Anticancer Activity of Dendriplexes against Advanced Prostate Cancer

Subscriber access provided by University of Winnipeg Library

Article

Anticancer Activity of Dendriplexes against Advanced Prostate Cancer from Protumoral Peptides and Cationic Carbosilane Dendrimers María Sanchez-Milla, Laura Muñoz-Moreno, Javier Sánchez-Nieves, Marek Maly, Rafael Gómez, Maria J. Carmena, and F. Javier de la Mata Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01632 • Publication Date (Web): 22 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Anticancer Activity of Dendriplexes against Advanced Prostate Cancer from Protumoral Peptides and Cationic Carbosilane Dendrimers María Sánchez-Milla,a,b,# Laura Muñoz-Moreno,b,c,d,# Javier Sánchez-Nieves,a,b,e Marek Malý,f Rafael Gómez,a,b,e María J. Carmena,c,* and F. Javier de la Mataa,b,e,*

a

Department of Química Orgánica y Química Inorgánica, Instituto de Investigación

Química "Andrés M. del Río" (IQAR), Campus Universitario; Universidad de Alcalá; E28805 Alcalá de Henares (Madrid) Spain; e-mail [email protected]; [email protected].

b

Networking Research Center for Bioengineering, Biomaterials and Nanomedicine

(CIBER-BBN).

c

Department of Biología de Sistemas, Universidad de Alcalá, Campus Universitario, E-

28805 Alcalá de Henares, Spain.

ACS Paragon Plus Environment

1

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

d

Page 2 of 60

Actual address: Institute for Health Research, Hospital Universitario La Paz, E- 28046

Madrid, Spain.

e

Institute Ramón y Cajal for Health Research (IRYCIS).

f

Faculty of Science, J. E. Purkinje University, České mládeže 8, 400 96 Ústí nad

Labem, Czech Republic.

#

These authors contributed equally to the work.

KEYWORDS: prostate cancer; VIP peptide; GHRH peptide; carbosilane dendrimer; molecular dynamics.

ABSTRACT.

The interaction of neuropeptides, vasoactive intestinal peptide (VIP) or growth hormonereleasing hormone (GHRH), with a cationic carbosilane dendrimer forms dendriplexes with antitumoral behavior in advanced prostate cancer cells PC3. At the concentrations used for dendriplexes formation, the free peptides were protumoral and prometastatic in


2

Page 3 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Advanced Prostate Cancer, whilst dendrimer only showed low cytotoxicity, but did not avoid the metastatic behavior of PC3 cells. However, these nanoplexes favoured also cell adhesion and avoided cell migration. Also, the dendriplexes were not toxic for no tumoral prostate cells (RPWE-1) or fibroblasts. The use of labelled GHRH peptide (rhodamine labelled) and a dendrimer (fluorescein labelled) allowed us to observe that both systems reach the intracellular milieu after dendriplex formation. The treatment of PC3 cells with the nanoplexes reduced expression of vascular endothelial growth factor (VEGF) and cyclic adenosine monophosphate (cAMP). Molecular modelling analysis highlights the important contribution of the carbosilane framework in the stabilization of the dendriplex, since dendrimer interacts with a peptide region where hydrophobic aminoacids are presented.


3


Page 4 of 60

1. Introduction

Prostate cancer is the second most frequent cancer among men.1 The 2012 statistics in Europe (38 countries) depicts around 417000 new cases and 96000 deaths2 and in USA (year 2014)3 over 172000 new cases and 28000 deaths. In its earlier stage, it can be adequately treated, i. e. by androgen deprivation therapy. However, if the tumor cells show a metastatic phenotype the treatment is nowadays only palliative. In this last stage, the castration resistant prostate cancer (CRPC) or advanced prostate cancer has become androgen-independent and, unfortunately, is even favored by several human neuropeptides, as vasoactive intestinal peptide (VIP)4 and growth hormone-releasing hormone (GHRH).5 In the particular case of VIP, this peptide is present in the human prostate gland and increases the expression of the major angiogenic factor, the vascular endothelial growth factor (VEGF). VEGF promotes vasculogenesis and angiogenesis but also metastatic progression. Since this peptide is secreted by cancer androgen-independent cells (PC3), VIP has autocrine and paracrine actions in CRPC.6 Regarding GHRH, it is produced by the hypothalamus and stimulates GH secretion from


4

Page 5 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

pituitary somatotrophs.7 In turn, GH favours the production of the insulin-like growth factor I (IGF-I), which is also involved in tumorigenesis and metastasis. As for VIP, GHRH has also autocrine and paracrine actions in CRPC.8

The biological action of these peptides is mediated by their corresponding receptors that for both peptides belong to the family B of the secretin receptor family. Whereas VIP receptors (VPAC1, VPAC2, PAC1) can also show affinity for other analogous peptides as pituitary adenylyl cyclase-activating peptide (PACAP), and vice versa,9 GHRH receptor (GHRH-R) is highly selective for this peptide.7 Nevertheless, in both cases, after peptide binding to receptors, similar signalling cascades are activated, involving the increase of cAMP as second messenger.

VIP and GHRH receptors and their splice variants (SVs) are overexpressed in numerous cancer cells.4,10,11 For that reason, VIP and GHRH receptor antagonists have been explored against different cancers with positive results, their activity being even improved in combination with antitumoral drugs. A different approach has taken advantage of the overexpression of these receptors by combining the peptides with


5


Page 6 of 60

antitumoral formulations, in this case the peptide being the drug carrier.12 One major drawback of these strategies is the peptide or antagonist stability in biological environment due to enzyme degradation. To solve this problem, combination of peptides with multivalent protecting nanosystems has been explored as a solution. These new systems could be more effective against cancer in vivo due to the EPR effect (Enhanced Permeability and Retention).13,14

As multivalent nanosystems, nanoparticles, polymers and dendrimers are widely employed. The first two derivatives are fast-grown synthesized but lead to polydisperse materials, which in the case of polymers are of random topology. However, dendrimers are multivalent macromolecules but monodisperse and with well-defined size and structure, as consequence of their step-by-step synthesis.15-17 Dendrimers and their derivatives have been also explored as drug carriers, increasing the solubility of the drug or its activity by supporting several active units on one macromolecule. Peptidedendrimers are dendrimers with a surface decorated with peptides.18 This type of macromolecules is of special interest since multipeptide functionalization is expected to


6

Page 7 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

increase their activity with respect to the peptide itself. For example, a dendrimer containing gonadotropin-releasing hormone (GnRH) agonist showed better behaviour for the treatment of hormone-resistant prostate cancer than the currently available GnRH agonists.19

Some of us have deeply studied the protumoral activity of VIP and GHRH in CRPC.20-24 With the aim to improve the stability of VIP and GHRH and to evaluate the ability of these peptides as carriers, the study of peptide/carbosilane dendrimers dendriplexes was proposed. The scaffold of carbosilane dendrimers is made of very apolar C-C and Si-C bonds, being highly hydrophobic, whilst widely employed PAMAM, PPI and polyester dendrimers contain a hydrophilic scaffold. The water solubility can be achieved by introducing ionic groups at the periphery, i. e. cationic groups.25,26 Moreover, the presence of ammonium functions at carbosilane dendrimer periphery has been useful to interact with anionic peptides27-30 and also as non-viral vectors for gene therapy.25,31 On the other hand, the hydrophobic framework of these dendrimers is also of relevance. Thus, cationic carbosilane dendrimers are able to cross the blood brain


7


Page 8 of 60

barrier32,33 and smaller systems penetrate into the double strand of siRNA, establishing interactions through the hydrophobic moieties of both dendrimers and the RNA.34 On the other hand, Hatano et al. reported that the surface of carbosilane dendrimers can also be modified with peptides, i. e. hemagglutinin binding peptide, showing antiinfluenza virus activity.35

Herein, we report the antitumoral activity of the dendriplexes formed between VIP or GHRH with cationic carbosilane dendrimers. The nanoplexes are formed at concentrations in which the peptides are protumoral and the dendrimers present scarce cytotoxicity. The effect of these dendriplexes in cellular adhesion, metastasis, expression of vascular endothelial growth factor (VEGF), and toxicity in no-tumoral prostate cells have been explored. Also, initial studies related with the mode of action of these dendriplexes have been carried. Thus, we have evaluated the effect of these systems in the expression of cyclic adenosine monophosphate (cAMP). Finally, molecular dynamics simulation has helped to understand the interaction between peptides and carbosilane dendrimers.


