Self-assembling ELR-based nanoparticles as smart drug-delivery systems modulating cellular growth via Akt

1 This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in [JournalTitle], copyright © American Chemical Society after peer review. To access the final edited and published work see [insert ACS Articles on Request author-directed link to Published Work, see http://pubs.acs.org/page/policy/articlesonrequest/index.html]. Self-assembling ELR-based nanoparticles as smart drugdelivery systems modulating cellular growth via Akt Juan Gonzalez-Valdivieso1, Alessandra Girotti1, Raquel Muñoz1, J. Carlos Rodriguez- Cabello1 and F. Javier Arias1*. 1BIOFORGE (Group for Advanced Materials and Nanobiotechnology), CIBER-BBN, University of Valladolid, 47011 Valladolid, Spain. *Corresponding author: [email protected] Abstract This work investigates the physicochemical properties and in vitro accuracy of a genetically engineered drug delivery system based on elastin-like block recombinamers. The DNA recombinant technics allowed us to create this smart complex polymer containing bioactive sequences for internalization, lysosome activation under acidic pH and blockage of cellular growth by a small peptide inhibitor. The recombinant polymer reversibly self-assembled, when temperature was increased above 15°C, into nanoparticles with a diameter of 72 nm and negative surface charge. Furthermore, smart nanoparticles were showed to enter in the cells via clathrin-dependent endocytosis, and properly blocked phosphorylation and consequent activation of Akt kinase. This system provoked apoptosis-mediated cell death in breast and colorectal cancer cells, which possess higher expression levels of Akt, whereas non-cancerous cells, such as endothelial cells, fibroblasts and mesenchymal stem cells, were 2 not affected. Hence, we conclude that the conformational complexity of this smart elastinlike recombinamer leads to achieve successful drug delivery in targeted cells and could be a promising approach as nanocarriers with bioactive peptides in order to modulate multiple cellular processes involved in different diseases. Keywords: drug delivery, nanoparticles, elastin-like recombinamers (ELRs), Akt, 1. Introduction One of the limitations of modern medicine is the lack of efficient drug carriers. Such carriers should accomplish their main function, namely release of a drug in a targeted tissue, so as to achieve two benefits: an increase in drug efficacy and a reduction in possible adverse side effects.1 The development of a good carrier for a specific drug is of particular importance as, in some cases, the therapeutic dose of a drug is so high that it cannot be used without causing severe damage to other organs.2 One of the most recent therapeutic approaches is based on smart advanced biomaterials. New biomaterials are thought to be an interesting alternative for drug delivery,3-4 as they are able to overcome the limitations of chemotherapy and improve the action of chemotherapeutic agents.5-6 Multifunctional carriers have been proposed to overcome these deficiencies.7 Although significant progress has been made in the field of synthetic devices with improved polymerization efficiency and lower polidispersities, genetically engineered polymers provide us the control to build advanced delivery carriers with acquired functionalities.8 Elastin-like recombinamers (ELRs) are one such biomaterial. ELRs are biopolymers based on short pentapeptide repeats found in the sequence of natural elastin, mainly the VPGXG pentapeptide, where X can be any amino acid except proline. The term ELR refers to those elastin-like polypeptides (ELPs) manufactured using genetic-engineering techniques. 3 Recombinant DNA technology allows us to design ELRs with full control over the amino acid sequence and include different functionalities and bioactive sequences.9-10 As such, ELRs presenting characteristic features are a novel alternative for the development of new biomedical devices because of their biological and mechanical properties, such as biocompatibility, biodegradability, and thermally and environmentally responsive behaviour.11 Moreover, ELRs have gained notable interest in the last years due to their lack of toxicity and immunogenicity as a consequence of their protein nature. ELRs exhibit an inverse temperature transition (ITT), which means that below a characteristic temperature, the so-called transition temperature (Tt), they remain soluble in a random coil conformation, self-assembling hydrophobically above this Tt and resulting in the reversible formation of coacervates due to a conformational reorganization at the molecular level. Coacervation of the polymer backbone can be triggered by different factors, including temperature, pH, light, ion