Multimerization increases tumor enrichment of peptide–photosensitizer conjugates

Article abstract of DOI:10.3390/molecules24040817

Photodynamic therapy (PDT) is an established therapeutic modality for the management of cancers. Conjugation with tumor-specific small molecule ligands (e.g., short peptides or peptidomimetics) could increase the tumor targeting of PDT agents, which is ve

Full text of DOI:10.3390/molecules24040817

molecules
Article
Multimerization Increases Tumor Enrichment of
Peptide–Photosensitizer Conjugates
Jisi Zhao 1,2,†, Shuang Li , Yingying Jin , Jessica Yijia Wang , Wenjing Li , Wenjie Wu * and
Zhangyong Hong *
2,†
2
3
2
1,
2
,
1
College of Material Science and Chemical Engineering, Tianjin University of Science and Technology,
Tianjin 300457, China; 13920398911@163.com
State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University,
2
Tianjin 300071, China; lishuang5258@163.com (S.L.); 18222912650@163.com (Y.J.); l800450@163.com (W.L.)
Tianjin Sirui International School, Sisui Road, Hexi District, Tianjin 300222, China;
3
jessica.Wang@students.tiseagles.com
*
Correspondence: wwjie@tust.edu.cn (W.W.); hongzy@nankai.edu.cn (Z.H.);
Tel.: +86-022-23502875 (W.W.); +86-022-23498707 (Z.H.)
These authors contributed equally to the project.
†
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 22 January 2019; Accepted: 19 February 2019; Published: 25 February 2019
Abstract: Photodynamic therapy (PDT) is an established therapeutic modality for the management
of cancers. Conjugation with tumor-specific small molecule ligands (e.g., short peptides or
peptidomimetics) could increase the tumor targeting of PDT agents, which is very important for
improving the outcome of PDT. However, compared with antibody molecules, small molecule
ligands have a much weaker affinity to their receptors, which means that their tumor enrichment
is not always ideal. In this work, we synthesized multimeric RGD ligand-coupled conjugates of
pyropheophorbide-a (Pyro) to increase the affinity through multivalent and cluster effects to improve
the tumor enrichment of the conjugates. Thus, the dimeric and trimeric RGD peptide-coupled Pyro
conjugates and the monomeric one for comparison were efficiently synthesized via a convergent
strategy. A short polyethylene glycol spacer was introduced between two RGD motifs to increase the
distance required for multivalence. A subsequent binding affinity assay verified the improvement of
the binding towards integrin αvβ receptors after the increase in the valence, with an approximately
3
20-fold improvement in the binding affinity of the trimeric conjugate compared with that of the
monomeric conjugate. In vivo experiments performed in tumor-bearing mice also confirmed a
significant increase in the distribution of the conjugates in the tumor site via multimerization, in which
the trimeric conjugate had the best tumor enrichment compared with the other two conjugates. These
results indicated that the multivalence interaction can obviously increase the tumor enrichment of
RGD peptide-conjugated Pyro photosensitizers, and the prepared trimeric conjugate can be used as a
novel antitumor photodynamic agent with high tumor enrichment.
Keywords: photodynamic therapy; pyropheophorbide-a; RGD peptide; integrin; photosensitizer
1
. Introduction
Photodynamic therapy (PDT) is an established therapeutic modality for the treatment of a variety
of premalignant and malignant diseases [
irreversible photodamage to tumor tissues [
as chemotherapy, radiotherapy, and surgery, PDT has the advantages of minimal invasiveness and
extremely low systemic toxicity [ ]. Furthermore, PDT agents can act as fluorescent probes for in vivo
cancer diagnoses and presentations, naturally combining fluorescence imaging and cancer therapy
1,
2]. It utilizes a photosensitizer activated by light to create
3,4]. Compared with traditional therapeutic methods, such
5
Molecules 2019, 24, 817; doi:10.3390/molecules24040817
www.mdpi.com/journal/molecules

