DA7R: A 7-Letter Zip Code to Target PDAC

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, and is among the most aggressive and still incurable cancers. Innovative and successful therapeutic strategies are extremely needed. Peptides represent a versatile and promising tool to achieve tumor targeting, thanks to their ability to recognize specific target proteins (over)expressed on the surface of cancer cells. A7R is one such peptide, binding neuropilin-1 (NRP-1) and VEGFR2. Since PDAC expresses these receptors, the aim of this study was to test if A7R-drug conjugates could represent a PDAC-targeting strategy. PAPTP, a promising mitochondria-targeted anticancer compound, was selected as the cargo for this proof-of-concept study. Derivatives were designed as prodrugs, using a bioreversible linker to connect PAPTP to the peptide. Both the retro-inverso (DA7R) and the head-to-tail cyclic (cA7R) protease-resistant analogs of A7R were tested, and a tetraethylene glycol chain was introduced to improve solubility. Uptake of a fluorescent DA7R conjugate, as well as of the PAPTP-DA7R derivative into PDAC cell lines was found to be related to the expression levels of NRP-1 and VEGFR2. Conjugation of DA7R to therapeutically active compounds or nanovehicles might allow PDAC-targeted drug delivery, improving the efficacy of the therapy and reducing off-target effects.


Introduction
Pancreatic cancer is one of the most common and fearsome malignant tumors of the digestive system. Worldwide, nearly half a million people were diagnosed with pancreatic cancer in 2020, accounting for about 3% of all cancers (https://www.cancer.net/cancertypes/pancreatic-cancer/statistics, accessed on 3 April 2023). Despite intensive research, it is still among the malignancies with the most unfavorable prognosis [1,2]. The general 5-year survival rate in the US is about 11%. The corresponding figure for nonmetastatic invasive breast cancer, for example, is 90% (https://www.cancer.net/cancer-types/breastcancer/statistics, accessed on 3 April 2023). Pancreatic ductal adenocarcinoma (PDAC) [3,4] is the most common and lethal type of pancreatic cancer, accounting for about 90% of total cases. The challenges posed by PDAC derive from multiple factors, including lack of symptoms and consequent late diagnosis, early metastasis, difficulties in surgical resection and, notably, resistance to pharmacological treatments [5]. The tumors generally present concurrent mutations in multiple, poorly druggable driver genes, the "signature" ones being KRAS, P53, CDKN2A, and SMAD4 [6,7]. The presence of a complex, immunosuppressive What makes PAPTP particularly interesting is its effectiveness in killing a variety of cancer cells, including those deriving from glioma [45] and PDAC [46], i.e., tumors still lacking effective therapeutic options. Since these drugs act at the level of mitochondria, their action is independent of the status of upstream oncoproteins, such as Bcl-2 or p53 [44].
In this work, we synthesized different variants of the PAPTP-A7R conjugate. PAPTP was modified with the addition of a short linker to one of the phenyl rings of the triphenylphosphonium group (PAPTPL), to allow conjugation with the peptide in a position not interfering with binding to and inhibition of Kv1.3, and at the same time to ensure the suitable stability of the resulting derivative [47]. PAPTPL was connected to DA7R or cA7R via a bioreversible linker, allowing release of the active principle according to the "prodrug" concept ( Figure 1). The different conjugates were preliminarily characterized for their stability in blood and for their hemolytic activity. Cell uptake of a fluorescent FITC-DA7R conjugate, as well as of a PAPTP-DA7R conjugate (PAPTPL-I-DA7R), was finally evaluated in PDAC cell lines.
the resulting derivatives (PAPTP and PCARBTP) bind to and inhibit the Kv1.3 channel residing in the inner mitochondrial membrane, causing oxidative stress and selectively killing cancer cells while sparing normal ones [44]. Mitochondrial targeting is essential in determining the anticancer effectiveness of the compounds, since PAP-1 is much less effective.
What makes PAPTP particularly interesting is its effectiveness in killing a variety of cancer cells, including those deriving from glioma [45] and PDAC [46], i.e., tumors still lacking effective therapeutic options. Since these drugs act at the level of mitochondria, their action is independent of the status of upstream oncoproteins, such as Bcl-2 or p53 [44].
In this work, we synthesized different variants of the PAPTP-A7R conjugate. PAPTP was modified with the addition of a short linker to one of the phenyl rings of the triphenylphosphonium group (PAPTPL), to allow conjugation with the peptide in a position not interfering with binding to and inhibition of Kv1.3, and at the same time to ensure the suitable stability of the resulting derivative [47]. PAPTPL was connected to DA7R or cA7R via a bioreversible linker, allowing release of the active principle according to the "prodrug" concept ( Figure 1). The different conjugates were preliminarily characterized for their stability in blood and for their hemolytic activity. Cell uptake of a fluorescent FITC-DA7R conjugate, as well as of a PAPTP-DA7R conjugate (PAPTPL-I-DA7R), was finally evaluated in PDAC cell lines.

