid was dissolved in dichloromethane and washed with water. The organic layer was dried (MgSO4) and concentrated under reduced pressure. The residue was purified using a silica gel column (CH2Cl2/MeOH = 10:1) to afford protected conjugate 12 in a 62% yield (3.6 mg, 0.0009 mmol). Deprotection was performed with 100 µL of the cleavage solution (TFA/Tis/water = 95:2.5:2.5) for 40 min at room temperature. The product was precipitated, washed three times with ethyl ether (0.5 mL), and purified with HPLC to produce Pyro-TriRGD in a 70% yield (3.0 mg, 0.97 µmol). ESI-HRMS: m/z = 1059.8714, calcd for C151H219N35O41 m/z = 1059.8735 [M + 3H]3+/3.
3.14. Determination of the Photophysical and Photochemical Properties
The UV–Vis absorption spectra and fluorescence spectra of the synthesized conjugates of Pyro-MonoRGD, Pyro-DiRGD, Pyro-TriRGD, and free Pyro were measured in DMSO. Absorption spectra were recorded from 300 to 800 nm. Fluorescence emission and excitation spectra were recorded from 550 to 800 nm upon excitation at approximately 668 nm and emission at 672–673 nm using a Hitachi model F-4500 FL spectrophotometer (Tokyo, Japan). The singlet oxygen quantum yields (ΦΔ) of the conjugates were measured in DMSO using DPBF as the quencher and Pyro as the reference.
3.15. Receptor Binding Assay
Human integrin αvβ3 (ITGAV&ITGB3) heterodimer protein was diluted to 2.0 μg/mL in PBS buffer (pH 7.4) and was then added to a 96-well plate (100 μL/well) and incubated overnight at 4 °C. The plate was blocked with 10% milk solution for an additional 2 h in a 37 °C shaker, followed by a 3 h incubation in the 37 °C shaker with various concentrations (10−3 to 10−11 M) of test compounds in the presence of biotinylated vitronectin (2.0 μg/mL). After washing, the plates were incubated for 1 h in the 37 °C shaker with streptavidin–HRP and then washed again, followed by 30 min of incubation with 100 μL/well TMB single-component substrate solution before stopping the reaction with the addition of 100 μL/well 2.0 N H2SO4. The absorbance at 450 nm was read on a microplate reader. Each data point represents the average of the triplicate wells, and data analysis was performed using nonlinear regression analysis with GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA.).
3.16. In Vivo Tumor Enrichment Analysis
Human U87-MG cells (4 × 106 cells/mouse) were subcutaneously inoculated on the axilla of female Balb/c nude mice. When the tumors grew to 200 mm3, the mice were divided into three groups with each group containing three mice. The mice were then intravenously injected with 50 nmol/mouse of Pyro-MonoRGD, Pyro-DiRGD, and Pyro-TriRGD, respectively. In vivo fluorescence analysis was performed before and after the injection at various time points using the IVIS Lumina imaging system (IVIS Lumina II, Xenogen, USA) with a Cy5.5 filter (excitation: 615–665 nm, emission: 695–770 nm). Alternatively, the mice were euthanized at 2 h postinjection, and major organs, including the heart, liver, spleen, lungs, kidneys, and muscles, as well as the tumor, were collected; fluorescence images were then acquired.