Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Mar 18.
Published in final edited form as: Photochem Photobiol. 2021 May 21;98(2):354–361. doi: 10.1111/php.13439

Structural Engineering of Fluorescent Self-Threaded Peptide Probes for Targeted Cell Imaging

Cynthia L Schreiber 1, Canjia Zhai 1, Bradley D Smith 1,*
PMCID: PMC8808266  NIHMSID: NIHMS1769773  PMID: 33934361

Abstract

Squaraine figure-eight (SF8) molecules are a new class of deep-red fluorescent probes that are well suited for fluorescence cell microscopy due to their very high fluorescence brightness and excellent stability. Three homologous SF8 probes, with peptidyl loops that differ by very minor changes in the peptide sequence, were synthesized and assessed for probe uptake by cancer cells. One of probes included the RGD motif that is recognized by many classes of integrin receptors that reside on the surface of the cancer cells, and it permeated the cells by receptor-mediated endocytosis. In contrast, cell microscopy showed that there was negligible cell uptake of the two homologous SF8 probes indicating differences in probe targeting capability. The synthetic method allows for easy alteration of the peptide sequence; thus, it is straightforward to develop new classes of peptidyl SF8 probes with loop sequences that target other cancer biomarkers.

INTRODUCTION

There is a need for next-generation fluorescent molecular probes for improved use in microscopy, diagnostics, and in vivo imaging.(1) (2) Deep-red fluorescent probes are highly desired because image contrast is relatively high due to low background absorption and autofluorescence by endogenous biomolecules. In addition, there is deeper penetration of the deep-red light through thick biological samples including living subjects.(3, 4) But long wavelength fluorophores are inherently reactive to both nucleophiles and electrophiles which limits their utility as high stability fluorescent probes.(1, 5, 6) In addition, the highly conjugated fluorochromes usually have large amounts of hydrophobic surface area which lowers water solubility and favors intermolecular aggregation processes that reduce molecular targeting performance.(7) (8) We and others are pursuing molecular design strategies that can simultaneously solve both technical problems.(9, 10) One synthetic approach is to chemically decorate the fluorophore with water solubilizing or chemical stabilizing groups.(11) An alternative idea is to develop molecular encapsulation strategies that surround a fluorophore with a protective cage. We have used this latter method to encapsulate fluorescent squaraine dyes within a surrounding macrocycle and create interlocked squaraine rotaxanes which exhibit excellent fluorescence brightness and high photostability.(12) (13) (14) More recently we developed a synthetic method that converts a mechanically bonded squaraine rotaxane into a single covalently connected probe structure that we call squaraine figure-eight (SF8).(15) Illustrated in Scheme 1 is the key macrocyclization reaction that produces a peptidyl SF8 probe with two equivalent loops. Peptidyl SF8 probes are well suited for fluorescence cell microscopy because they exhibit very bright deep-red fluorescence, high chemical stability including protease resistance, and excellent photostability.

Scheme 1.

Scheme 1.

Open in a new tab

Synthetic conversion of SR precursor into SF8 molecule; the second synthetic step removes side chain protecting groups from residues, P, to reveal amino acid residues, A.

To date, we have evaluated the biological imaging properties of two sets of SF8 probes. One set is a series of uncharged molecules that were able to directly permeate cells and accumulate within intracellular organelles.(16) A second set of SF8 probes had longer peptide-containing loops that contained charged side chains and exploited electrostatics to achieve probe association with biological surfaces.(15) For example, a polycationic SF8 probe with loops containing multiple arginine residues was found to strongly interact with cell membranes, and a polyanionic SF8 probe with loops containing multiple aspartates was found to strongly bind to the Ca2+ cations within the mineral component of bone. In contrast, a charge balanced SF8 probe with peptide loops containing an equal number of cationic and anionic residues showed little affinity for biological surfaces.(15)

The goal of this present study was to assess if we could engineer the sequence of the peptide loops within the charge balanced probes to favor targeting and uptake of the probe by a cancer cell. Our chemical synthesis approach is reminiscent of literature studies that used site-directed mutagenesis to alter the loop region within a lasso peptide to produce a sequence (epitope) that improved affinity for a cell surface receptor.(17) (18) (19) In this present case, we synthesized the three homologous peptidyl SF8 analogues in Scheme 2 with peptide loops that differ by very minor changes in the peptide sequence. One of these peptidyl probes included the RGD motif that is the consensus recognition sequence for many classes of integrin receptors that reside on the surface of cancer cells.(20) (21) We find that fluorescent probe entry into the cells by receptor-mediated endocytosis depends on the peptidyl loop sequence.