Molecules 2019, 24, 817
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in a “see and treat” manner [
6,
7]. 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 [ ]. For these reasons, PDT has emerged as an important therapeutic option for
8,9
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 [13,14]. 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 [18–21].
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 αvβ integrin receptor [23–31]. This conjugate showed improved
3
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 αvβ integrin receptor compared with antibody molecules.
3
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 αvβ integrin targeting,
3
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 [32,33]. Multivalent interactions are thought to be a very practical strategy for amplifying

Molecules 2019, 24, 817
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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.
2
. Results and Discussion
2
.1. Molecular Design
Pyro is a very promising photosensitizer with ideal photoactivity properties, including a high
4
−1 −1
extinction coefficient (3.79
5
×
10 L mol cm ) and a high singlet oxygen quantum yield (over
0%) [36]. However, Pyro has very limited tumor localization ability. Solving the tumor enrichment
problem is very important for its clinical application. Here, to further increase the affinity of
RGD peptide-coupled Pyro conjugates to the receptor αvβ integrin, we synthesized multimeric
3
RGD-coupled Pyro conjugates to increase the affinity via multivalent and cluster effects, thereby
improving the uptake selectivity and tumor enrichment of the conjugates by tumor tissues. Thus,
in this study, we synthesized the dimeric and trimeric RGD peptide-coupled Pyro conjugates and the
monomeric one to compare and optimize the effect of multivalence (Figure 1). Since an exchange of
valine in the cyclic pentapeptide cyclo(-RGDfV-) for another amino acid has no significant influence
on the activity or selectivity [37], we used cyclo(-RGDfD-), which offers a free carboxylic acid
group for peptide coupling reactions, as the ligand for the convenience for molecular and functional
modifications. The distance between two RGD motifs in multimeric RGD peptides is very important for
multivalence [38]. It must be long and flexible enough to achieve simultaneous integrin αvβ binding.
3
Thus, we introduced a polyethylene glycol moiety of similar length as reported in the literature [39
]
as the spacer. The use of a polyethylene glycol spacer is desirable because of its high hydrophilicity,
which is helpful to increase the solubility of peptide conjugates and for extending the ligand out for
receptor binding and for reducing the influence of the Pyro macrocycle on the receptor affinity of the
RGD ligand.
To synthesize these conjugates, we adopted a convergent strategy in which we used a solid-phase
strategy based on Fmoc chemistry to prepare the cyclic RGD peptide molecule with a carboxylic group
that is free for conjugation and with all other groups protected. Then, we used solution reactions to
assemble the building blocks of Pyro, multivalent/monomeric linkers, and the side chain-protected
RGD peptide ligand together. This convergent synthesis strategy affords these conjugates in a very
efficient manner.

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Figure 1. Structures of the Pyro-MonoRGD, Pyro-DiRGD, and Pyro-TriRGD conjugates.
2
.2. Solid-Phase Synthesis of Side Chain-Protected Cyclic RGD Pentapeptide 1 and 2
The synthesis started from the preparation of the cyclic RGD peptide ligands (Scheme 1). Here,
we used the strategy of Fmoc solid-phase peptide synthesis to prepare the side chain-protected cyclic
RGD peptide ligand: cyclic pentapeptide cyclo(-Arg[Pbf]GlyAsp[tBu]-D-Phe-Asp-) . It conveniently
1
contains a free carboxylic acid group for the following derivatization. For its synthesis, Fmoc-Asp-OAll
was first attached to the 2-chlorotrityl chloride resin, followed by coupling with D-Phe, Asp, Gly, and
Arg. After removing the Fmoc group using piperidine and the OAll group by Pd(PPh ) , the peptide
3
4
was cyclized on the resin. Then, the peptide was cleaved from the resin suing 1% TFA in CH Cl with
2
2
all side chain groups protected, and it was purified using a silica gel column to produce the protected
pentapeptide cyclo(-Arg[Pbf]GlyAsp[tBu]-D-Phe-Asp-) in a yield of 42%. Then, to add a hydrophilic
polyethylene glycol (PEG) linker as a spacer between Pyro and the RGD ligand, the pentapeptide
was reacted with 4,7,10-Trioxa-1,13-tridecanediamine to produce the amino group-modified RGD
peptide in an 81% yield. It is worth mentioning that both RGD peptides and could be easily
1
1
2
1
2
purified with a conventional silica gel column. Through this on-resin cyclization and the following
amino group derivatization in-solution reaction, the side chain-protected RGD ligand was obtained
very easily with one free carboxylic acid or one amino group for the following derivatization, offering
a very simple method for synthesis of RGD-coupled conjugates.