8

Page 9 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

2. Matherials and Methods

General Considerations. Reactions were carried out under inert atmosphere. Solvents were purified with MBraun-SPS purification system and storage into ampoules containing molecular sieves. NMR spectra were recorded on a Varian Unity VXR-300 (300.13 (1H), 75.47 (13C) MHz) or on a Bruker AV400 (400.13 (1H), 100.60 (13C), 79.49 (29Si) MHz). Chemical shifts (δ) are given in ppm. 1H and 13C resonances were measured relative to solvent peaks considering TMS = 0 ppm, meanwhile 29Si resonances were measured relative to external tetramethylsilane. When necessary, assignment of resonances was done from HSQC, HMBC, COSY and TOCSY NMR experiments. Elemental analyses were performed on a LECO CHNS-932. The UVvisible absorption measurements were performed using a Perkin-Elmer Lambda 18 spectrophotometer. The spectra were recorded by measuring dilute samples in a quartz cell with a path length of 1 cm. Compounds [GnO3(S-NMe2(C2H4OH))m(I)m] (1-3),26 [GnO3(S-NMe3)m(I)m] (4-6)26 and [G2O3(S-NMe2)11(NHFlu)]33 were synthesized as


9


Page 10 of 60

published. All other compounds were obtained from commercial sources. VIP and GHRH(1-29)NH2 peptides were purchased from PolyPeptide (Strasbourg, France).

Synthesis of G2O3(S-NMe2-OH+)11(S-NHFlu) (7). A solution of [G2O3(S-NMe2)11(NHFlu)] (0.110 g, 0.038 mmol) and I(CH2)2OH in THF:DMF (10 ml:0.8 ml) was irradiated with microwaves at 100 ºC for 2 h under inert atmosphere. Then, the volatiles were removed under vacuum and the residue was washed once with acetonitrile (10 ml) and once with ether (10 ml). The remaining solid was dialyzed first with NaI, to avoid interaction of unreacted FITC with dendrimer, and then with distilled water. After drying, compound 7 was obtained as orange solid (0.1 g, 58 %). Data for 7: 1H-NMR (D2O): δ 0.03 (s, 9 H, SiMe), 0.05 (s, 18 H, SiMe), 0.63 (m, 30 H, OCH2CH2CH2CH2Si and SiCH2CH2CH2Si), 0.88 (m, 24 H, SiCH2CH2S), 1.37 (m, 18 H, OCH2CH2CH2CH2Si and SiCH2CH2CH2Si), 1.65 (m, 6 H, OCH2CH2CH2CH2Si), 2.60 (m, 24 H, SiCH2CH2S), 2.96 (m, 22 H, SCH2CH2N), 3.11 (s, 66 H, NMe2CH2CH2OH), 3,43 (m, 22 H, NMe2CH2CH2OH), 3.58 (m, 24 H, SCH2CH2N), 3.85 (t, 28 H, OCH2CH2CH2CH2Si and NMe2CH2CH2OH), 5.29 (s, 11 H, NMe2CH2CH2OH), 6.02 (s, 3 H, C6H3O3), 6.54 and 6.66 (m, 8 H, FITC); 13C-


10

Page 11 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

NMR (D2O): δ -4.7 (SiCH3), 14.6 (OCH2CH2CH2CH2Si), 18.4 (SiCH2CH2S), 20.5 (OCH2CH2CH2CH2Si and SiCH2CH2CH2Si), 23.9 (SCH2CH2N), 27.2 (SiCH2CH2S), 31.1 (OCH2CH2CH2CH2Si), 41.9 (NHC(S)NH), 51.2 (NMe2CH2CH2OH), 55.4 (NMe2CH2CH2OH), 64.1 (SCH2CH2N), 65.0 (NMe2CH2CH2OH), 67.5 (OCH2CH2CH2CH2Si), 93.7 (C6H3O3; C-H), 101.9, 112.2, 128.4, 151.3 and 158.8 (FITC), 160.0 (C6H3O3; C-O); 15N-NMR (D2O): -324.3 (–NMe2CH2CH2OH); 29SiNMR (D2O): 3.2 (G1–SiMe). MS- ESI: q = 6 (561.98 [M-6I-]6+), q = 8 (421.48 [M-8I-]8+). U.VVis: λ = 489.50 nm. Anal. Calcd. for C158H320I11N13O19S13Si9 (4771.87 g/mol): C, 56.21; H, 9.55; N, 5.39; S, 12.35. Experimental: C, 56.50; H, 9.61; N, 5.30; S, 12.29.

Dendriplexes characterization. Zeta potential and dynamic light scattering were carried out in a a Zetasizer Nano ZS (Malvern Instruments Ltd., UK). Circular dichroism was done using a J-715 CD spectrometer (Jasco, Japan). For transmission electron microscopy (TEM), a Zeiss EM-10 transmission electron microscope was used. Complete information about the procedure can be found in the Supporting Information.


11


Page 12 of 60

Cell culture. The androgen-unresponsive cell line PC3 was obtained from the American Type Culture Collection (Manassas, VA) and is related to recurrent prostate cancers that have achieved androgen independence. All culture media were supplemented with 1% penicillin/streptomycin/amphotericin B (Life Technologies, Barcelona, Spain) and 10% fetal bovine serum (FBS). The culture was performed in a humidified 5% CO2 environment at 37ºC. After the cells reached 70–80% confluence, they were washed with PBS, detached with 0.25% trypsin/0.2% EDTA and seeded at 30000–40000 cells/cm2. Every 3 days, the culture medium was changed.

Toxicity assays. PC-3 1.5 x 105 cells/ml, RWPE 2.0 x 104 cells/ml or fibroblast 1.5 x 104 were grown in 24 well plates. After 24 h, cells were treated with the corresponding concentrations of the systems (peptides, dendrimer 2 and dendriplexes 2/VIP and 2/GHRH) for 24 h. For dendriplexes, the dendrimer was added first to the well and then the peptide. Then medium was removed and cells were incubated with 500 μl cell culture and 25 μl thiazolyl blue tetrazolium bromide 5mg/ml in the last 1.5 h. After


12

Page 13 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

incubation, to cells were added cold isopropanol and the absorbance at 540 nm with a reference wavelength at 630 nm was reported.

Cell adhesion assay. Concentrated type-I collagen solution was diluted in 10 mM glacial acetic acid and coated onto 96-well plates for 1 h at 37ºC. Plates were washed 4 times with PBS (pH 7.4). On the other hand, a cells suspension (2.5 x 104 cells/ml), were treated with the corresponding concentrations of the systems (peptides, dendrimer 2 and dendriplexes 2/VIP and 2/GHRH) for 1 h. Then, cells plated 100 μl on plate preparated previously. The assay was terminated at 40 min by aspiration of the wells. Cell adhesion was quantified by adding 0.25 mg/ml of thiazolyl blue tetrazolium bromide (MTT) followed by incubation for 1.5 h. Isopropanol (50 μl) was added to each well to dissolve the formazan precipitate and absorbance at 540 nm with a reference wavelength at 630 nm was reported.