. Materials and Instruments
Fmoc-protected amino acids, preloaded 2-Cl-Trytil resin, and coupling reagents (HBTU and HOBt) were purchased from Iris Biotech (Marktredwitz, Germany). Other reagents and solvents were purchased from Sigma Aldrich (Milan, Italy), and were used as received.
Preparative HPLC was performed using a Shimadzu LC-8 system (Shimazdu, Kyoto, Japan) with a C18 column (Vydac 218TP1022, 10 µm, 250 × 22 mm). The column was perfused at a flow rate of 12 mL/min with a mobile phase consisting of eluent A (0.05% TFA in water) and B (0.05% TFA in 9:1 v/v acetonitrile:water); eluent B was increased over time following a linear gradient.
Analytical HPLC analyses were performed on a Shimadzu LC-10 instrument fitted with a Jupiter C18 column (10µm, 250 × 4.6 mm Phenomenex, Torrance, CA, USA) using the above eluent system (eluents A and B), flow rate of 1 mL/min, detection at 216 nm. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded with a Bruker 500 Avance III operating at 500 MHz (for 1 H NMR) and 126 MHz (for 13 C NMR). Chemical shifts (δ) are given in parts per million (ppm) relative to the signal of the solvent. The following abbreviations are used to indicate multiplicities: d, doublet; t, triplet; m, multiplet.
Electrospray ionization-mass spectrometry (ESI-MS) analysis was carried out with a Mariner mass spectrometer (PerSeptive Biosystem, Framingham, MA, USA) or a 1100 Series Agilent Technologies system (Agilent Technologies, Milan, Italy). The ESI source operated in full-scan positive ion mode, applying the following ESI parameters: nebulizer pressure 20 psi, dry gas flow 5 L/min, dry gas temperature 325 • C. The flow rate was 0.05 mL/min.
Microwave-assisted syntheses were performed in a CEM Discover ® monomode reactor (CEM Srl, Cologno Al Serio (BG), Italy) with the temperature monitored by a built-in infrared sensor.

Peptide Synthesis
Peptides were synthesized by manual solid phase using Fmoc chemistry in 0.06 mmole scale using a preloaded (L-Arg or D-Ala) 2-Cl-Trt-resin. HBTU/HOBt activation employed a three-fold molar excess (0.24 mmol) of Fmoc-amino acids in DMF (N,N-Dimethylformamide) for each coupling cycle, unless otherwise stated. Coupling time was 40 min. Fmoc deprotection was performed with 20% piperidine. Coupling yields were monitored on aliquots of peptide-resin by evaluation of Fmoc displacement. The cleavage of peptide from the resin was performed by treatment with 1% TFA in DCM (Dichloromethane) (4 × 5 mL) followed by neutralization with pyridine, to preserve the side chain protecting groups. Deprotection of peptides was achieved by treatment with a TFA-triisopropylsilane-H 2 O (96:3:1 v/v) mixture for 45 min at room temperature. Pra-A7R peptide was cyclized in dilute DMF solution (1 mM) by the addition of 2 eq. of PyBOP in the presence of HOBt (2 eq.) and DIPEA (4 eq.). Peptides were purified by preparative reversed-phase HPLC. The fractions containing the desired products were collected and lyophilized to constant weight. All peptides showed less than 1% impurities. Molecular weights of compounds were determined by ESI-MS on a Mariner mass spectrometer. The mass was assigned using a mixture of neurotensin, angiotensin, and bradykinin at a concentration of 1 pmol/µL as external standard.