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Scheme 1. Synthetic route for cyclic RGD pentapeptide
1 and amino-modified cyclic RGD
peptapeptide 2.
2
.3. Synthesis of Mono-RGD Conjugate Pyro-MonoRGD
The Pyro molecule has one free carboxylic acid group, which can be used for coupling with
amino-modified RGD ligand via the typical condensation reaction between a carboxylic group and an
amino group (Scheme 2). The condensation between RGD peptide and Pyro afforded compound in
2
2
3
an 80% yield. After removing the protective group on the RGD moiety with 95% TFA, the monomeric
RGD peptide-coupled Pyro conjugate (Pyro-MonoRGD) was obtained as a solid in a 71% yield after
HPLC purification. The product was characterized by mass spectrometry and HPLC (Supplementary
Materials, Figures S1 and S4). Mass spectrometry showed that it has a molecular weight of 1309.6694,
which is consistent with its theoretical mass of 1309.6734. HPLC showed that the product was of a
very good purity.

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Scheme 2. Synthetic route for the mono-RGD conjugate, Pyro-MonoRGD.
2
.4. Synthesis of the Dimeric-RGD Conjugate, Pyro-DiRGD
For the synthesis of the dimeric RGD-coupled Pyro conjugate Pyro-DiRGD (Scheme 3), a proper
dimeric linker was first prepared. Here, 2-amino-1,3-propanediol was first tested to directly condense
with tert-butyl acrylate in the presence of NaOH as a base; however, we could not obtain the desired
product. We could only obtain the addition product in which the amino group reacted with tert-butyl
acrylate. The amino group seems to have a higher reaction rate than the hydroxyl group to react with
tert-butyl acrylate in 2-amino-1,3-propanediol. Thus, 2-amino-1,3-propanediol was first protected
with the tert-butyloxycarbonyl (Boc) group to afford derivative
with tert-butyl acrylate to afford in a 58% yield. After deprotection of the Boc and tert-butyl groups,
the obtained compound was first assembled with the preformed activated ester of Pyro at room
temperature to afford the Pyro coupled dimeric linker in a yield of 92% and then subsequently coupled
with amino-modified RGD peptide to produce the side chain-protected dimeric RGD-coupled Pyro
conjugate in a yield of 72%. It is worth mentioning that both Pyro-coupled dimeric linker and
dimeric RGD-coupled Pyro conjugate can be easily purified with a conventional silica gel column,
4 in a 62% yield and then condensed
5
6
7
2
8
7
8
making this method very convenient for amplifying the synthesis. The desired dimeric RGD conjugate
Pyro-DiRGD was then obtained in a 67% yield after simple deprotection and HPLC purification.
The product was characterized via mass spectrometry and HPLC, and the relative data are presented
in the Supplementary Materials (Figures S1 and S5).

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Scheme 3. Synthetic route for the di-RGD conjugate, Pyro-DiRGD.
2
.5. Synthesis of the Trimeric-RGD Conjugate, Pyro-TriRGD
The synthesis of the trimeric RGD-coupled Pyro conjugate, Pyro-TriRGD, began with the
preparation of the trimeric linker with the functional amino and carboxylic groups for coupling
Scheme 4). Here, tris(hydroxymethyl)aminomethane (Tris base) was condensed with tert-butyl
acrylate in the presence of NaOH without protection of the amino group to afford the trimeric linker
(
9
in a 38% yield. In contrast to 2-amino-1,3-propanediol, the free amino group in the Tris base seemed to
be not able to compete with the hydroxyl group for the condensation with tert-butyl acrylate, possible
due to the strong steric hindrance. Thus, there is no necessity to protect the free amino group in the
Tris base for this condensation. Trimeric linker
Pyro to obtain the Pyro-coupled trimer Linker 10 in a yield of 92%. Deprotection with TFA afforded
, which was then subsequently coupled with amino group-modified RGD peptide to produce the
side chain-protected trimeric RGD-coupled Pyro conjugate 12 in 62% yield. Here, both Pyro-coupled
9 was assembled with the preformed activated ester of
1
1
2
a
trimer Linker 10 and side chain-protected RGD-coupled with the Pyro conjugate 12 could also be easily
purified with a silica gel column. Then, the desired product, Pyro-TriRGD, was obtained in a 70% yield
after simple deprotection and HPLC purification. The product was characterized by mass spectrometry
and HPLC, and the relative data are presented in the Supplementary Materials (Figures S1 and S6).