Wound-healing assay. PC3 cells were incubated in 24-well plates and a small wound area was made with a scraper in the confluent monolayer. Afterwards, cells were incubated in the absence or presence of the corresponding concentrations of the


13


Page 14 of 60

systems (peptides, dendrimer 2 and dendriplexes 2/VIP and 2/GHRH). Three representative fields of each wound were captured using a Nikon Diaphot 300 inverted microscopy at different times (0, 6 and 24 h). Wound areas of untreated samples were averaged and assigned a value of 100%.

Determination of VEGF. VEGF levels were determined in extracellular medium of treated or untreated PC3 by ELISA (human VEGF DuoSet, R&D Systems, Madrid, Spain) according to the manufacturer’s instructions. Data were normalized to the protein concentration in each sample.

Determination of cAMP. PC3 cells, 1 million per 100 plate, were grown. After 24 h, cells were treated with the corresponding concentrations of the systems (peptides, dendrimer 2 and dendriplexes 2/VIP and 2/GHRH) for 24 h. Then medium was removed. Cells were scraped, lysed and centrifuged at 1000 rpm for 10 min. The AMPc levels were determined in supernatant by ELISA (cyclic AMP ELISA Kit, Caiman Chemical Company, Michigan, USA) according to the manufacturer’s instructions.


14

Page 15 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Cell cycle. PC3 (7.5 x 104cells/ml) were grown in 6-well plates. After 24 h, the culture medium was removed and replaced with RPMI-1640 medium containing 0% FBS and 1% antibiotic/antimycotic (penincillin/streptomycin/amphotericin B) for 24 h. Then, cells were treated with the corresponding systems (peptides, dendrimer 2 and dendriplexes 2/VIP and 2/GHRH) for 24h. After incubation, the cells were washed with PBS, fixed with ice-cold absolute ethanol at 4ºC for 5 days. Afterwards, cells were centrifuged and washed with PBS. Finally, cells were resuspended in a solution of PBS, 50 mg/ml propidium iodide (PI) and 10 μg/ml RNasa. The amount of DNA distribution in the difference phases of cell cycle was analized with the use of the Cyfogic program (Version 1.2.1.)

Statistics. The Shapiro-Wilk test was used to check the normality of distribution. Variance homogeneity was verified using Levene’s test. Results are presented as Mean±S.D. (standard deviation), N = 6. Data were analyzed by ANOVA test with post-

hoc tests (indicated at the figures and tables).


15


Page 16 of 60

Computational details. Computer model of dendrimer 2 was created using dendrimer builder, as implemented in the Materials Studio software package from BIOVIA (formerly Accelrys). Structure of simulated VIP peptide was adopted from RCSB Protein Data Bank (PDB: 2RRI), just slightly modified at the C-terminal by substituting terminal Glycine with -NH2 capping unit (according to request from experimental team) so the final primary sequence was "HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH2". The starting Histidine was considered in charged form with total charge +2 for simulations at neutral pH (protonated imidazol group plus protonated terminal amine). On the other hand, we did not find any tertiary structure in RCSB Protein Data Bank corresponding to GHRH peptide primary sequence provided by experimental team i.e. "YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL" and so here, the tertiary structure was obtained using molecular dynamics simulation where the major part was done using fast implicit solvent approach. The RESP technique36 was used for calculation of dendrimer atoms partial charges. For this charge parameterization the R.E.D.-IV tools37 was used. The necessary QM calculations were done using GAMESS.38,39 The default, HF/6-31G*, level of theory was used for all charge-related


16

Page 17 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

QM calculations and the MEP potential was fitted on Connolly molecular surface. Generalized Amber Force Field (GAFF)40 was used for parameterization of dendrimer. Missing “Si containing“ force field parameters were fitted by minimizing the differences between QM and force field based relative energies of different configurations. QM energies were calculated at MP2/HF/6-31G** level of theory using GAMESS and fitting was accomplished using paramfit routine from AMBER14 software.41 Slightly adjusted van der Waals parameters for Si atoms from MM3 force field were used in this study.42 For simulations of VIP and GHRH peptides, force field ff14SB was used. In case of longer and more flexible GHRH peptide, two the most and similarly representative conformations were selected (to obtain more representative results), using cluster analysis of implicit solvent MD trajectory,43 and consequently simulated in explicit solvent, to obtain final peptide conformations for simulations of dendrimer/peptide complexes. The individual molecules (VIP, GHRH (both conformations obtained from implicit solvent simulation) and dendrimer 2) were solvated in explicit water (TIP3P model).44 The initial complexes were created from these equilibrated molecules using the UCSF Chimera software.45 The hydrogens were constrained with the SHAKE


17


Page 18 of 60

algorithm46 to allow 2 fs time steps, and a Langevin thermostat47 with a collision frequency of 2 ps-1 was used for all MD runs. Dendrimer/peptide complexes were simulated for 175 ns (molecular dynamics simulation in explicit solvent, T = 310 K and P = 0.1 MPa) using pmemd.cuda module from the Amber14 package.48 Molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) methodology, as implemented in the Amber14 routine MMPBSA.py,49 was used to obtain estimates of the free energies of binding. PBSA calculations were performed using Adaptive PoissonBoltzmann Solver sander. APBS from Amber14.50 Please see supporting information for more details.

3. Results

Dendrimers

The dendrimers employed in this work26 were dendrimers of three generations (first, second and third) derived from a polyphenoxo core (1,3,5-(HO)3C6H3), containing 6, 12 and 24 peripheral ammonium groups of two types: [GnO3(S-NMe2(C2H4OH))m]m+ (n = 1,


18

Page 19 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

m = 6 (1); n = 2, m = 12 (2); n = 3, m = 24 (3); Figure 1 and S1) and [GnO3(S-NMe3)m]m+ (n = 1, m = 6 (4); n = 2, m = 12 (5); n = 3, m = 24 (6); Figure 1 and Figure S2).

OH N

OH N

HO

OH

N S

S Si

N

S

S

Si

Si

Si

OH

N

S

O HO

Si

N S

S

O

Si

N

O

OH

S Si

N

S

Si

N

OH

Si

HO

S

N

HO

S S

N

N

OH

OH G2O3(S-NMe2-OH+)12 (2) OH N

OH HO

N

OH

N S

S Si

N

S

S

Si

Si

S

Si

O HO

Si

N S

S

S

O

Si

N H

O

OH

N

HO

NH

O

S

OH

Si

N

S

Si

N

Si

HO HO

N

S

N

OH O

S S

O

N OH

OH G2O3(S-NMe2-OH+)11(S-NHFlu) (7)

Figure 1. Drawing of second generation dendrimers used for nanoplexes formation. Anions are omitted for clarity.


19


Page 20 of 60

For the studies related with dendriplex internalization, it was necessary to use a labelled dendrimer with a fluorescein moiety. Taking into account the results obtained (see below), for this procedure was chosen a second generation derivative covered with – [NMe2(C2H4OH)]+ groups. The corresponding labelled dendrimer was synthesized from the reaction of a neutral dimethylamino dendrimer containing one fluorescein unit [G2O3(S-NMe2)11(NHFlu)], previously described,33 with the alkylating agent I(CH2)2OH, after heating at 100 ºC for 2 hours under microwave irradiation (Scheme S1). With this procedure, compound [G2O3(S-NMe2(C2H4OH))11(NHFlu)]11+ (7) was obtained as an orange solid (Figure 1). Compound 7 was characterized by 1H and 13C-NMR, ultraviolet and infrared spectroscopy and elemental analysis (Figures S3-S5). These techniques corroborated the alkylation of the terminal amino groups and that the fluorescein moiety remains attached to the carbosilane dendrimer after this process (see DOSY 1H NMR, Figure S5). The main data from NMR are the shifting of the resonances belonging to the methyl groups bound to the nitrogen atom after quaternization (i. e. in the 1H NMR spectrum from ca. 2.20 ppm in neutral compound to ca. 3.20 in cationic derivative 7). 1H NMR spectrum indicates the presence of one fluorescein unit per dendrimer, although


20

Page 21 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

since the synthesis of 7 implies the heterofunctionalization of a dendrimer, this value has to be considered as a mean value.