Protein Extraction
Cells were washed with cold PBS, detached with trypsin, and centrifuged at 2000× g for 5 min. The resulting pellet was then resuspended in 0.2 mL RIPA lysis buffer containing protease and phosphatase inhibitors (Merck Life Science S.r.l., Milano, Italy). Each sample was incubated for 30 min on ice and then physically disaggregated with three freeze-andthaw cycles using liquid nitrogen. The lysates were then centrifuged at 20,000× g for 15 min at 4 • C, and finally transferred to a new tube. Quantification of the total protein content in each lysate was performed using the BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

Blood Stability
Procedures involving animals were approved by the University of Padova Ethical Committee for Animal Welfare (OPBA) and by the Italian Ministry of Health (Permit Number 846/2021-PR), and conducted with the supervision of the Central Veterinary Service of the University of Padova, in compliance with Italian Law DL 26/2014, embodying UE Directive 2010/63/EU. Mice were anesthetized and blood was withdrawn from the jugular vein and heparinized. Blood samples (1 mL) were spiked with compound (5 µM; dilution from a 5 mM stock solution in DMSO) and incubated at 37 • C for 4 h (the maximum period allowed by blood stability). Aliquots (100 µL) were taken after 10 min, 30 min, 1 h, 2 h, and 4 h, kept on ice, and treated as follows: blood was mixed with 5 volumes of 0.1%TFA in CH 3 CN, mixed and sonicated for 2 min, and finally centrifuged for 7 min at 12,000× g; the supernatant was collected and analyzed by UHPLC. Quantitative analyses were carried out by UHPLC (1290 Infinity LC System, Agilent Technologies, Milan, Italy) using a reversed-phase column (Zorbax RRHT Extend C18, 1.8 µm, 50 × 3.0 mm, Agilent Technologies) and a UV diode array detector (190-500 nm), at a flow rate of 0.6 mL/min. Solvents A and B were 0.1% TFA in water and CH 3 CN, respectively. The gradient for B was as follows: 10% for 1 min, then from 10 to 100% in 8.0 min, 100% for 0.5 min, then from 100% to 10% in 1.5 min. The injection volume was 5 µL. The eluate was preferentially monitored at 312 nm (corresponding to an absorbance maximum of psoralenic compounds). The temperature of the column was kept at 25 • C. Concentration of PAPTPL or its derivatives was determined using the following calibration curve, which correlates peak area at 312 nm (y) with compound concentration in the sample (x): y = 6.3321x. The stability in blood was expressed as % of PAPTPL released relative to the amount of the derivative initially spiked in the sample.