concentration, etc. In addition, their stimulus-responsive behaviour can be tuned as Tt is controlled by the amino-acid composition of the recombinamer.12 Given their cell-friendly behaviour, tunable mechanical properties, thermal sensitivity, and ability to self-assemble, they are useful biomaterials for most applications in the fields of nanotechnology and biomedicine and, specifically, for controlled drug delivery.13-17 Cancer is one of the potential applications of drug delivery systems as it is one of the most common diseases worldwide. Indeed, according to the World Health Organization, 14.1 million new cases of cancer are reported each year, with around 8.2 million cancer-related deaths. As cancer is a very complex disease with multiple origins and evolution states, different approaches are needed to tackle this illness. Although different therapies have been developed for cancer treatment, chemotherapy suffers from numerous problems, such as cytotoxicity, poor tumour accumulation and dose-limiting side effects.18 Consequently, new 4 therapeutic approaches are needed to improve the diagnosis at earlier stages and increase the rate of treatment success. Advanced drug delivery systems enable to control the release of drugs in a specific cell or tissue, so smart bioresponsive biomaterials able to self-assemble and act under certain stimuli emerge as promising approaches for the achievement of reduced doses of the drugs and limited side effects. Due to the poor tumour accumulation of standard drugs used in chemotherapy, carriers have become an interesting approach for drug-delivery purposes.19 Nanoparticle-based delivery can reduce side effects by redistributing drug accumulation away from critical organs, such as the kidney or liver, thus allowing the administration of larger doses than is possible with free drugs.20 As tumours have a porous vasculature, aberrant vascular endothelium and enhanced vascular permeability, 10–100 nm sized nanoparticles accumulate in tumours because of the enhanced permeability and retention effect (EPR).21 This effect arises due to the fact that tumours have no functional lymphatic vessels, thus resulting in inefficient drainage from tumour tissue.22-23 As such, nanoparticles are able to enter into the interstitial space but are not efficiently removed,24 thus being retained in the tumor tissue. The choice of the specific target in drug delivery makes the difference between whether healthy tissues are affected or not.25 In this regard, cancer markers, such as overexpressed receptors and cytoplasmic proteins, are the most widely used targeting systems due to their higher expression in cancerous cells when compared to non-cancerous cells.18, 22 Consequently, novel strategies that target overexpressed proteins could be of interest when determining how to stop uncontrolled cell proliferation, which is markedly faster in cancerous cells. Of these proteins, Akt stands out due to its important activity in controlling multiple signalling pathways and processes in cells.26 Akt is a protein kinase that plays a central role in the regulation of multiple cellular processes, enhancing cellular proliferation, 5 metabolism and motility and inhibiting apoptosis.26 It has three differentiated functional regions, namely the N-terminal pleckstrin homology domain (PH), the central catalytic domain and, finally, the C-terminal hydrophobic region.27 In response to growth factors, Akt is activated by products of phosphatidyl inositol triphosphate generated by PI3K. These lipid products bind to the PH domain of Akt, thereby inducing a conformational change and allowing PDK1 to phosphorylate threonine 308. Phosphorylation of serine 473 and membrane anchoring are also required after threonine 308 phosphorylation for final activation of Akt kinase.28 There are three different isoforms (Akt1, Akt2 and Akt3) in mammalian cells. Akt1 is the most abundant isoform and is overexpressed in multiple types of cancer, such as colon, pancreatic, breast, ovarian and lung neoplastic diseases.29 As such, Akt is an attractive target for drug design and development. In light of this, Hiromura et al. have developed a small peptide (Akt-in) that accurately prevents phosphatidyl inositol species from binding to the pleckstrin homology domain (PH) of Akt by causing conformational changes, thereby inhibiting membrane translocation and Akt activation. This inhibitor prevents Akt kinase activity and, consequently, a biological response downstream. Moreover, Akt-in inhibits both in vitro proliferation and anti-apoptosis action as well as in vivo tumor progression.30 The objectives of this study were to synthesize and characterize a smart stimulus-responsive therapeutic system that can be modulated for application in different cells depending. Only recombinant technology allows us to create multi-functional block copolymers that can selfassembly into versatile NPs carrying the peptidic inhibitor of Akt, in a targeted and protected manner, and are specifically released in the intracellular environment. In light of the above, we have developed ELR nanoparticles carrying the peptidic inhibitor of protein kinase Akt (Akt-in). Moreover, we have determined in vitro the therapeutic window in which tumor cells 6 are affected and normal ones not, and have studied their internalization pathway and intracellular trafficking. 