Toxicity assays of peptides, dendrimers and dendriplexes in advanced prostate cancer cells PC3

The cytotoxicity of dendrimers 1-6 in advanced prostate cancer cells PC3 was evaluated by MTT. As can be seen in Figure 2 and Figure S6, at concentrations below the toxicity threshold (10-6 M)33 all the dendrimers reached 50% of activity. In the particular case second generation derivative 2, its activity at lower concentrations was slightly higher than for the rest of dendrimers and this dendrimer was selected for next experiments. For them, we chose a concentration of 10-9 M, which is the lowest value were significant viability reduction was observed but far away from the toxicity limit.


21


Page 22 of 60

Figure 2. Effect of dendrimers 1-6 on the cell proliferation of PC3 prostate cancer. Results are the means of 6 SE experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. corresponding control.

In the case of VIP and GHRH, the peptides raised PC3 viability at 10-6 M and 10-7 M, respectively (Figure 3), that is, both peptides are protumoral. These concentrations were the lowest to which significant variation of viability was observed.

Figure 3. Effect of peptides VIP (10-6 M) or GHRH (10-7 M), dendrimer 2 (10-9 M) and dendriplexes 2/VIP (10-9/10-6 M) or 2/GHRH (10-9/10-7 M) on the cell proliferation of PC3 prostate cancer. Results are the means 6 SE of 5–7 experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. corresponding control. #p < 0.05; ##p < 0.01; ###p < 0.001 vs. 2.


22

Page 23 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

For the nanoplexes studies, the corresponding peptide and dendrimer were mixed previously in the well at the chosen concentrations (see experimental). These concentrations of peptides (VIP 10-6 M, GHRH 10-7 M), dendrimer 2 (10-9 M) and dendriplexes 2/VIP (10-9 M/10-6 M) and 2/GHRH (10-9 M/10-7 M) are the chosen for all the experiments described in this paper.

Regarding the PC3 viability in the presence of dendriplexes 2/VIP and 2/GHRH, the proliferation of this line cell was reduced to ca. 50%. That is, while both peptides provoked PC3 proliferation and dendrimer 2 reduced the cell survival only 25%, the dendriplexes were able to reach ca. 50% of mortality with respect to control conditions.

Toxicity of peptides, dendrimer and dendriplexes in non-neoplastic prostate RWPE-1 and fibroblast cells


23


Page 24 of 60

Figure 4. Effect of peptides VIP or GHRH, dendrimer 2 and dendriplexes 2/VIP or 2/GHRH on the cell viability of non-tumoral human prostate RWPE-1 cells. Results are the means 16 SE of 3 experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. corresponding control. #p < 0.05; ##p < 0.01; ###p < 0.001 vs. 2.

In order to evaluate whether dendriplexes can differentiate between tumoral and nontumoral prostate cells, we tested the effect of these systems in non-tumoral human prostate RWPE-1 cells (Figure 4). Peptides VIP and GHRH promoted slightly the proliferation in this cell line, whilst dendrimer 2 produced an important increase of viability (over 140%). When these cells were treated with the nanoplexes 2/VIP and 2/GHRH, the number of cells with respect to the control significantly increased, reaching values of around 140% and 130% respectively. This behaviour of dendrimer 2 and


24

Page 25 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

dendriplexes in RWPE-1 cells is clearly different to that observed for tumoral PC3 cells, in which survival was reduced after treatment with these systems. In addition, we also tested these systems in other no tumoral cells as fibroblasts, observing the same behaviour that in RPWE-1 (Figure S7).

One reason that could explain the differences in the toxicity of dendrimers toward tumoral PC3 and non-tumoral RPWE-1 cells is the presence of both peptides in the environment of PC3 cells. This situation could lead to formation of the corresponding dendriplexes that, as we have shown, are active against PC3 cells.

Cell adhesion and migration

The PC3 cell adhesion on a collagen I matrix was studied (Figure 5). Peptides by themselves reduced the adhesion, dendrimer 2 did not affect this process but nanoplexes 2/VIP and 2/GHRH were beneficial, increasing the adhesion to this matrix.


25


Page 26 of 60

Figure 5. Effect of peptides VIP or GHRH, dendrimer 2 and dendriplexes 2/VIP or 2/GHRH on the cell adhesion of PC3 prostate cancer. Results are the means 6 SE of 5– 7 experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. corresponding control.

Figure 6. PC3 cell migration after treatment with peptides VIP or GHRH, dendrimer 2 and dendriplexes 2/VIP or 2/GHRH by recovery of monolayer wounds assays.


26

Page 27 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figures 6 and S8 show the effect of peptides, dendrimer 2 and dendriplexes in a wound scratch of PC3 cell monolayer, after treatment at different times. The comparison with control (untreated cells) show that both peptides speeded up the migration and closing of injure, that dendrimer barely affect the cell migration compared with control and that the dendriplexes 2/VIP and 2/GHRH clearly slowed down the migration, keeping the injure even after 24 h.

These data suggest that the dendriplexes change the behaviour of the peptides, since VIP and GHRH show a metastatic profile in the PC3 cell line and dendrimer 2 keeps adhesion and migration levels similar to the control, but dendriplexes 2/VIP and 2/GHRH diminish in an important way the metastatic ability of this cell line.

Cell cycle

The flow cytometric assays carried out to determine any changes in cell cycle of PC3 did not show any variations in the percentage of cells in each step upon PC3 treatments with peptides, dendrimer 2 or nanoplexes (Figure S9).


27


Page 28 of 60

Uptake of peptides, dendrimer and dendriplexes by PC3 cells

Evaluation of dendriplexes uptake by PC3 cells was studied by confocal microscopy. For these experiments, we formed the nanoplexes by mixing the rhodamine labelled peptide GHRH-Rho with the fluorescein modified dendrimer 7. As can be seen in Figure 7, GHRH-Rho and dendrimer 7 were internalized into the cells when they were added in an individual manner. Dendriplex 7/GHRH-Rho was also internalized into the cells, confirming the continuance of these dendriplexes after cellular uptake. None of the systems penetrated into the cell nuclei and neither remained attached to the cell or nuclei membrane. These results point out that the prevalence of dendriplexes inside the cell is the cause of the different behaviour of them with respect to peptides or dendrimer. However, a comparison of confocal images also reveals that the yellow intensity has been reduced after 24 h, meaning separation of nanoplex components at longer times.


28

Page 29 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 7. Confocal images after addition of compound 7 alone (green), GHRH-Rho alone (red) and dendriplex 7/GHRH-Rho (merge) after 10 min (A) and 24 h (B). DAPIstained nuclei are shown in blue.

cAMP determination


29


Page 30 of 60

ELISA tests to quantify the variation of cyclic adenosine monophosphate (cAMP), the second universal messenger of VIP and GHRH, upon treatment with the systems here studied were carried out (Figure 8). With respect to control (100%), both peptides produced an increase of this monophosphate, around 170% for VIP and 130% for GHRH, whereas dendrimer 2 reduced the level to ca. 90%. Diminish of cAMP production by dendriplexes was higher, observing values of 81% for 2/VIP and of 76% for 2/GHRH.