Hemolysis Assay
Hemolysis studies were conducted as described in [48]. Briefly, freshly withdrawn blood samples were centrifuged at 2000× g for 10 min to obtain erythrocytes; the pellet was then washed three times with sterile PBS (pH 7.4). Erythrocytes (5 × 10 7 cells in 1 mL of PBS for each experimental condition) were incubated with the tested compounds at the concentrations of 1, 0.5, 0.25, or 0.125 nmoles/10 7 cells for 15 min. The samples were then centrifuged and the absorbance (Abs) of the supernatant was measured at 540 nm. Triton X-100 1% and PBS were used as positive and negative control, respectively. The percentage of hemolysis was calculated with the following equation:

Cell Uptake of PAPTPL-I-DA7R
HPAFII and Pan02 cells were seeded in 6-well plates at a density of 300,000 and 150,000 cells/well, respectively, and grown for 48 h. They were then washed twice with PBS and incubated for 15 min at 37 • C with a solution of 5 µM PAPTPL-I-DA7R in DMEM without Phenol Red and serum. At the end of the incubation, cells were washed twice, 100 µL of PBS were added, and finally the cells were scraped and collected. A volume of 100 µL of CH 3 CN + 0.1%TFA was added, and the mixture was vortexed and sonicated for 5 min. Samples were finally centrifuged for 5 min at 12,000× g, and the supernatant was collected and the amount of PAPTPL-I-DA7R was quantified by HPLC/UV, as described in Section 2.5 (blood stability). The remaining cell pellets were resuspended in 200 µL of SDS 2%, and the total protein amount of each sample was quantified with the BCA assay. Cell uptake of PAPTPL-I-DA7R was expressed as nmoles of PAPTPL-I-DA7R/total protein.

Statistics
Significance in comparisons was assessed using the Mann-Whitney-Wilcoxon test.

Chemistry
We first synthesized a fluorescent derivative of the retro-inverso peptide variant (FITC-DA7R), to follow peptide uptake into PDAC cells. We then proceeded to link the same peptide to the chemotherapeutic compound PAPTP, using a carbamate bond to allow the release of the active drug over time. The first derivative (PAPTPL-I-DA7R) was produced attaching the peptide to the molecule through an Isoleucine linker. An analog comprising a tetraethylene glycol chain, inserted to increase the water solubility of the construct, was then synthesized using click chemistry (PAPTPL-Teg-DA7R). Following the same procedures, we also synthesized an analog of this latter compound comprising the cyclic instead of the retro-inverso peptide (PAPTPL-Teg-cA7R).

Peptide Design and Synthesis
Linear peptides (A7R and DA7R (Figure 2)) were synthesized by manual solid phase peptide synthesis starting from a preloaded 2-Cl-Trytil resin using the Fmoc/HBTU chemistry. To prevent the formation of byproducts due to deletion reactions either in the Pro-Pro or in the Xaa-Pro sequences, double coupling of either Fmoc-Pro-OH or Fmoc-Xaa-OH residue was performed using HATU as a coupling agent.
ido analogue (see below). The Pra-A7R peptide was detached from the resin by treatment with 1% trifluoroacetic acid (TFA) in DCM, and the crude peptide was cyclized in a diluted DMF solution (final peptide concentration 1 mM) by the addition of PyBop as a coupling reagent in the presence of HOBt and DIEA. After treatment with TFA to remove side chain protecting groups and RP-HPLC purification, the peptide was obtained in good yield with a purity of >95%.

Synthesis of PAPTPL-I-DA7R
PAPTPL-I (Figure 1) was synthesized according to the procedure described in [47], and then coupled to DA7R with the peptide still attached to the 2-Cl-Trytil resin following standard solid-phase synthetic procedures. After detachment from the resin by TFA treatment, the PAPTPL-I-DA7R conjugate was purified by preparative HPLC, and characterized by ESI-MS (Supplementary Figure S1).