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Scheme 4. Synthetic route for the Pyro-TriRGD conjugate.
2
.6. Photophysical and Photochemical Properties
The photophysical and photochemical properties of Pyro-MonoRGD, Pyro-DiRGD, Pyro-TriRGD,
and free Pyro were measured and re summarized in Table 1. All these conjugates exhibited very
similar UV–Vis spectra and fluorescence spectra compared with free Pyro with an intense and sharp
Q-band at 667–668 nm, a fluorescence excitation band at approximately 668 nm and an emission band
at 672–673 nm. The singlet oxygen quantum yields (Φ ) of these conjugates were in the range of 0.47
∆
to 0.49, which were also comparable with that of free Pyro (Φ∆ = 0.52). These data show that the RGD
peptide conjugation did not obviously affect the photophysical or photochemical properties of Pyro.

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Table 1. Photophysical and photochemical data for Pyro-MonoRGD, Pyro-DiRGD, Pyro-TriRGD, and
Pyro in DMSO.
Compound
λmax (nm)
λem (nm)
Stokes Shift (nm)
Φ∆
Pyro
668
668
668
668
673
672
673
673
5
4
5
5
0.52
0.49
0.47
0.48
Pyro-MonoRGD
Pyro-DiRGD
Pyro-TriRGD
2
.7. Receptor Binding Assay
To evaluate the influence of multivalent conjugation on the binding capability of the RGD
conjugate, the ability of the three conjugates—Pyro-MonoRGD, Pyro-DiRGD, and Pyro-TriRGD—to
inhibit the binding of biotinylated vitronectin to human isolated αvβ integrin receptors in vitro was
3
determined by a competitive binding assay (Figure 2 and Table 2). Here, compared with the monovalent
RGD conjugate of Pyro-MonoRGD (IC50 = 950.7
Pyro-DiRGD (IC50 = 313.2 23.1 nM) to integrin αvβ increased. An even better improvement
was observed for the trivalent conjugate, Pyro-TriRGD (IC50 = 43.8
±
39.6 nM), the binding of the divalent conjugated
±
3
±
8.2 nM), which binds integrin
αvβ 20-fold better than the monovalent counterpart, Pyro-MonoRGD, and 7-fold better than the
3
divalent Pyro-DiRGD. These values suggest that the multivalent interactions play important roles in
increasing the binding affinity of conjugates. It can enhance the binding strength to a greater extent
than the sum of the affinity of the involved ligands, which is somewhat proportional to the number of
conjugated ligands.
Figure 2. Comparison of the inhibitory action of Pyro-MonoRGD, Pyro-DiRGD, and Pyro-TriRGD on
binding of integrin αvβ and vitronectin. Data are expressed as the means ± SD (n = 3).
3
Table 2. Inhibition of biotinylated vitronectin binding to αvβ receptors. IC50 values were
3
calculated as the concentration of compound required for 50% inhibition of biotinylated vitronectin
binding as estimated by GraphPad Prism 5.0. All values are the means (
triplicate determinations.
±
standard deviation) of
Compounds
Pyro-TriRGD
Pyro-DiRGD
Pyro-MonoRGD
IC50 (nM)
43.8 ± 8.2
313.2 ± 23.1
950.7 ± 39.6
2
.8. In Vivo Distribution Analysis
To examine the tumor enrichment of these conjugates in vivo, we prepared a xenograft tumor
6
model by subcutaneously inoculating mice with U87-MG tumor cells (1.0
×
10 cells per mouse) on the