Figure 8. Determination of cAMP in PC3 cells after treatment with peptides VIP or GHRH, dendrimer 2 and dendriplexes 2/VIP or 2/GHRH. Results are the means 2 SE of


30

Page 31 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

3 experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. corresponding control. #p < 0.05; ##p < 0.01; ###p < 0.001 vs. free peptide.

Expression of angiogenic factor VEGF

The expression of the angiogenic factor vascular endothelial growth factor (VEGF) in PC3 cells was analysed by an ELISA test (Figure 9). VIP and GHRH peptides produced and increase over 150% of this protein compared to non-treated cells. On the other hand, the expression of VEGF was rather modified upon treatment with dendrimer 2 but was reduced to ca. 80% after treatment with nanoplex 2/VIP and, more noticeable, to

ca. 60% after treatment with 2/GHRH. Again, the behaviours of dendriplexes are opposite to those of the corresponding peptides, while variation with dendrimer 2 is insignificant.


31


Page 32 of 60

Figure 9. Effect of peptides VIP or GHRH, dendrimer 2 and dendriplexes 2/VIP or 2/GHRH on the expression of VEGF in PC3 cells. Results are the means 6 SE of 5–7 experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. corresponding control. . #p < 0.05; ##p < 0.01; ###p < 0.001 vs. free peptide.

Study of interaction between second generation dendrimer 2 and peptides

A) Zeta potential, Dynamic Light Scattering (DLS), Circular Dichroism (CD), Transmission electron microscopy (TEM)

The variation of zeta potential of a solution of GHRH peptide (10-7 M) was measured in the presence of increasing concentration of dendrimer 2. In this experience, it was


32

Page 33 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

observed the stabilization of the zeta potential value when the dendrimer concentration was close to 10-10 M (Figure S10A), that is, the nanoplex 2/GHRH is formed at GHRH concentration of 10-7 M and at dendrimer concentrations over 1.0 x 10-10 M. Regarding VIP peptide, we could not measure the zeta potential, probably because to VIP size is below the detection limit of the device.

DLS was also helpful to determine formation of 2/GHRH nanoplex (Figure S10B). The initial size of GHRH measured by DLS was around 1.13 nm. After addition of dendrimer 2, the size of the dendriplex rised until a value ca. 295 nm, observing then a decrease of the nanoplex size with the increasing dendrimer concentrations. The final value reached was around 117 nm at the concentration employed for biomedical assays. Probably, at lower dendrimer concentration the dendrimer interacts with more peptides and then the size of the dendriplex is bigger. As for Z potential measurements, we could not obtain an adequate graphic for VIP peptide.

Regarding CD spectra for VIP and GHRH interactions with dendrimer 2, in both cases we observed significant variations with respect to spectra of free peptides (Figure S10C


33


Page 34 of 60

and S11). Since CD spectrum of peptides is related with their secondary structure, the alterations of their spectra involved that dendrimer 2 interact with them, modifying this secondary structure.

TEM images of free peptides and their corresponding dendriplexes with dendrimer 2 were done (Figure S12). These images showed the change in the particle size after dendriplex formation at the concentration used for the biomedical assays.

B) Molecular modelling

Molecular dynamic simulations of dendrimer/peptide complexes were done to provide detail insight into interaction of these molecules in water environment. In case of longer and more flexible GHRH peptide, three representative conformations were used for this study (Figure S13) to obtain more representative results. Dendrimer-peptide interactions were quantitatively characterized using the free energy of binding ΔG.


34

Page 35 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 10. Simulated complexes of dendrimer 2 with GHRH peptide, corresponding to two different initial peptide conformations. Complexes A, B correspond to initial peptide conformations K1, K2 in Figure S11, respectively. Magenta color denotes the N-terminal part of the peptide and cyan color highlights dendrimer core, other dendrimer atoms are colored as follows: C - black, H - white, N - blue, Si - tan (beige), O - red, S - yellow.

In all cases, simulations demonstrated the ability of dendrimer [G2O3(SNMe2(C2H4OH))12]12+ (2) to create stable complexes with peptides. Simulated complexes are shown in Figures 10 and S14-S16 and binding energies, including individual components, are shown in Table S1. Since dendrimer is cationic with net


35


Page 36 of 60

charge +12 and both peptides are also positively charged (GHRH charge +4, VIP charge +5) the complexes are stabilized mainly by interactions of hydrophobic dendrimer skeleton with hydrophobic amino acids, while charged dendrimer terminals rather prefer interaction with surrounding polar molecules of water.

In case of GHRH peptide, dendrimer 2 prefers interaction mainly with N-terminus part (Figure 10 and S15) though also C-terminal Leucine may contribute to stability of the complex (see complex B in Fig. S15), whilst in VIP case, the main interaction is with hydrophobic C-terminal part (capped with a –NH2 group) (Figures S14 and S16). Simulations suggests that the interaction of dendrimer 2 with GHRH is stronger than with VIP, -62.82 kcal/mol (average value) vs. -31.73 kcal/mol, and in both cases is quite sufficient for good stabilization of the dendrimer/peptide complex. Regarding eventual H-bonds between the dendrimer 2, due to the presence of terminal hydroxyl moieties,34 and peptides, they were really sporadic (typically 0 or 1) in both peptide cases.

4. Discussion


36

Page 37 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

The neuropeptides VIP and GHRH have a remarkable protumoral profile in advanced prostate cancer.20,51 Once tumour reaches the castrate resistant stage, these peptides favour the spreading of the cancer through the overexpression of their receptors in tumoral cells, and since VIP and GHRH are involved in the synthesis of different growing factors the process become metastatic. Human VIP and GHRH peptides are 28 and 44 aminoacid peptides, respectively. These two peptides, and others of the glucagon-secretin family, share important similarities in the N-terminal portion, which is the region required for receptor binding in humans. Particularly, the tridimensional structure in this region presents an amphiphilic α-helix, which is the key in the stabilization of the peptides interaction with their receptors. Once the peptides are bound to the receptors, different signalling cascades are activated,7,52 involving stimulation of adenylyl cyclase protein, increase of cAMP levels and subsequent production of growing factors that facilitates angiogenesis and, thus, the overproduction of growing factors can be clearly related with tumor formation.


37


Page 38 of 60

According to data obtained in the experiments comparing the behaviour of peptides VIP or GHRH, dendrimer 2 and dendriplexes 2/VIP or 2/GHRH, peptides favour protumoral characteristics of advanced prostate cancer cells PC3 (increasing proliferation, reducing adhesion and accelerating migration); dendrimer 2 reduces PC3 viability but modifies little adhesion and migration; meanwhile dendriplexes 2/VIP or 2/GHRH are nontumoral, as can be inferred from their reduction on the viability of PC3 cells, their increase of cell adhesion and the slowing down of cell migration. That dendriplex formation is important is confirmed by confocal microscopy, which proved the uptake of the systems inside PC3 cells.

Related with these observations are also the differences observed on the expresion of the second messenger cAMP. The protumoral peptides VIP or GHRH notably favour its production, whilst dendrimer 2 and dendriplexes 2/VIP or 2/GHRH reduce the expresion of cAMP.

Also, the presence of the vascular endothelial growth factor (VEGF) was evaluated, since this factor has been related with tumor growing and survival and favouring


38

Page 39 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

metastasis. Moreover, in recent years VEGF has been associated with chemotherapy resistance and tumor recurrence.53 As expected, peptides increase VEGF release, which was reduced in the presence of dendrimer 2 and in higher degree in the presence of nanoplexes 2/VIP or 2/GHRH.