Synthesis of PAPTPL-Teg-DA7R and PAPTPL-Teg-cA7R
Synthesis of PAPTPL-Teg-N3 The synthesis of PAPTPL-Teg-N3 is illustrated in Scheme 1. In the first step, 14-azido-3,6,9,12-tetraoxatetradecan-1-amine was converted to the 4-nitrophenyl carbamate derivative 1 by reaction with bis(4-nitrophenyl) carbonate in the presence of DMAP. In the second step, PAPTPL (synthesized according to the procedure described in [47]) was coupled to the intermediate 1 in the presence of DMAP to afford PAPTPL-Teg-N3 in excellent yield (93%). The Teg chain was introduced into the structure of PAPTPL-Teg-N3 to improve the water solubility of the resulting derivative. In the synthesis of cA7R (Figure 2), an additional propargylglycine residue (Pra) was added to the N-terminal amino group of the resin-attached peptide, before its final cyclization. The introduction of this residue provides an alkyne moiety in the side chain of the cyclic peptide to be used for the subsequent condensation reaction to the PAPTP azido analogue (see below). The Pra-A7R peptide was detached from the resin by treatment with 1% trifluoroacetic acid (TFA) in DCM, and the crude peptide was cyclized in a diluted DMF solution (final peptide concentration 1 mM) by the addition of PyBop as a coupling reagent in the presence of HOBt and DIEA. After treatment with TFA to remove side chain protecting groups and RP-HPLC purification, the peptide was obtained in good yield with a purity of >95%.

Synthesis of PAPTPL-I-DA7R
PAPTPL-I (Figure 1) was synthesized according to the procedure described in [47], and then coupled to DA7R with the peptide still attached to the 2-Cl-Trytil resin following standard solid-phase synthetic procedures. After detachment from the resin by TFA treatment, the PAPTPL-I-DA7R conjugate was purified by preparative HPLC, and characterized by ESI-MS (Supplementary Figure S1).

Synthesis of PAPTPL-Teg-DA7R and PAPTPL-Teg-cA7R Synthesis of PAPTPL-Teg-N 3
The synthesis of PAPTPL-Teg-N 3 is illustrated in Scheme 1. In the first step, 14azido-3,6,9,12-tetraoxatetradecan-1-amine was converted to the 4-nitrophenyl carbamate derivative 1 by reaction with bis(4-nitrophenyl) carbonate in the presence of DMAP. In the second step, PAPTPL (synthesized according to the procedure described in [47]) was coupled to the intermediate 1 in the presence of DMAP to afford PAPTPL-Teg-N 3 in excellent yield (93%). The Teg chain was introduced into the structure of PAPTPL-Teg-N 3 to improve the water solubility of the resulting derivative.

Synthesis of PAPTPL-Teg-cA7R
PAPTPL-Teg-cA7R (Figure 1) was obtained by copper(I)-catalyzed azide-alkyne cycloaddition between PAPTPL-Teg-N 3 and cA7R-alkyne. The reaction was performed in a microwave reactor to speed up the process. The product was purified by preparative HPLC and characterized by ESI-MS (Supplementary Figure S1).

Neuropilin-1 and VEGFR2 Expression in PDAC Cell Lines
Since A7R is recognized by NRP-1 and VEGFR2, we evaluated whether the two receptors are expressed in vitro in a panel of PDAC cell lines (five human and one murine). The results obtained by Western blot analysis highlighted a great variability in the expression of NRP-1 and VEGFR2 in the different cell lines ( Figure 3A). A PDAC tissue lysate was used as positive control and confirmed high expression levels of both receptors in an orthotopically-implanted Pan02-PDAC. HPAFII cells clearly showed expression of both the receptors, and were thus used for the uptake studies. Cultured Pan02 cells, on the other hand, express low levels of both proteins (near/below the detection limit of Western blot), and were thus used as "negative" controls ( Figure 3B,C).

Synthesis of PAPTPL-Teg-cA7R
PAPTPL-Teg-cA7R (Figure 1) was obtained by copper(I)-catalyzed azide-alkyne cycloaddition between PAPTPL-Teg-N3 and cA7R-alkyne. The reaction was performed in a microwave reactor to speed up the process. The product was purified by preparative HPLC and characterized by ESI-MS (Supplementary Figure S1).