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axilla. U87-MG cells have high expression levels of the αvβ integrin receptor. When the tumors grew
3
3
to approximately 200 mm in volume, the conjugates (50 nmol per mouse) were intravenously injected
into the mice via the tail vein. The fluorescence signal and intensity distribution were continuously
monitored using an in vivo fluorescence imaging system (IVIS Lumina II, Xenogen, Alameda, CA,
USA). As shown in Figure 3A, these conjugates were rapidly distributed throughout the body, and an
intense fluorescence signal was primarily located in the organs of the liver, kidney, and tumor one hour
postinjection. At two hours postinjection, the fluorescence signals in the liver obviously decreased.
For the Pyro-MonoRGD conjugate, at two hours postinjection, the tumor-to-background ratios
increased, and accumulation in the tumor could be observed; however, the signals for the fluorescence
in the liver were still very strong. Afterwards, the signals both in the liver and tumor disappeared
quickly, and almost disappeared at four hours postinjection. For the Pyro-DiRGD conjugate, the
tumor distribution was significantly different. At two hours postinjection, the fluorescence signal
of Pyro-DiRGD in the tumor site was already stronger than the one in the liver, and this strong
fluorescence intensity in the tumor site lasted until 4 h postinjection. However, relatively weak
fluorescence signals in the tumor site could be detected. For the Pyro-TriRGD conjugate, after two
hours postinjection, it was mainly concentrated in the tumor site, and its signal became even stronger
at four hours postinjection, at which time the signals were almost completely located in the tumor area
and only negligible signals could be found in other tissues. More importantly, the signal in the liver
region sequentially weakened from Pyro-MonoRGD, Pyro-DiRGD, to Pyro-TriRGD. The increased
water hydrophilicity of the divalent and trivalent conjugates may contribute to the reduction in the
liver accumulation.
Figure 3. Drug accumulation and retention studies in U87-MG tumor-bearing nude mice. (
fluorescence imaging of Pyro-MonoRGD (a), Pyro-DiRGD (b), and Pyro-TriRGD (c) obtained at 0, 1, 2, 4,
, and 9 h after injection of the corresponding conjugates. Fluorescent signals of the different conjugates
were imaged ( ) and quantitative detected ( ) in the organs and tumors 2 h post-administration (a, b,
A) In vivo
6
B
C
and c represent Pyro-MonoRGD, Pyro-DiRGD, and Pyro-TriRGD, respectively). The results are shown
as averages ±SD of triplicates.
To further verify the tumor targeting ability of these conjugates, the mice were euthanized 2 h
postinjection, and their organs were collected for imaging. As shown in Figure 3B (heart, liver, spleen,
lungs, and tumor from left to right), when the valence of the conjugates increased, the fluorescence
signal in the tumor region increased. Pyro-TriRGD exhibited the strongest fluorescence intensity in
the tumor. The fluorescence intensity in these organs was also calculated and is shown in Figure 3C.

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Pyro-TriRGD exhibited the highest fluorescence intensity at the tumor site and the lowest intensity at
the liver compared with the other two conjugates.
These initial data provide strong evidence for high-tumor targeting of multivalent RGD conjugates,
especially the trimeric one. The trivalent conjugate, Pyro-TriRGD, exhibited much improved tumor
enrichment ability with excellent tumor accumulation at 2–6 h postinjection, and it may have good
potential for tumor targeting PDT. Additionally, the accumulation of the conjugates in the liver
significantly reduced because of the multimeric conjugation, probably due to the increased water
hydrophilicity of the divalent and trivalent conjugates.
3
. Experimental
3
.1. Instruments and Materials
t
Fmoc-Asp-OAll, Fmoc-Dphe-OH, Fmoc-Asp(O Bu)-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(PPh ) ), benzotriazol-1-yl-
3
4
oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), diisopropylethylamine (DIPEA),
trifluoroacetic acid (TFA), and N,N-dimethylformamide (DMF) were purchased from Heowns (Tianjin,
1
13
China). H and C NMR spectra were recorded in CDCl or in D O solutions on a Bruker AV400
3
2
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,
1
0 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 (CH Cl :MeOH:DIPEA = 17:1:2, 20 mL) was used to seal the unreacted chlorine group
2
2
for 30 min. The Fmoc protecting group was removed by treatment with 20% piperidine in DMF (20 mL)
t
for 30 min. Coupling reactions of the other four amino acids (Fmoc-D-Phe-OH, Fmoc-Asp(O Bu)-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 PhSiH (24 equiv) and Pd(PPh )
3
3 4
(
0.3 equivalents) in dichloromethane for 1 h under N protection for selective alpha-carboxyl
2
deprotection of the allyl groups. Cyclization between
α-NH of Arg and α-CO H of Asp was conducted
2 2
overnight in DMF using PyBOP (2 equiv)/HOBt (1.5 equiv)/DIPEA (5 equiv). The resin was washed