The computer simulation of dendrimer/peptides interactions confirmed, first of all, that formation of these dendriplexes is viable, in spite the fact that dendrimer 2 and both peptides present a positive net charge. Also was shown that complexes are stabilized mainly by interaction of hydrophobic carbosilane framework with hydrophobic regions of peptides. Previously it has been reported that dendrimers with hydrophilic skeleton, as polyamidoamine PAMAM, were able to interact with VIP only in the case of high generation (G5.NH2, 128 amine groups) but not in the case of second generation (G2.NH2, 16 amine groups). In this case, it was suggested that the complex was mainly formed by inclusion of the peptide into the interior cavities of the G5-PAMAM, being this process impossible for the smaller G2-PAMAM.54


39


Page 40 of 60

Dendrimer 2 occupies the N-terminal region of GHRH. Since this region is the responsible of the interaction with the cell receptors, the blocking of this part could be considered as the responsible of the modification in the behaviour observed for the dendriplexes with respect to the free peptides. On the other hand, dendrimer 2 showed preference for the hydrophobic C-terminal part, leaving free the N-terminal region. Taking into account that both dendriplexes had similar activities and that VIP is smaller than GHRH, we believe that the interaction of 2 with VIP also difficult the interaction of VIP with the corresponding membrane receptors. Additionally, due to the smaller size of VIP, in its dendriplex would prevail the positive charge of dendrimer that could favour interaction with the most negatively charged parts of the cell surface, which would also modify the peptide behaviour. As consequence, in both cases the normal performance of these peptides on PC3 cells is altered showing antitumoral behaviour instead of protumoral behaviour.

It is important to note that the action of dendriplexes is consequence of the formation of them outside the cells. Although PC3 cells contain these peptides into their cytoplasm,


40

Page 41 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

the interaction of dendrimer 2 with them can be considered impossible, since the presence of other biomacromolecules would hamper this interaction. Moreover, as can be seen in confocal images, the presence of dendriplexes diminish at longer times, probably by competition with other biomacromolecules due the weak nature of the interactions stablished between dendrimer and peptides.28 Regarding uptake of dendriplexes, the classical way described for cationic macromolecules is by endocytosis.55 This mechanism has been observed in nanoconjugates of different cationic dendrimers.56,57

5. Conclusions

The interaction of the protumoral peptides VIP or GHRH in CRPC cells (PC3) with a compound with scarce antitumoral activity, a cationic carbosilane dendrimer, generates dendriplexes with very relevant antitumoral properties in this cell line. This result is consequence of the jamming of the active site of peptides by the dendrimer, which would avoid their interactions with receptors. Similarly to the CRPC, triple negative breast cancer does not respond to hormone treatment and the survival rate of women


41


Page 42 of 60

with this cancer is very low. Taking into account that this cancer and that other malignant neoplasms also overexpress receptors for neuropeptides as those studied in this work, VIP and GHRH, we can propose that formation of dendriplexes between these peptides, and others of the same family like PACAP, with cationic carbosilane dendrimers could be evaluated as potential anticancer systems. Research on this issue and also on further understanding of the processes involve in this behavior are currently in progress.

ASSOCIATED CONTENT

Supporting Information. Reaction scheme, spectra of compound 7 (NMR, UV, IR) and of dendriplexes (CD), Z potential and DLS graphs, TEM images, and additional figures related with toxicity, migration and molecular modelling.

AUTHOR INFORMATION

Corresponding Authors Javier Sánchez-Nieves, email: [email protected]


42

Page 43 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

F. Javier de la Mata, email: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. # These authors contributed equally to the work.

ACKNOWLEDGMENTS This work was supported by grants from CTQ2017-86224-P (MINECO), Consortium NANODENDMED II-CM ref B2017/BMD-3703 (CAM), Consortium IMMUNOTHERCANCM B2017/BMD-3733 (CAM) and CCGP2017-EXP/023 (UAH). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. This work was also supported from ERDF/ESF project "UniQSurf - Centre of biointerfaces and hybrid functional materials" (No. CZ.02.1.01/0.0/0.0/17_048/0007411) and from the Research Infrastructure NanoEnviCz, supported by the Ministry of Education, Youth and Sports of


43


Page 44 of 60

the Czech Republic under project No. LM2015073. M. S.-M. wishes to thank UAH for a fellowship (FPI 2015).

REFERENCES (1)

Siegel, R.; Ma, J.; Zou, Z.; Jemal, A. Cancer statistics, 2014. Cancer J.

Clin. 2014, 64, 9-29.

(2)

Ferlay, J.; Steliarova-Fouche, E.; Lortet-Tieulent, J.; Rosso, S.; Coebergh,

J. W. W.; Comber, H.; Forman, D.; Bray, F. Cancer incidence and mortality patterns in Europe: Estimates for 40 countries in 2012. Eur. J. Cancer 2013, 49, 1374– 1403.

(3)

U.S. Cancer Statistics Working Group (http://www.cdc.gov/uscs); Atlanta

(GA): Department of Health and Human Services, Centers for Disease Control and Prevention, and National Cancer Institute. United States Cancer Statistics: 1999–2014

Incidence and Mortality Web-based Report 2017.


44

Page 45 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(4)

Moody, T. W.; Nuche-Berenguer, B.; Jensen, R. T. Vasoactive intestinal

peptide/pituitary adenylate cyclase activating polypeptide, and their receptors and cancer. Curr. Opin. Endocrinol. Diabetes Obes. 2016, 47, 23-38.

(5)

Schally, A. V. New approaches to the therapy of various tumors based on

peptide analogues. Horm. Metab. Res. 2008, 40, 315-322.

(6)

Gutiérrez-Cañas, I.; Rodríguez-Henche, N.; Bolaños, O.; Carmena, M. J.;

Prieto, J. C.; Juarranz, M. G. VIP and PACAP are autocrine factors that protect the androgen-independent prostate cancer cell line PC-3 from apoptosis induced by serum withdrawal. Br. J. Pharmacol. 2003, 139, 1050-1058.

(7)

Malagón, M. M.; Vázquez-Martinez, R.; Martínez-Fuentes, A. J.; Gracia-

Navarro, F.; Castaño, J. P. GHRH. Handbook of Biologically Active Peptides (Second

Edition) 2013, Chapter 105, 784-791.

(8)

Busto, R.; Schally, A. V.; Varga, J. L.; García-Fernández, M. O.; Groot, K.;

Armatis, P.; Szepeshazi, K. The expression of growth hormone-releasing hormone


45


Page 46 of 60

(GHRH) and splice variants of its receptor in human gastroenteropancreatic carcinomas. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 11866-11871.

(9)

Moody, T. W.; Jensen, R. T. VIP/PACAP Receptors. Handbook of

Biologically Active Peptides (Second Edition) 2013, Chapter 76, 556-561.

(10)

Nadji, M.; Block, N. L.; Schally, A. V.; Lara, J. F.; Michaelson, R. A.;

Pulinthanathu, R.; Tortora, M.; Ali, S. M.; Leitzel, K.; Rizvi, S. M.; Al-Marrawi, M. Y.; Lipton, A. GHRH-receptor as a new targetable biomarker in breast cancer and its correlation with ER/PR/HER2 status. J. Clin. Oncol. 2015, 33, 576-576.

(11)

Schally, A.; Varga, J.; Engel, J. Antagonists of growth-hormone-releasing

hormone: an emerging new therapy for cancer. Nat. Clin. Pract. Endocrinol. Metab. 2008, 4, 33-43.

(12)

Dissanayake, S.; Denny, W. A.; Gamage, S.; Sarojini, V. Recent

developments in anticancer drug delivery using cell penetrating and tumor targeting peptides. J.Controll. Release 2017, 250, 62–76.