Scheme 2.

Scheme 2.

Open in a new tab

Chemical structures of the three peptidyl molecular probes that were tested for uptake by cancer cells. Color-coded within each single molecule are three separate structural components; encapsulated squaraine dye (blue); surrounding tetralactam macrocycle (red), and peptidyl loops (green).

MATERIALS AND METHODS

Probe synthesis:

Materials, chemical synthesis, and compound characterization are described in the Supporting Materials.

Cell culture:

Cells were cultured and maintained in a humidified incubator at 37 °C under 5% CO2. The cell medium (EMEM, F12-K, RPMI-1640) were purchased from American Type Culture Collection (ATCC), the fetal bovine serum was purchased from Atlanta Biologicals, and all other supplies were purchased from Sigma Aldrich. HT-1080 (ATCC CCL-121) human fibrosarcoma cells were maintained in EMEM medium supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate, and 1% penicillin/streptomycin. A549 (ATCC CCL-185) human non-small cell lung adenocarcinoma cells were maintained in F-12K medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. OVCAR-4 (CVCL_1627) human ovarian adenocarcinoma cells were maintained in RPMI-1640 media supplemented with 10% fetal bovine serum, 1% L-glutamine, and 0.1 mM non-essential amino acids.

Fluorescence cell microscopy:

The A549, OVCAR-4, and HT-1080 cells were seeded into 8-well chambered slides (Lab-Tek, Nunc, USA) and were grown to 70% confluency. Cells were incubated with 1 μM probe (SF8(DRADG)2, SF8(DDGRG)2, or SF8(DRGDG)2) in media for 30 min at 37 °C. The cells were washed three times with phosphate buffered saline and fixed with 4% cold paraformaldehyde for 20 min at room temperature. Cells were then washed one time with phosphate buffered saline, co-stained with 3 μM Hoechst 33342 for 10 min at room temperature, washed two times with phosphate buffered saline, and imaged.

Colocalization studies:

The A549 cells were seeded into 8-well chambered slides (Lab-Tek, Nunc, USA) and were grown to 70% confluency. Cells were co-incubated with 1 μM Cy3-cRGDfK (Molecular Targeting Technologies Inc.) and 1 μM SF8(DRGDG)2 in media for 30 min at 37 °C. The live cells were then washed three times with phosphate buffered saline, co-stained with 3 μM Hoechst 33342 for 10 min at room temperature, washed two times with phosphate buffered saline, and imaged.

Cold blocking studies:

The A549, OVCAR-4, and HT-1080 cells were seeded into 8-well chambered slides (Lab-Tek, Nunc, USA) and were grown to 70% confluency. Prior to probe incubation, the cells were placed either in the fridge (4 °C) or a cell incubator (37 °C) for 30 min. The cells were then washed with phosphate buffered saline and incubated at the same temperature as before with 1 μM SF8(DRGDG)2 in media for 30 min. The cells were washed three times with phosphate buffered saline, co-stained with 3 μM Hoechst 33342 for 10 min at room temperature, washed two times with phosphate buffered saline, and imaged.

Competitive blocking studies:

A549 or OVCAR4 cells were grown to 70% confluency on 8-well microscopy slides. The cells were then incubated with 1 μM SF8(DRGDG)2 probe in media for 30 minutes at 37 °C. For blocking, 200 μM cRGDfK or 50 μM cilengitide was added to the cells for 5 minutes prior to probe treatment and remained present during probe incubation. The cells were washed three times with phosphate buffered saline and fixed with 4% cold paraformaldehyde for 20 min at room temperature. Cells were then washed one time with phosphate buffered saline, co-stained with 3 μM Hoechst 33342 for 10 min at room temperature, washed two times with phosphate buffered saline, and imaged.