Molecules 2019, 24, 817
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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, CH Cl -MeOH, 10:1) to afford pure
2
2
peptapeptide
1 (57 mg, 42%). HRMS-ESI: m/z = 899.3956, calcd for C H N O S m/z = 899.3973
42 59 8 12
+
[M + H] .
3
.3. Synthesis of Amino-Modified Cyclic Pentapeptide
Cyclo(-Arg[Pbf]-Gly-Asp[tBu]-D-Phe-Asp[PEG-amine]-)
Peptapeptide
.10 mmol, 2.0 eq), and DIPEA (41
then 4,7,10-trioxa-1,13-tridecanediamine (220
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 Na SO .
1
(44 mg, 0.050 mmol, 1.0 eq), HOBt (10 mg, 0.075 mmol, 1.5 eq), HBTU (38 mg,
L, 0.25 mmol, 5.0 eq) were dissolved in 500 L of DMF, and
L, 1.0 mmol, 20 eq) was added. The mixture was
0
µ
µ
µ
2
4
After concentration, the mixture was purified by column chromatography (silica gel:CH Cl -MeOH,
2
2
1
0
0:1) to produce amino-modified cyclic pentapeptide 2 as a light yellow solid in an 81% yield (44 mg,
+
.041 mmol). ESI-HRMS: m/z = 1101.5633, calcd for C H N O S m/z = 1101.5654 [M + H] .
52
81 10 14
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,
.0 eq) were dissolved in 150 L of DMF. DIPEA (7.4 L, 0.045 mmol, 5.0 eq) and amino-modified
(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 (MgSO ), concentrated under reduced pressure,
2
µ
µ
pentapeptide
2
4
and purified via a silica gel column (CH Cl /MeOH = 15:1) to afford condensed compound
3
in an
L 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
2
2
80% yield (12.0 mg, 7.2 µmol). Deprotection was performed with 100 µ
(
7
1% yield (6.7 mg, 5.1
µ
mol). ESI-HRMS: m/z = 1309.6694, calcd for C H N O m/z = 1309.6734
68
89 14 13
+
[M + H] .
3
.5. Synthesis of Tert-butyl (1,3-dihydroxypropan-2-yl)carbamate
-Amino-1,3-propanediol (1.62 g, 17.8 mmol, 1.0 eq) was dissolved in 60 mL of tetrahydrofuran/
H O (1/1). Di-tert-butyl dicarbonate (Boc O) (5.82 g, 26.7 mmol, 1.5 eq) and K CO (6.15 g, 44.5 mmol,
2
2
2
2
3
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 Na SO , evaporated
2
4
in vacuo, and purified by column chromatography (silica gel:petroleum ether/ethyl acetate, 1:1) to
1
produce compound
4
as a white solid in a 62% yield (2.1 g, 11.0 mmol). H-NMR (400 MHz, CDCl )
3
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].
0
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 H O and then with saturated brine, and dried over anhydrous
2
Na SO . After evaporation, the residue was purified by column chromatography (silica gel, petroleum
2
4