2. Materials and methods 2.1 Chemical reagents and cell lines Genes for LAEL, Cathepsin D sensitive peptide, H5 peptide and Akt-in were acquired from NZYTECH (Portugal). Escherichia coli BLR (DE3) strain was supplied by Novagen. Chloroquine, filipin, amiloride, and monodansylcadaverine were purchased from Sigma- Aldrich. Pepstatin A was acquired from Apollo Scientific. Primary antibodies against Akt (#9272), ρ-Akt Ser473 (#9271) and GAPDH (sc-32233) were purchased from Cell Signaling and Santa Cruz Biotechnology. Goat secondary antibodies against rabbit (ab6721) and mouse (ab205719) were supplied by Abcam. Cell lysis buffer and Bradford reagent were supplied by Sigma-Aldrich. Human adipose-derived mesenchymal stem cells (hMSCs, R7788-115), basal medium Dulbecco’s Modified Eagle’s Medium (DMEM), Minimum Essential Medium (MEM), Fetal Bovine Serum (FBS), penicillin streptomycin solution, trypsin-EDTA, DPBS, glutamine, non-essential amino acids (NEAA) and LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells were supplied by Invitrogen (USA). Human umbilical vein endothelial cells (HUVEC cc-2517), medium 200, low serum growth supplement (LSGS), L-15 medium and gentamicin/amphotericin solution were purchased from Gibco. Human foreskin fibroblasts (HFF-1, SCRC-1041) were purchased from the American Type Culture Collection (ATCC, USA). Human breast cancer (MCF-7, 86012803 ECACC) and human colorectal cancer (Caco-2, 86010202 ECACC) cell lines were supplied by Sigma-Aldrich. 2.2 ELR design, bioproduction and purification 7 The elastin-like recombinamers (ELR) used in this work were obtained as described elsewhere.31 The final fusion genes with a fully controlled composition and chain length were constructed by sequential introduction of the monomer gene segments in a stepwise manner using the recursive directional ligation method (RDL). The DNA sequence of every cloning step was corroborated by DNA sequencing. Expression vectors containing the selected ELR genes were transformed into Escherichia coli BLR (DE3) strain (Novagen) for production. The ELR was then bioproduced in Escherichia coli in a 15-L bioreactor (Applikon Biotechnology, Netherlands) and purified by several cooling and heating purification cycles (inverse transition cycling) following centrifugation, taking advantage of the ability of these recombinamers to aggregate above their transition temperature. Endotoxins were removed from the ELR by way of additional NaCl and NaOH treatments.32 Finally, the polymer was dialyzed against ultrapure water type I and sterilized by filtration (0.22 μm filters Nalgene), and freeze-dried prior to storage. The molecular weight and purity of the recombinamers were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and mass spectrometry (MALDI-TOF/MS), respectively. The amino acid composition was further verified by high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy by the Instrumental Techniques Laboratory of the University of Valladolid. Endotoxin levels were measured using the Endosafe-PTSTM test (Charles River, USA). 2.3 Differential Scanning Calorimetry (DSC) DSC experiments were performed using a Mettler Toledo 822e with liquid-nitrogen cooler. Both temperature and enthalpy were calibrated against an indium standard. Solutions were prepared by dissolving the ELRs in PBS (pH 7.4) at 50 mg/mL. A 20 μL aliquot of each 8 solution and its corresponding PBS reference were subjected to an initial isothermal stage (5 min at 0°C to stabilize the temperature and state of the samples), followed by heating from 0 to 60°C at 5°C/min. The enthalpy values for endothermic processes were taken as negative and exothermic values as positive. 