Flow cytometry:

The A549 cells (1.5x105) were dissociated from the plate with a non-enzymatic cell dissociation solution (Sigma Aldrich). The cells were incubated with 100 nM SF8 probe (SF8(DRADG)2, SF8(DDGRG)2, or SF8(DRGDG)2) for 15 min at 37 °C. Then the cells were washed twice with flow cytometry buffer, fixed with IC fixation buffer, and transferred to flow cytometry tubes. The cell fluorescence intensity was measured using a BD LSRFortessa X-20 flow cytometer (ex:640 nm, em: 730/45 nm, events: 15,000). The geometric mean fluorescence intensity was then calculated by applying a standard gate (full width at half maximum) using FlowJo. Averages and SEM were plotted using GraphPad Prism.

Imaging parameters:

All microscopy images were collected with a Zeiss Axiovert 100 TV epifluorescence microscope equipped with a UV filter (ex: 387/11, em: 447/60), TxRed filter (ex: 562/40, em: 624/40), and Cy5.5 filter (ex: 655/40, em: 716/40).

Statistical analysis:

Image processing for each micrograph was conducted using ImageJ2 software with a 200-pixel rolling ball radius background subtraction. An auto-threshold method on ImageJ2 was then chosen based on the best fit for determining the mean fluorescence intensity of cell micrographs. The averages and SEM were calculated and plotted in GraphPad Prism. For colocalization experiments, a 10-pixel rolling ball radius background subtraction was applied and a colocalization threshold program (JaCoP) using ImageJ2 software determined the Pearson’s correlation coefficient. Averages and SEM were plotted in GraphPad Prism.

RESULTS AND DISCUSSION

Synthesis and molecular structure

The synthetic methods and compound characterization are described in the supplementary materials. In each case, a common squaraine rotaxane precursor with protected amino acid side chains was converted to an SF8 probe in 25-55% isolated yield by conducting a double macrocyclization reaction using copper catalyzed azide/alkyne cycloaddition (CuAAC) conditions followed by TFA deprotection (Scheme 1).(15) The naming scheme for the figure-eight probes is SF8 followed by an acronym for the two loop components. For example, the pentapeptidyl SF8 probe SF8(DRGDG)2 has two identical DRGDG loops and it was prepared assuming that it would have affinity for αvβ5 and αvβ3 integrin receptors which are commonly overexpressed by cancer cells.(22),(23-25) Furthermore, integrin receptor binding would subsequently trigger cell uptake by receptor mediated endocytosis. For comparison, two control pentapeptidyl SF8 probes were prepared; namely, SF8(DDGRG)2 with each loop containing a scrambled peptide sequence, and SF8(DRADG)2 with each peptidyl loop containing an alanine residue instead of a glycine. These two control probes lack the RGD motif, but they have the same number of oppositely charged residues as SF8(DRGDG)2 and the same log P values of −1.4 (Figure S1).

Cell microscopy

To investigate the cell targeting capabilities of SF8(DRGDG)2, three cell lines were chosen (A549, OVCAR-4, and HT-1080) for cell microscopy studies due to their overexpression of integrin receptors that bind RGD sequences, their clinical relevance, and their representation of different cancer types. The A549 (human non-small cell lung adenocarcinoma) cell line is a well-established system for cancer imaging(26),(27) and was previously reported to overexpress the αvβ5 protein.(28) The OVCAR-4 (human high-grade serous ovarian carcinoma) cell line is a clinically relevant cancer model(29) and has a high expression of αvβ3 integrin receptors.(30) Lastly, the HT-1080 (human fibrosarcoma) cell line was shown by immunofluorescence to overexpress αvβ3 integrin receptors but not the β5 protein (Figure S2).(31, 32) The cell microscopy experiments employed a standard procedure of 30 minute incubation with 1 μM peptidyl SF8 probe. Cell toxicity was assumed to be negligible under these limited exposure conditions since previous studies had found that a 24 hour incubation of 10 μM peptidyl SF8 probes did not induce cell toxicity.(15) (16)