46

Page 47 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(13)

Azzopardi, E. A.; Ferguson, E. L.; Thomas, D. W. The enhanced

permeability retention effect: a new paradigm for drug targeting in infection. J.

Antimicrob. Chemother. 2013, 68, 257-274.

(14)

Fang, J.; Nakamura, H.; Maeda, H. The EPR effect: Unique features of

tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv. Drug Deliv. Rev. 2011, 63, 136-151.

(15)

Newkome, G. R.; Moorefield, C. N.; Vögtle, F. Dendrimers and Dendrons:

Concepts, Syntheses, Applications. Dendrimers and Dendrons: Concepts, Syntheses,

Applications. Wiley-VCH, Weinheim, Germany. 2001.

(16)

Tomalia, D. A.; Fréchet, J. M. J. Discovery of dendrimers and dendritic

polymers: A brief historical perspective. J. Polym. Sci., Part A: Pol. Chem. 2002, 40, 2719-2728.

(17)

Fréchet, J. M. J.; Tomalia, D. A. Dendrimers and Other Dendritic

Polymers. Dendrimers and Other Dendritic Polymers; Wiley VCH, Weinheim 2002.


47


(18)

Page 48 of 60

Wan, J.; Alewood, P. F. Peptide-Decorated Dendrimers and Their

Bioapplications. Angew. Chem. Int. Ed. 2016, 55, 5124 – 5134.

(19)

Varamini, P.; Rafiee, A.; Giddam, A. K.; Mansfeld, F. M.; Steyn, F.; Toth, I.

Development of New Gonadotropin-Releasing Hormone-Modified Dendrimer Platforms with Direct Antiproliferative and Gonadotropin Releasing Activity. J. Med. Chem. 2017,

60, 8309-8320.

(20)

Muñoz-Moreno, L.; Bajo, A. M.; Prieto, J. C.; Carmena, M. J. Growth

hormone-releasing hormone (GHRH) promotes metastatic phenotypes through EGFR/HER2 transactivation in prostate cancer cells. Mol. Cell. Endocrinol. 2017, 446, 59-69.

(21)

Muñoz-Moreno, L.; Arenas, M. I.; Carmena, M. J.; Schally, A. V.; Prieto, J.

C.; Bajo, A. M. Growth hormone-releasing hormone antagonists abolish the transactivation of human epidermal growth factor receptors in advanced prostate cancer models. Invest. New Drugs 2014, 32, 871-882.


48

Page 49 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(22)

Fernández-Martínez, A. B.; Bajo, A. M.; Isabel Arenas, M.; Sánchez-

Chapado, M.; Prieto, J. C.; Carmena, M. J. Vasoactive intestinal peptide (VIP) induces malignant transformation of the human prostate epithelial cell line RWPE-1. Cancer

Lett. 2010, 299, 11-21.

(23)

Collado, B.; Carmena, M. J.; Clemente, C.; Prieto, J. C.; Bajo, A. M.

Vasoactive intestinal peptide enhances growth and angiogenesis of human experimental prostate cancer in a xenograft model. Peptides 2007, 28, 1896-1901.

(24)

García-Fernández, M. O.; Solano, R. M.; Carmena, M. J.; Busto, R.;

Bodega, G.; Ruiz-Villaespesa, A.; Prieto, J. C.; Sánchez-Chapado, M. Expression of functional PACAP/VIP receptors in human prostate cancer and healthy tissue. Peptides 2003, 24, 893-902.

(25)

Ortega, P.; Bermejo, J. F.; Chonco, L.; de Jesús, E.; de la Mata, F. J.;

Fernández, G.; Flores, J. C.; Gómez, R.; Serramía, M. J.; Muñoz-Fernández, M. A. Novel water-soluble carbosilane dendrimers: Synthesis and biocompatibility. Eur. J.

Inorg. Chem. 2006, 1388-1396.


49


(26)

Page 50 of 60

Fuentes-Paniagua, E.; Hernández-Ros, J. M.; Sánchez-Milla, M.; Camero,

M. A.; Maly, M.; Pérez-Serrano, J.; Copa-Patiño, J. L.; Sánchez-Nieves, J.; Soliveri, J.; Gómez, R.; de la Mata, F. J. Carbosilane cationic dendrimers synthesized by thiol–ene click chemistry and their use as antibacterial agents. RSC Adv. 2014, 4, 1256–1265.

(27)

Pion, M.; Serramía, M. J.; Díaz, L.; Bryszewska, M.; Gallart, T.; García, F.;

Gómez, R.; de la Mata, F. J.; Muñoz-Fernández, M. Á. Phenotype and functional analysis of human monocytes-derived dendritic cells loaded with a carbosilane dendrimer. Biomaterials 2010, 31, 8749-8758.

(28)

Ionov, M.; Ciepluch, K.; Klajnert, B.; Glinska, S.; Gomez-Ramirez, R.; de

la Mata, F. J.; Muñoz-Fernández, M. A.; Bryszewska, M. Complexation of HIV derived peptides with carbosilane dendrimers. Colloids Surf. B: Biointerfaces 2013, 101, 236– 242.

(29)

Ionov, M.; Ciepluch, K.; Rasines-Moreno, B.; Applehans, D.; Sánchez-

Nieves, J.; Gómez, R.; de la Mata, F. J.; Muñoz-Fernández, M. A.; Bryszewska, M.


50

Page 51 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Biophysical Characterization of Glycodendrimers as Nanocarriers for HIV Peptides.

Curr. Med. Chem. 2013, 20, 3935-3943.

(30)

Melikishvili, S.; Poturnayova, A.; Ionov, M.; Bryszewska, M.; Vary, T.;

Cirak, J.; Muñoz-Fernández, M. Á.; Gomez-Ramirez, R.; de la Mata, F. J.; Hianik, T. The effect of polyethylene glycol-modified lipids on the interaction of HIV-1 derived peptide–dendrimer complexes with lipid membranes. Biochim. Biophys. Acta 2016,

1858, 3005–3016.

(31)

Jiménez, J. L.; Gómez, R.; Briz, V.; Madrid, R.; Bryszewsk, M.; de la Mata,

F. J.; Muñoz-Fernández, M. A. Carbosilane dendrimers as carriers of siRNA. J. Drug

Deliv. Sci. Technol. 2012, 22, 75-82.

(32)

Serramía, M. J.; Álvarez, S.; Fuentes-Paniagua, E.; Clemente, M. I.;

Sánchez-Nieves, J.; Gómez, R.; de la Mata, F. J.; Muñoz-Fernández, M. A. In vivo delivery of siRNA to the brain by carbosilane dendrimer. J. Control. Release 2015, 200, 60-70.


51


(33)

Page 52 of 60

Fuentes-Paniagua, E.; Serramía, M. J.; Sánchez-Nieves, J.; Álvarez, S.;

Muñoz-Fernández, M. A.; Gómez, R.; de la Mata, F. J. Fluorescein labelled cationic carbosilane dendritic systems for biological studies. Eur. Polym. J. 2015, 71, 61-72.

(34)

Sánchez-Milla, M.; Pastor, I.; Maly, M.; Serramía, M. J.; Gómez, R.;

Sánchez-Nieves, J.; Ritort, F.; Muñoz-Fernández, M. Á.; de la Mata, F. J. Study of noncovalent interactions on dendriplex formation: influence of hydrophobic, electrostatic and hydrogen bonds interactions. Colloids Surf. B: Biointerfaces 2018, 162, 380-388.