Neuropilin-1 and VEGFR2 Expression in PDAC Cell Lines
Since A7R is recognized by NRP-1 and VEGFR2, we evaluated whether the two receptors are expressed in vitro in a panel of PDAC cell lines (five human and one murine). The results obtained by Western blot analysis highlighted a great variability in the expression of NRP-1 and VEGFR2 in the different cell lines ( Figure 3A). A PDAC tissue lysate was used as positive control and confirmed high expression levels of both receptors in an orthotopically-implanted Pan02-PDAC. HPAFII cells clearly showed expression of both the receptors, and were thus used for the uptake studies. Cultured Pan02 cells, on the other hand, express low levels of both proteins (near/below the detection limit of Western blot), and were thus used as "negative" controls ( Figure 3B,C).

Blood Stability and Hemolysis
Since the derivatives were designed as prodrugs, regeneration of the active compound (i.e., PAPTPL) with suitable kinetics once in circulation is essential for future applications. We thus evaluated blood stability and hemolytic activity of the three PAPTPL derivatives synthesized in this study. PAPTPL-I-DA7R turned out to be the most stable, with about 30% of the conjugate being hydrolyzed within 4 h. PAPTPL-Teg-DA7R and PAPTPL-Teg-cA7R underwent a slightly faster hydrolysis, with about 50% hydrolysis in 4 h (Figure 4).
Since the derivatives were designed as prodrugs, regeneration of the active compound (i.e., PAPTPL) with suitable kinetics once in circulation is essential for future applications. We thus evaluated blood stability and hemolytic activity of the three PAPTPL derivatives synthesized in this study. PAPTPL-I-DA7R turned out to be the most stable, with about 30% of the conjugate being hydrolyzed within 4 h. PAPTPL-Teg-DA7R and PAPTPL-Teg-cA7R underwent a slightly faster hydrolysis, with about 50% hydrolysis in 4 h (Figure 4). Previous studies have shown that PAPTP-peptide constructs can be hemolytic [29,47,49], and this can heavily impact their future in vivo applications, since the derivatives are expected to reach the bloodstream upon administration. Hemolysis assays revealed a quite different behavior of the three PAPTPL conjugates: PAPTPL-I-DA7R proved to be completely safe and did not induce any hemolysis, while the addition of Teg caused an increase in the ability of the derivative (PAPTPL-Teg-DA7R) to lyse erythrocytes. Finally, the presence of the cyclic peptide caused a dramatic increase in the hemolysis induced by PAPTPL-Teg-cA7R ( Figure 5). cA7R itself was completely ineffective towards erythrocytes. Based on these results, we thus considered for further experiments only PAPTPL-I-DA7R.  Previous studies have shown that PAPTP-peptide constructs can be hemolytic [29,47,49], and this can heavily impact their future in vivo applications, since the derivatives are expected to reach the bloodstream upon administration. Hemolysis assays revealed a quite different behavior of the three PAPTPL conjugates: PAPTPL-I-DA7R proved to be completely safe and did not induce any hemolysis, while the addition of Teg caused an increase in the ability of the derivative (PAPTPL-Teg-DA7R) to lyse erythrocytes. Finally, the presence of the cyclic peptide caused a dramatic increase in the hemolysis induced by PAPTPL-Teg-cA7R ( Figure 5). cA7R itself was completely ineffective towards erythrocytes. Based on these results, we thus considered for further experiments only PAPTPL-I-DA7R. pound (i.e., PAPTPL) with suitable kinetics once in circulation is essential for future applications. We thus evaluated blood stability and hemolytic activity of the three PAPTPL derivatives synthesized in this study. PAPTPL-I-DA7R turned out to be the most stable, with about 30% of the conjugate being hydrolyzed within 4 h. PAPTPL-Teg-DA7R and PAPTPL-Teg-cA7R underwent a slightly faster hydrolysis, with about 50% hydrolysis in 4 h (Figure 4). Previous studies have shown that PAPTP-peptide constructs can be hemolytic [29,47,49], and this can heavily impact their future in vivo applications, since the derivatives are expected to reach the bloodstream upon administration. Hemolysis assays revealed a quite different behavior of the three PAPTPL conjugates: PAPTPL-I-DA7R proved to be completely safe and did not induce any hemolysis, while the addition of Teg caused an increase in the ability of the derivative (PAPTPL-Teg-DA7R) to lyse erythrocytes. Finally, the presence of the cyclic peptide caused a dramatic increase in the hemolysis induced by PAPTPL-Teg-cA7R ( Figure 5). cA7R itself was completely ineffective towards erythrocytes. Based on these results, we thus considered for further experiments only PAPTPL-I-DA7R.