Initial fluorescence microscopy experiments were conducted using integrin-positive A549 cells incubated with the three different SF8 probes (Figure 1A). The mean fluorescence intensity (MFI) of the epifluorescence micrographs indicated significantly higher probe uptake of SF8(DRGDG)2 compared to the control peptidyl probes (Figure 1B, see Figure S8 for a fluorescence image merged with light transmitted micrograph that indicates the cell limits) and this trend was also observed when the same fluorescence microscopy experiments were conducted using integrin-positive OVCAR-4 and HT-1080 cell lines (Figure 2). Furthermore, flow cytometry utilizing A549 cells confirmed significantly higher uptake of the targeted SF8(DRGDG)2 compared to the control SF8(DRADG)2 and SF8(DDGRG)2 (Figure 1C).

Figure 1.

Figure 1.

Open in a new tab

Enhanced uptake of SF8(DRGDG)2 by integrin-positive A549 cells. (A) Representative epifluorescence cell micrographs with (B) calculated mean fluorescence intensity showing probe uptake by A549 cells after a 30 min incubation with SF8 probes (1 μM) using fluorescence cell microscopy. SF8 probes in red; Hoechst nuclear stain in blue. Scale bar = 30 μm. (C) Mean fluorescence intensity showing probe up by A549 cells after a 15 min incubation with SF8 probes (0.1 μM) using flow cytometry. Asterisks represent * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001.

Figure 2.

Figure 2.

Open in a new tab

Enhanced uptake of SF8(DRGDG)2 by integrin-positive cells. Representative epifluorescence cell micrographs for probe uptake after a 30 min incubation of SF8 probes (1 μM) with (A) OVCAR-4 and (B) HT-1080 cells. The SF8 probes are shown as red; Hoechst nuclear stain as blue. Scale bar = 30 μm. (C) and (D) show the calculated mean fluorescence intensity. Asterisks represent * p<0.05, ** p<0.01, and **** p<0.0001.

Three sets of cell microscopy experiments were conducted in order to gain insight concerning the cell uptake mechanism of SF8(DRGDG)2. The first mechanistic experiment incubated the three integrin-positive cell lines with SF8(DRGDG)2 at either 37 °C or 4 °C. In each case, a significantly lower amount of probe uptake was observed at 4 °C, indicating active membrane transport as the probe uptake mechanism (Figure 3).(33)

Figure 3.

Figure 3.

Open in a new tab

Cold block of SF8(DRGDG)2 uptake by integrin-positive cells. The cells were incubated for 30 min with SF8(DRGDG)2 (1 μM) at either 37 °C or 4 °C. Representative epifluorescence micrographs qualitatively show intracellular probe uptake, with SF8(DRGDG)2 in red and Hoechst nuclear stain in blue. The bar graphs show mean cell fluorescence intensities for: (A) A549 cells, (B) OVCAR-4 cells, and (C) HT-1080 cells. Mean cell fluorescence intensity was normalized to probe uptake at 37 °C. Scale bar = 30 μm. Asterisks represent * p<0.05 and ** p<0.01.

The second mechanistic experiment was a competitive probe uptake fluorescence microscopy assay. A population of integrin-positive A549 cells was co-incubated with a binary 1:1 mixture of deep-red emitting SF8(DRGDG)2 and Cy3-cRGDfK (1 μM each), a commercially available orange-fluorescent probe with an appended cyclic RGDfK peptide sequence that is well-known to target αvβ5 and αvβ3 integrin receptors with high affinity.(21) As shown in Figure 4A the two probes co-localized within A549 cells with a Pearson’s correlation coefficient of 0.70. However, the presence of the Cy3-cRGDfK did not reduce cell uptake of SF8(DRGDG)2 (Figure S3), a result that contrasts with the observations of a previous study that showed that the presence of Cy3-cRGDfK reduced cell uptake of deep-red fluorescent cRGDfK probe by factor of 2.2.(30)

Figure 4.