(35)

Hatano, K.; Matsubara, T.; Muramatsu, Y.; Ezure, M.; Koyama, T.;

Matsuoka, K.; Kuriyama, R.; Kori, H.; Sato, T. Synthesis and Influenza Virus Inhibitory Activities of Carbosilane Dendrimers Peripherally Functionalized with HemagglutininBinding Peptide. J. Med. Chem. 2014, 57, 8332–8339.

(36)

Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. A well-behaved

electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem. 1993, 97, 10269.


52

Page 53 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(37)

Dupradeau, F.-Y.; Pigache, A.; Zaffran, T.; Savineau, C.; Lelong, R.;

Grivel, N.; Lelong, D.; Rosanski, W.; Cieplak, P. The R.E.D. Tools, Advances in RESP and ESP charge derivation and force field library building. Phys. Chem. Chem. Phys. 2010, 12, 7821.

(38)

Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.; Elbert, S. T.; Gordon, M. S.;

Jensen, J. H.; Koseki, S.; Matsunaga, N.; Nguyen, K. A.; Su, S.; Windus, T. L.; Dupuis, M.; Montgomery, J. A. General Atomic and Molecular Electronic Structure System. J.

Comput. Chem. 1993, 14, 1347-1363.

(39)

Gordon, M. S.; Schmidt, M. W. Advances in electronic structure theory:

GAMESS a decade later. Theory and Applications of Computational Chemistry: the First

Forty Years 2005, Elsevier Science, 1167–1189.

(40)

Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollamn, P. A.; Case, D. A.

Development and testing of a general Amber force field. . J. Comput. Chem. 2004, 25, 1157–1174.


53


(41)

Page 54 of 60

Case, D. A.; Babin, V.; Berryman, J. T.; Betz, R. M.; Cai, Q.; Cerutti, D. S.;

III, T. E. C.; Darden, T. A.; Duke, R. E.; Gohlke, H.; Goetz, A. W.; Gusarov, S.; Homeyer, N.; Janowski, P.; Kaus, J.; Kolossváry, I.; Kovalenko, A.; Lee, T. S.; LeGrand, S.; Luchko, T.; Luo, R.; Madej, B.; Merz, K. M.; Paesani, F.; Roe, D. R.; Roitberg, A.; Sagui, C.; Salomon-Ferrer, R.; Seabra, G.; Simmerling, C. L.; Smith, W.; Swails, J.; Walker, R. C.; Wang, J.; Wolf, R. M.; Wu, X.; Kollman, P. A. AMBER 14, University of

California, San Francisco 2014.

(42)

Lii, J.-H.; Norman, A.; Allinger, L. The MM3 force field for amides,

polypeptides and proteins. J. Comput. Chem. 1991, 12, 186-199.

(43)

Nguyen, H.; Roe, D. R.; Simmerling, C. L. Improved Generalized Born

Solvent Model Parameters for Protein Simulations. J. Chem. Theory Comput. 2013, 9, 2020–2034.

(44)

Jorgensen, W. L.; Chandrasekhar, J.; Madura, J.; Klein, M. L. Comparison

of simple potential functions for simulating liquid water. J. Chem. Phys. 1983, 79, 926– 935.


54

Page 55 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(45)

Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt,

D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605–1612.

(46)

Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C. Numerical integration of

the cartesian equations of motion of a system with constraints: Molecular dynamics of nalkanes. . J. Comput. Phys. 1977, 23, 327-341.

(47)

Wu, X.; Brooks, B. R. Self-guided Langevin dynamics simulation method.

Chem. Phys. Lett. 2003, 381, 512–518.

(48)

Götz, A. W.; Williamson, M. J.; Xu, D.; Poole, D.; Grand, S. L.; Walker, R.

C. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 1. Generalized born. J. Chem. Theory Comput. 2012, 8, 1542–1555.

(49)

Miller III, B. R.; McGee Jr., T. D.; Swails, J. M.; Homeyer, N.; Gohlke, H.;

Roitberg, A. E. MMPBSA.py: An Efficient Program for End-State Free Energy Calculations. J. Chem. Theory Comput. 2012, 8, 3314-3321.


55


(50)

Page 56 of 60

Baker, N. A.; Sept, D.; Joseph, S.; Holst, M. J.; McCammon, J. A.

Electrostatics of nanosystems: Application to microtubules and the ribosome. Proc. Natl.

Acad. Sci. USA 98 2001, 10037-10041.

(51)

Fernández-Martínez, A. B.; Bajo, A. M.; Sánchez-Chapado, M.; Prieto, J.

C.; Carmena, M. J. Vasoactive intestinal peptide behaves as a pro-metastatic factor in human prostate cancer cells. Prostate 2009, 69, 774-786.

(52)

Passemard, S.; Abad-Rabat, C.; Gressens, P.; Lelièvre, V. VIP. Handbook

of Biologically Active Peptides (Second Edition) 2013, Chapter 128, 966-974.

(53)

Goel, H. L.; Mercurio, A. M. VEGF targets the tumour cell. Nat Rev Cancer

2013, 13, 871-882.

(54)

Dribek, M.; Le Potier, I.; Rodrigues, A.; Pallandre, A.; Fattal, E.; Taverna,

M. Determination of binding constants of vasoactive intestinal peptide to poly(amidoamine) dendrimers designed for drug delivery using ACE. Electrophoresis 2007, 28, 2191–2200.


56

Page 57 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(55)

Bareford, L. M.; Swaan, P. W. Endocytic mechanisms for targeted drug

delivery. Adv Drug Deliv Rev 2007, 59, 748–758.

(56)

Restani, R. B.; Conde, J.; Baptista, P. V.; Cidade, M. T.; Bragança, A. M.;

Morgado, J.; Correia, I. J.; Aguiar-Ricardo, A.; Bonifácio, V. D. B. Polyurea dendrimer for efficient cytosolic siRNA delivery. RSC Adv 2014, 4, 54872-54878.

(57)

Urbiola, K.; Blanco-Fernández, L.; Navarro, G.; Rödl, W.; Wagner, E.;

Ogris, M.; Tros de Ilarduya, C. Evaluation of improved PAMAM-G5 conjugates for gene delivery targeted to the transferrin receptor. Eur J Pharm Biopharm 2015, 94, 116-122.


57


Page 58 of 60

For Table of Contents Use Only:

Anticancer Activity of Nanoplexes against Advanced Prostate Cancer from Protumoral Peptides and Cationic Carbosilane Dendrimers

María Sánchez-Milla,a,b,# Laura Muñoz-Moreno,b,c,d,# Javier Sánchez-Nieves,a,b,e Marek Malý,f Rafael Gómez,a,b,e María J. Carmena,c,* and F. Javier de la Mataa,b,e,*

a

Department of Química Orgánica y Química Inorgánica, Instituto de Investigación

Química "Andrés M. del Río" (IQAR), Campus Universitario; Universidad de Alcalá; E28805 Alcalá de Henares (Madrid) Spain; e-mail [email protected]; [email protected].

b

Networking Research Center for Bioengineering, Biomaterials and Nanomedicine

(CIBER-BBN).

c

Department of Biología de Sistemas, Universidad de Alcalá, Campus Universitario, E-

28805 Alcalá de Henares, Spain.


58

Page 59 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

d

Actual address: Institute for Health Research, Hospital Universitario La Paz, E- 28046

Madrid, Spain.

e

Institute Ramón y Cajal for Health Research (IRYCIS).

f

Faculty of Science, J. E. Purkinje University, České mládeže 8, 400 96 Ústí nad

Labem, Czech Republic.

#

These authors contributed equally to the work.


59


Page 60 of 60

Dendriplexes obtained from protumoral peptides in advanced prostate cancer (VIP or GHRH peptides) with a cationic carbosilane dendrimer generate antitumoral systems.


60

Anticancer Activity of Dendriplexes against Advanced Prostate Cancer

Recommend Documents