Molecules 2019, 24, 817
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ether/ethyl ester, 8:1), and compound
5
was obtained as a colorless oil in a 58% yield (273 mg,
1
0
3
.61 mmol). H-NMR (400 MHz, CDCl )
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,
13
1
H). C 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,
+
3
6.27, 28.36, 28.08. LC-MS: m/z = 448.5, calcd for C H NO m/z = 448.3 [M + H] .
22
43
8
0
3
.7. Synthesis of Dimeric Linker 3,3 -((2-Aminopropane-1,3-diyl)bis(oxy))dipropionic acid hydrochloride
Compound (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
5
room temperature and then concentrated under reduced pressure. Dimeric linker
6 was obtained as a
1
white solid in a 93% yield (75 mg, 0.28 mmol). H-NMR (300 MHz, D O)
δ
3.93–3.76 (m, 6H), 3.73 (dd,
2
J = 9.4, 6.1 Hz, 3H), 2.73 (t, J = 5.8 Hz, 4H). 13C NMR (100.6 MHz, D O):
δ 176.33, 67.21, 66.50, 50.54,
2
+
3
4.23. LC-MS: m/z = 236.4, calcd for C H NO m/z = 236.1 [M + H] .
9
18
6
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 Na SO .
(
2
4
After evaporation, the activated ester was obtained. The activated ester (10 mg, 0.016 mmol, 1.0 eq) was
dissolved in 300 L of DMF, and compound (5 mg, 0.019 mmol, 1.2 eq) and DIPEA (13 L, 0.08 mmol,
.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
µ
6
µ
5
residue was purified via silica gel (DCM/MeOH = 10:1) to produce Pyro-conjugated dimeric linker
7
1
as a black solid in a 92% yield (11.0 mg, 0.055 mmol). H-NMR (300 MHz, CDCl )
δ 9.23 (s, 1H), 9.15 (s,
3
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 C H N O m/z = 752.3659 [M + H] .
42
50
5
8
3
.9. Synthesis of Conjugate Pyro-DiRGD
Pyro-conjugated dimeric linker (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 L of DMF. Then, DIPEA (5.5 L,
.033 mmol, 11 eq) and amino-modified RGD peptide (8.0 mg, 0.0073 mmol, 2.4 eq) were added.
7
µ
µ
0
2
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 (MgSO ), concentrated under reduced pressure, and purified via silica gel (DCM/MeOH = 10:1)
4
to afford protected conjugate
with 100 L 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 mol). HRMS-ESI: m/z = 1151.0880, calcd
8 in a 72% yield (6.5 mg, 0.002 mmol). Deprotection was performed
µ
µ
2+
for C112H159N O m/z = 1151.0893 [M + 2H] /2.
25
28
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 (Na SO ), concentrated, and purified by silica gel column chromatography (petroleum
2
4
ether/ethyl ester, 1:2) to afford compound
9 as a pale yellow oil in a 38% yield (3.86 g, 7.63 mmol).

Molecules 2019, 24, 817
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1H-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 (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 L, 1 mmol, 5.0 eq) were added to the solution followed
9
(
µ
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
Na SO . After concentration, the residue was purified by silica gel column chromatography (CH Cl /
2
4
2
2
MeOH = 50:1) to afford compound 10 as a blue-purple solid in a 90% yield (180 mg, 0.176 mmol).
1
H-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,
H), 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,
00.6 MHz): 196.35, 172.41, 171.72, 170.91, 161.02, 154.96, 150.58, 148.96, 144.81, 141.34, 137.75, 136.01,
35.90, 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,
3
1
1
−
δ
7
6.68, 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,
+
1
2.00, 11.19. ESI-HRMS: m/z = 1022.5861, calcd for C H N O m/z = 1022.5854 [M + H] .
58
80
5
11
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 MgSO . Concentration under reduced pressure afforded deprotected
4
1
Pyro-conjugated trimeric linker 11 as a purple solid in an 85% yield (75 mg, 0.088 mmol). H-NMR
(
6
3
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),
.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),
.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 C H N O m/z = 854.3976
46 55 5 11
+
[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 eq), and HBTU (3.5 mg, 0.009 mmol, 6.6 eq) were dissolved in 50 L of DMF. DIPEA (3.8 L,
.023 mmol, 16.5 eq) and amino-modified RGD peptide (5.0 mg, 0.0046 mmol, 3.3 eq) were added.
6
0
µ
µ
2
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 (MgSO ) and concentrated under reduced pressure. The residue was purified using a silica
4
gel column (CH Cl /MeOH = 10:1) to afford protected conjugate 12 in a 62% yield (3.6 mg, 0.0009
2
2
mmol). Deprotection was performed with 100
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 mol).
µL of the cleavage solution (TFA/Tis/water = 95:2.5:2.5)
µ
3+
ESI-HRMS: m/z = 1059.8714, calcd for C151H219N O m/z = 1059.8735 [M + 3H] /3.
35
41
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