Figure 4.

Open in a new tab

Colocalization of SF8(DRGDG)2 with Cy3-cRGDfK in A549 cells after co-incubation with the two probes (1 μM each) for 30 min. (A) Representative epifluorescence micrographs depict qualitative colocalization. SF8(DRGDG)2 in red and Cy3-cRGDfK in green; yellow shows colocalization and a Pearson’s correlation coefficient of 0.70 was determined by quantitative analysis. Scale bar = 30 μm. Bar graphs showing mean cell fluorescence intensities due to uptake of (B) Cy3-cRGDfK or (C) SF8(DRGDG)2 by A549 cells in the presence and absence of blocking agents.

The third set of mechanistic experiments included additional competition experiments that pretreated cells with excess amounts of the cyclic peptide cRGDfK or the analogous peptidyl drug Cilengitide (cRGDfVMe). As expected, control experiments showed that pre-incubating A549 cells with cRGDfK (200 μM) reduced cell uptake of Cy3-cRGDfK (Figure 4B and Figure S4) consistent with competitive binding for the RGD recognition site within the cell surface integrin receptor(s). Surprisingly, the same pre-incubation experiments found that neither cRGDfK (200 μM) or Cilengitide (50 μM) were able to block uptake of SF8(DRGDG)2 by A549 or OVCAR4 cells (Figure 4B and Figure S5-S7). These competition imaging experiments were repeated many times using fixed and living cells, and in all cases the cell uptake of SF8(DRGDG)2 was not reduced.

The results of these probe uptake studies lead to the following conclusions. The three SF8 probes have very similar chemical structures and the same log P values, yet there is much higher cell uptake of SF8(DRGDG)2 compared to the other two, indicating selective recognition of its peptide loop sequence by cell surface receptors. Cell uptake of SF8(DRGDG)2 is diminished at low temperature which is consistent with receptor-mediated endocytosis. Co-incubation of SF8(DRGDG)2 and Cy3-cRGDfK produces a high level of intracellular colocalization suggesting that both probes target largely the same cell surface integrin receptor(s). However, cell uptake of SF8(DRGDG)2 is not blocked by Cy3-cRGDfK, free cRGDfK peptide, or Cilengitide suggesting that access of SF8(DRGDG)2 to the RGD recognition site within the integrin receptor(s) is not needed for cell uptake. Integrins are known to recognize a wide range of extracellular matrix ligands beyond the RGD motif,(34) (35) and also bind small molecules at receptor locations that are proximal to the RGD recognition site.(36) (37) (38) (39) A goal for future studies is to determine the precise location of the receptor binding site for SF8(DRGDG)2.

CONCLUSIONS

Squaraine figure-eight (SF8) probes exhibit very high deep-red fluorescence and high stability and are very well suited for fluorescence cell microscopy. The fluorescent squaraine dye is encapsulated within the self-threaded structure which makes SF8 probes especially attractive for intracellular imaging because the buried dyes do not alter cell penetration properties. A comparison of three closely related SF8 probes with peptidyl loops shows that probe entry into cancer cells depends on the peptidyl loop sequence. The probe SF8(DRGDG)2 exhibited greatly enhanced cell uptake by receptor-mediated endocytosis. Since the SF8 synthetic method allows for ready alteration of the peptide loops, it will be easy to develop new probes with loop sequences that target other cancer biomarkers(40) such as NGR (targets aminopeptidase N),(41) HAIYPRH (targets transferrin receptor),(42) and SE (targets peptide transporter).(43) It should also be possible to create unsymmetrical SF8 probes with structures that have two different peptidyl loop sequences and thus possess dual targeting capabilities.(41, 44, 45)

Supplementary Material

Supp Material

ACKNOWLEDGMENTS:

This work has been supported in part by the US NIH (R35GM136212 and T32GM075762).