DA7R-FITC Cell Uptake
To establish whether DA7R is taken up by cells, we performed flow cytometry experiments using the fluorescent construct FITC-DA7R; fluorescence increase was monitored over time (15 min and 2 h) in HPAFII and Pan02 cells. HPAFII cells showed a significant increase of the fluorescence signal (median of the fluorescence of the cell population) compared to the control. This increase was related to NRP-1 and VEGFR2 expression, since it was significantly different from that observed in cultured Pan02 cells, which express non-detectable levels of these proteins (Figure 6).
To establish whether DA7R is taken up by cells, we performed flow cytometry experiments using the fluorescent construct FITC-DA7R; fluorescence increase was monitored over time (15 min and 2 h) in HPAFII and Pan02 cells. HPAFII cells showed a significant increase of the fluorescence signal (median of the fluorescence of the cell population) compared to the control. This increase was related to NRP-1 and VEGFR2 expression, since it was significantly different from that observed in cultured Pan02 cells, which express non-detectable levels of these proteins ( Figure 6).

Cellular Uptake of PAPTPL-I-DA7R
Uptake of PAPTPL-I-DA7R was evaluated in HPAFII and Pan02 cell lines. Cells were incubated with the conjugate for 15 min, and then medium and cells were collected, extracted and analyzed by HPLC/UV. The results showed that uptake of PAPTPL-I-DA7R by HPAFII cells, which express both NRP-1 and VEGFR2, was roughly twice as much as that by cultured Pan02 (in which the levels of the receptors are under the detection limit) (Figure 7).
The results suggest that conjugation with the DA7R peptide confers a certain selectivity in cell uptake, which is related to the expression levels of NRP-1 and VEGFR2 in the cells.

Cellular Uptake of PAPTPL-I-DA7R
Uptake of PAPTPL-I-DA7R was evaluated in HPAFII and Pan02 cell lines. Cells were incubated with the conjugate for 15 min, and then medium and cells were collected, extracted and analyzed by HPLC/UV. The results showed that uptake of PAPTPL-I-DA7R by HPAFII cells, which express both NRP-1 and VEGFR2, was roughly twice as much as that by cultured Pan02 (in which the levels of the receptors are under the detection limit) (Figure 7). nificant increase of the fluorescence signal (median of the fluorescence of the cell population) compared to the control. This increase was related to NRP-1 and VEGFR2 expression, since it was significantly different from that observed in cultured Pan02 cells, which express non-detectable levels of these proteins (Figure 6).

Cellular Uptake of PAPTPL-I-DA7R
Uptake of PAPTPL-I-DA7R was evaluated in HPAFII and Pan02 cell lines. Cells were incubated with the conjugate for 15 min, and then medium and cells were collected, extracted and analyzed by HPLC/UV. The results showed that uptake of PAPTPL-I-DA7R by HPAFII cells, which express both NRP-1 and VEGFR2, was roughly twice as much as that by cultured Pan02 (in which the levels of the receptors are under the detection limit) (Figure 7).
The results suggest that conjugation with the DA7R peptide confers a certain selectivity in cell uptake, which is related to the expression levels of NRP-1 and VEGFR2 in the cells. The results suggest that conjugation with the DA7R peptide confers a certain selectivity in cell uptake, which is related to the expression levels of NRP-1 and VEGFR2 in the cells.