Molecules 2019, 24, 817
15 of 18
Hitachi model F-4500 FL spectrophotometer (Tokyo, Japan). The singlet oxygen quantum yields (Φ∆
)
of the conjugates were measured in DMSO using DPBF as the quencher and Pyro as the reference.
3
.15. Receptor Binding Assay
Human integrin αvβ (ITGAV&ITGB3) heterodimer protein was diluted to 2.0
µ
g/mL in PBS
3
◦
buffer (pH 7.4) and was then added to a 96-well plate (100
µ
L/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
M) of test compounds in the
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
◦
◦
−3
−11
incubation in the 37 C shaker with various concentrations (10 to 10
presence of biotinylated vitronectin (2.0
◦
with 100
µL/well TMB single-component substrate solution before stopping the reaction with the
addition of 100
µ
L/well 2.0 N H SO . The absorbance at 450 nm was read on a microplate reader.
2
4
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
6
Human U87-MG cells (4
×
10 cells/mouse) were subcutaneously inoculated on the axilla of
3
female Balb/c nude mice. When the tumors grew to 200 mm , the mice were divided into three
groups with each group containing three mice. The mice were then intravenously injected with
5
0 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:
6
95–770 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.
4
. Conclusions
To improve the tumor enrichment of small molecule ligand-conjugated photosensitizers, we
designed and synthesized multivalent cyclic RGD peptide-conjugated Pyro molecules. These conjugates
were prepared using a convergent strategy, and the synthesis was carefully characterized. Their
UV–Vis absorption spectra, fluorescence spectra, and singlet oxygen quantum yields were measured.
The subsequent binding affinity assay verified the improvement in the binding towards the integrin
αvβ receptors after the increase in the valence, which was a nearly 20-fold improvement in the binding
3
affinity of Pyro-TriRGD compared with that of monomeric conjugate Pyro-MonoRGD. The in vivo
distribution experiments on the tumor-bearing mice also confirmed a significant increase in the
distribution of the conjugates in the tumor sites via multimerization, where the trimeric conjugate,
Pyro-TriRGD, had the best tumor enrichment compared with the other two conjugates. These
results indicated that the multivalence interaction may be used to increase the tumor enrichment of
RGD peptide-conjugated Pyro photosensitizers for tumor targeting PDT, and the trimeric conjugate
Pyro-TriRGD may be used as a novel antitumor photodynamic agent with high tumor enrichment.
Supplementary Materials: The following are available online. HPLC analysis, Mass spectrometry analysis, and
UV-Vis and fluorescence spectrum analysis of the reported conjugates, NMR spectra of the intermediates.
Author Contributions: J.Z., S.L., W.W. and Z.H. conceived and designed the experiments; J.Z., S.L., Y.J. and J.Y.W.
performed the experiments; J.Z., S.L. and W.L. analyzed the data; J.Z., S.L., W.W. and Z.H. wrote the paper.
Funding: This research was funded by the Major Program of the National Natural Science Foundation of China
(
No. 31527801), the Natural Science Foundation of Tianjin (No. 18YFZCSY00280) and by the Science and
Technology Support Funding from Jinnan District of Tianjin (No. 201805013).
Acknowledgments: This work utilized core resources supported by Nankai University.

Molecules 2019, 24, 817
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Conflicts of Interest: There are no conflicts of interest to declare.
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[
Sample Availability: Samples of the compounds Pyro, Pyro-MonoRGD, Pyro-DiRGD, and Pyro-TriRGD are
available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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