REFERENCES

  • 1.Zheng Q, Juette MF, Jockusch S, Wasserman MR, Zhou Z, Altman RB and Blanchard SC (2014) Ultra-stable organic fluorophores for single-molecule research. Chem. Soc. Rev 43, 1044–1056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Jradi FM and Lavis LD (2019) Chemistry of photosensitive fluorophores for single-molecule localization microscopy. ACS Chem. Biol 14, 1077–1090. [DOI] [PubMed] [Google Scholar]
  • 3.Yi X, Wang F, Qin W, Yang X and Yuan J (2014) Near-infrared fluorescent probes in cancer imaging and therapy: an emerging field. Int. J. Nanomedicine 9, 1347–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hong G, Antaris AL and Dai H (2017) Near-infrared fluorophores for biomedical imaging. Nat. Biomed. Eng 1, 0010. [Google Scholar]
  • 5.Ilina K, MacCuaig WM, Laramie M, Jeouty JN, McNally LR and Henary M (2020) Squaraine dyes: molecular design for different applications and remaining challenges. Bioconjug. Chem 31, 194–213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Escobedo JO, Rusin O, Lim S and Strongin RM (2010) NIR dyes for bioimaging applications. Curr. Opin. Chem. Biol 14, 64–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Cavaco M, Pérez-Peinado C, Valle J, Silva RDM, Correia JDG, Andreu D, Castanho MARB and Neves V (2020) To what extent do fluorophores bias the biological activity of peptides? A practical approach using membrane-active peptides as models. Front. Bioeng. Biotechnol 8, 552035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Simpson JD, Monteiro PF, Ediriweera GR, Prior AR, Sonderegger SE, Bell CA, Fletcher NL, Alexander C and Thurecht KJ (2021) Fluorophore selection and incorporation contribute to permeation and distribution behaviors of hyperbranched polymers in multi-cellular tumor spheroids and xenograft tumor models. ACS Appl. Bio Mater 4, 2675–2685. [DOI] [PubMed] [Google Scholar]
  • 9.Li D, Schreiber CL and Smith BD (2020) Sterically shielded heptamethine cyanine dyes for bioconjugation and high performance near‐infrared fluorescence imaging. Angew. Chem. Int. Ed 59, 12154–12161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Luciano MP, Crooke SN, Nourian S, Dingle I, Nani RR, Kline G, Patel NL, Robinson CM, Difilippantonio S, Kalen JD, Finn MG and Schnermann MJ (2019) A nonaggregating heptamethine cyanine for building brighter labeled biomolecules. ACS Chem. Biol 14, 934–940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Bernardin A, Chipon B, Bouteiller C, Clavé G, Massonneau M, Romieu A and Renard P-Y (2007) Novel water-soluble near-infrared cyanine dyes: synthesis, spectral properties, and use in the preparation of internally quenched fluorescent probes. Bioconjug. Chem 18, 1303–1317. [DOI] [PubMed] [Google Scholar]
  • 12.Cole EL, Arunkumar E, Xiao S, Smith BA and Smith BD (2012) Water-soluble, deep-red fluorescent squaraine rotaxanes. Org Biomol Chem 10, 5769–5773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Podgorski K, Terpetschnig E, Klochko OP, Obukhova OM and Haas K (2012) Ultra-Bright and -Stable Red and Near-Infrared Squaraine Fluorophores for In Vivo Two-Photon Imaging. PLoS One 7, e51980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Gassensmith JJ, Baumes JM and Smith BD (2009) Discovery and early development of squaraine rotaxanes. Chem. Commun 6329–6338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zhai C, Schreiber CL, Padilla‐Coley S, Oliver AG and Smith BD (2020) Fluorescent self-threaded peptide probes for biological imaging. Angew. Chem. Int. Ed 59, 23740–23747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Schreiber CL, Zhai C and Smith BD (2021) Chiral figure-eight molecular scaffold for fluorescent probe development. Org. Biomol. Chem DOI: 10.1039/D1OB00306B. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Knappe TA, Manzenrieder F, Mas-Moruno C, Linne U, Sasse F, Kessler H, Xie X and Marahiel MA (2011) Introducing lasso peptides as molecular scaffolds for drug design: Engineering of an integrin antagonist. Angew. Chem. Int. Ed 50, 8714–8717. [DOI] [PubMed] [Google Scholar]