Discussion
We have reported the successful development and preliminary testing of a peptidedrug conjugate directed against PDAC, one of the most terrible cancers on the clinical scene. The concept underlying the new molecule is that of a "prodrug", comprising a targeting portion and an apoptosis-inducing chemotherapeutic drug to be released. The former is based on peptide DA7R, expected to target the NRP-1/VEGFR2 receptor complex in PDAC cells, as it does in other systems. As payload, i.e., chemotherapeutic active principle, we used PAPTP, a mitochondriotropic drug under development in our group [44][45][46][50][51][52].
The segment connecting these two key parts of the molecule comprises a linker needed to separate the hydrolysis-prone carbamate bond system from the activating positive charge of the triphenylphosphonium moiety. We followed, in this respect, the same approach used to build a conjugate of PAPTP and peptide Angiopep-2 [47]. As a labile joint conferring the prodrug character to the construct, we considered using a carboxyester group, but preliminary experiments showed that in blood it was hydrolyzed too rapidly to be useful. The more solid carbamate, which we had already used in other cases (e.g., [47]), was confirmed to be a more suitable choice, hydrolyzing with a t 1/2 of about 4-6 h ( Figure 4).
We also synthesized variants comprising, besides PAPTPL and cA7R or DA7R, a Teg chain, inserted to increase the water solubility of the construct, as well as a triazole ring produced by the coupling of the two halves of the construct by a "click" reaction as a final step in the synthesis. In all the derivatives, the Arg side chain of the peptide was free and not involved in conjugation; given the literature regarding the interaction of DA7R and cA7R with VEGFR2 and NRP-1 [19,20], this is expected to be sufficient to retain a certain affinity for the receptors. Furthermore, the "cargo" (i.e., PAPTPL-I, PAPTPL-Teg) has a flexible hydrophobic structure which could well accommodate structural changes of the construct for an optimal interaction with the receptors. This strategy was already proven to be successful with a DA7R derivative with myristic acid: even if the conjugation involved the N-terminal Arg amino group of DA7R, the derivative was shown to retain (and also have improved) cell uptake and glioma-homing properties compared to DA7R itself [53].
The PAPTP constructs comprising a Teg chain were not utilized in uptake studies because they proved hemolytic in preliminary studies ( Figure 5). Their behavior confirms that elaborations, such as the attachment of "cargo" groups or linker moieties, may confer the ability to cause lysis of erythrocytes [47], even though the peptide itself shows no, or little, such activity. Such controls are therefore clearly needed for each construct intended to be transported by the bloodstream.
The results of the uptake experiments validate the idea that DA7R may be a good candidate as a peptide targeting PDAC. The data obtained with the FITC-DA7R and PAPTPL-I-DA7R (Figures 6 and 7) clearly show that the delivery of "cargo" to the cellular interior is strongly favored by the expression of the receptor molecules on the cell surface. Since PDAC cells and the tumoral stroma express high levels of these receptors in comparison with healthy tissue [30,35], this in vitro selectivity may well be reflected in vivo.
So far, A7R has been mainly used to enhance transport across the BBB and antiglioma/glioblastoma activity. The constructs we developed may therefore be useful also in that field, especially in view of the current understanding of the role of neuropilin in those cancers [24,54].
In principle, A7R might be used to guide to their target the major drugs currently used-with little success-against PDAC, namely, 5-FU and gemcitabine. Indeed, since these are generic "anti-metabolite" drugs, focusing their delivery may be advantageous, also to reduce undesirable side-effects. They are more hydrophilic than PAPTP, thus they may be less problematic than PAPTP constructs from this point of view. These drugs, also including PAPTP and drug combinations such as FOLFIRINOX [3,55], may be steered using conjugates or DA7R-decorated nanovehicles.