  • 18.Hegemann JD, De Simone M, Zimmermann M, Knappe TA, Xie X, Di Leva FS, Marinelli L, Novellino E, Zahler S, Kessler H and Marahiel MA (2014) Rational improvement of the affinity and selectivity of integrin binding of grafted lasso peptides. J. Med. Chem 57, 5829–5834. [DOI] [PubMed] [Google Scholar]
  • 19.Saito F and Bode JW (2017) Synthesis and stabilities of peptide-based [1]rotaxanes: molecular grafting onto lasso peptide scaffolds. Chem. Sci 8, 2878–2884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Hatley RJD, Macdonald SJF, Slack RJ, Le J, Ludbrook SB and Lukey PT (2018) An αv-RGD integrin inhibitor toolbox: drug discovery insight, challenges and opportunities. Angew. Chem. Int. Ed 57, 3298–3321. [DOI] [PubMed] [Google Scholar]
  • 21.Danhier F, Breton A,Le Préat V,Le Breton A and Preát V (2012) RGD-Based strategies to target alpha(v) beta(3) integrin in cancer therapy and diagnosis. Mol. Pharm 9, 2961–2973. [DOI] [PubMed] [Google Scholar]
  • 22.Barczyk M, Carracedo S and Gullberg D (2010) Integrins. Cell Tissue Res. 339, 269–280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Huang R, Harmsen S, Samii JM, Karabeber H, Pitter KL, Holland EC and Kircher MF (2016) High precision imaging of microscopic spread of glioblastoma with a targeted ultrasensitive SERRS molecular imaging probe. Theranostics 6, 1075–1084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Lucie Sancey, Elisabeth Garanger, Stéphanie Foillard, Guy Schoehn, Amandine Hurbin, Corinne Albiges-Rizo, Didier Boturyn, Catherine Souchier, Alexeï Grichine, Pascal Dumy, and J.-L. C (2009) Clustering and internalization of integrin alpha v beta 3 with a tetrameric RGD-synthetic peptide. Mol. Ther 17, 837–843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Spitaleri A, Mari S, Curnis F, Traversari C, Longhi R, Bordignon C, Corti A, Rizzardi G-P and Musco G (2008) Structural basis for the interaction of iso DGR with the RGD-binding site of αvβ3 integrin. J. Biol. Chem 283, 19757–19768. [DOI] [PubMed] [Google Scholar]
  • 26.Song Y, Li W, Meng S, Zhou W, Su B, Tang L, Zhao Y, Wu X, Yin D, Fan M and Zhou C (2018) Dual integrin αvβ 3 and NRP-1-targeting paramagnetic liposome for tumor early detection in magnetic resonance imaging. Nanoscale Res. Lett 13, 380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Song C, Li F, Guo X, Chen W, Dong C, Zhang J, Zhang J and Wang L (2019) Gold nanostars for cancer cell-targeted SERS-imaging and NIR light-triggered plasmonic photothermal therapy (PPTT) in the first and second biological windows. J. Mater. Chem. B 7, 2001–2008. [DOI] [PubMed] [Google Scholar]
  • 28.Shaw SK, Liu W, Gómez Durán CFA, Schreiber CL, Betancourt Mendiola M. de L., Zhai C, Roland FM, Padanilam SJ and Smith BD (2018) Non-covalently pre-assembled high-performance near-infrared fluorescent molecular probes for cancer imaging. Chem. – A Eur. J 24, 13821–13829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Domcke S, Sinha R, Levine DA, Sander C and Schultz N (2013) Evaluating cell lines as tumour models by comparison of genomic profiles. Nat. Commun 4, 2126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Shaw SK, Schreiber CL, Roland FM, Battles PM, Brennan SP, Padanilam SJ and Smith BD (2018) High expression of integrin alpha v beta 3 enables uptake of targeted fluorescent probes into ovarian cancer cells and tumors. Bioorg. Med. Chem 26, 2085–2091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Lin RY, Dayananda K, Chen TJ, Chen CY, Liu GC, Lin KL and Wang YM (2012) Targeted RGD nanoparticles for highly sensitive in vivo integrin receptor imaging. Contrast Media Mol. Imaging 7, 7–18. [DOI] [PubMed] [Google Scholar]
  • 32.Wang X, Qiao X, Shang Y, Zhang S, Li Y, He H and Chen S (2017) RGD and NGR modified TRAIL protein exhibited potent anti-metastasis effects on TRAIL-insensitive cancer cells in vitro and in vivo. Amino Acids, 49, 931–941. [DOI] [PubMed] [Google Scholar]
  • 33.Yang C, Bromma K and Chithrani D (2018) Peptide mediated in vivo tumor targeting of nanoparticles through optimization in single and multilayer in vitro cell models. Cancers 10, 84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Lafoya B, Munroe JA, Miyamoto A, Detweiler MA, Crow JJ, Gazdik T and Albig AR (2018) Beyond the matrix: The many non-ECM ligands for integrins. Int. J. Mol. Sci 19, 1–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Kapp TG, Rechenmacher F, Neubauer S, Maltsev OV, Cavalcanti-Adam EA, Zarka R, Reuning U, Notni J, Wester HJ, Mas-Moruno C, Spatz J, Geiger B and Kessler H (2017) A comprehensive evaluation of the activity and selectivity profile of ligands for RGD-binding integrins. Sci. Rep 7, 1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Cody V, Davis PJ and Davis FB (2007) Molecular modeling of the thyroid hormone interactions with αvβ3 integrin. Steroids 72, 165–170. [DOI] [PubMed] [Google Scholar]
  • 37.Rajabi M, Godugu K, Sudha T, Bharali DJ and Mousa SA (2019) Triazole modified tetraiodothyroacetic acid conjugated to polyethylene glycol: High affinity thyrointegrin αvβ3 antagonist with potent anticancer activities in glioblastoma multiforme. Bioconjug. Chem 30, 3087–3097. [DOI] [PubMed] [Google Scholar]
  • 38.Li J, Liu H, Zuo R, Yang J and Li N (2020) Competitive binding assays for measuring the binding affinity of thyroid-disrupting chemicals for integrin αvβ3. Chemosphere 249, 126034. [DOI] [PubMed] [Google Scholar]
  • 39.Ruoslahti E (1996) RGD and other recognition sequences for integrins. Annu. Rev. Cell Dev. Biol 12, 697–715. [DOI] [PubMed] [Google Scholar]
  • 40.Zhao N, Qin Y, Liu H and Cheng Z (2017) Tumor-targeting peptides: ligands for molecular imaging and therapy. Anticancer Agents Med. Chem 18, 74–86. [DOI] [PubMed] [Google Scholar]
  • 41.Schreiber CL and Smith BD (2018) Molecular imaging of aminopeptidase n in cancer and angiogenesis. Contrast Media Mol. Imaging 2018, 5315172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Zhang Y, Wang Z, Wu L, Zong S, Yun B and Cui Y (2016) Dual peptides modified fluorescence-SERS dual mode imaging nanoprobes with improved cancer cell targeting efficiency. RSC Adv. 6, 81046–81052. [Google Scholar]
  • 43.Dai T, Li N, Zhang Y, Liu Q and Zhang L (2016) A new target ligand Ser-Glu for PEPT1-overexpressing cancer imaging. Int. J. Nanomedicine 11, 203–212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Wen Y, Schreiber CL and Smith BD (2020) Dual-targeted phototherapeutic agents as magic bullets for cancer. Bioconjug. Chem 31, 474–482. [DOI] [PubMed] [Google Scholar]
  • 45.Ehlerding EB, Sun L, Lan X, Zeng D and Cai W (2018) Dual-targeted molecular imaging of cancer. J. Nucl. Med 59, 390–395. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Material
NIHMS1769773-supplement-Supp_Material.pdf (3.1MB, pdf)

RESOURCES