2.4 Particle Size and Zeta Potential The particle size and zeta potential of the polymers were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK) at a temperature of 37°C. Solutions of both ELRs were prepared by dissolving the ELRs in PBS (pH 7.4) or ultrapure water type I (pH 7.4), when indicated. The solutions were stored at 4°C overnight to allow complete dissolution of the recombinamers and filtered using a 0.45 μm PVDF syringe filter. The samples were then incubated for 30 minutes at 37°C to allow supramolecular assembly to occur and then introduced into polystyrene cuvettes and stabilized for 2 min at the desired temperature. For BSA interaction experiments, ELRs were incubated in 5% BSA PBS for 1, 2 or 3 hours at 37°C after overnight dissolution and then filtered, introduced into polystyrene cuvettes and stabilized for 2 min at the desired temperature. Autocorrelation functions were used to obtain the size distribution and polydispersity index. Z-average mean (nm) and zeta potential (mV) were used for data analysis. Three different samples were analyzed. 2.5 Transmission electron microscopy (TEM) Solutions were prepared by dissolving the ELRs in ultrapure water type I and kept at 4 °C overnight to allow complete dissolution of the polymers. The sample was incubated for 30 minutes at 37 °C to allow supramolecular assembly to occur, stained with uranyl acetate solution (1,0 wt %) to enhance the contrast of the nanoparticles on a carbon-coated copper 9 grid, followed by solvent evaporation. Samples were observed using a JEM-2200 electron microscope operating at 200 kV. 2.6 Surface Tension by Pendant Drop Technique The critical micellar concentration (CMC) of the different ELR solutions in both PBS and ultrapure water type I was determined from surface tension measurements derived from a drop-shape analysis using the pendant drop technique.33 The changes in the shape of the resulting drop at the air/water interface upon increasing the ELR concentration previously stabilized at 37 °C for 15 min from a blank solution to 20 μM were monitored using the SCA 20 software of a Data Physics OCA20 instrument, which scaled the profile of the drop hanging from a straight precision dosing needle. The drops (4 μL at 0.5 μL/s) were infused using a 500 μL Gastight Hamilton syringe. Three drops were analyzed per condition and the CMC was determined from the point of slope change after plotting the change in surface tension values versus log(concentration) of the ELRs. 2.7 Cell culture MCF-7 and Caco-2 cells were maintained in MEM supplemented with 10% FBS, 2 mM glutamine, 1% NEAA, 100 U/mL penicillin and 0.1 mg/mL streptomycin at 5% CO2 and 37 °C. MDA-MB-231 cells were cultured in L-15 medium supplemented with 10% FBS, 100 U/mL penicillin and 0.1 mg/mL streptomycin at 0% CO2 and 37 °C. hMSC and HFF-1 cells were cultured in DMEM supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin and 10% or 15% FBS, respectively, at 10% CO2 and 37 °C. HUVEC cells were grown in Medium 200 supplemented with 1% gentamicin/amphotericin and LSGS at 5% CO2 and 37 °C. When required, cells were detached using a solution of 0.05% Trypsin-EDTA. Cells were seeded onto 96-well plates at a quantity of 2×104 cells per cm2 for tumor cells and 1×104 cells 16 despite their different composition, thus meaning that the bioactive sequences added to the polymer carrying the Akt inhibitor do not affect their transition temperature. This means that they could be used for biomedical applications because, at body temperature of 37°C, both ELR-based polymers self-assemble. Once the Tt of both polymers had been determined, nanoparticle formation had to be checked because the non-ELR block could prevent selfassembly of the polymers. Nanoparticle Size (nm) PdI Zeta Potential (mV) CMC mg/mL µM Control NP 65.60 ± 3.73 0.087 -27.8 ± 1.5 0.25 4.49 Akt-in NP 72.46 ± 3.52 0.079 -26.2 ± 1.2 0.21 4.21 Table 2. Characterization of ELR nanoparticles. Size and polydispersity index of self-assembled polymers dissolved in PBS measured by dynamic light scattering (DLS). Surface charge of self-assembled polymers dissolved in ultrapure water type I measured by dynamic light scattering (DLS). The CMC was calculated from the surface tension using the pendant drop technique. Mean ± SD. The nanotechnological approach to cancer therapy takes advantage of the multiple abnormalities inherent to tumor vasculature, such as hypervascularization, aberrant vascular architecture, enhanced production of vascular permeability factors, and the lack of lymphatic drainage.21-23 Thus, nanocarriers can selectively extravasate into tumor tissues due to their abnormal vascular nature and are subsequently not efficiently removed, thus remaining retained therein.44 The ideal size for a nanoparticle depends on several factors. First of all, for significant extravasation from fenestrations in the tumor vasculature, nanocarriers need to be smaller than 400 nm.24 Secondly, particles bigger than 200 nm are likely to be sensitive to macrophages and undergo opsonization.45 As such, in order to avoid specific capture by the liver, they should be less than 100 nm in size46 but larger than 10 nm to avoid filtration 17 by the kidneys.47 For all these reasons, nanoparticles with a size of 10-100 nm are preferred because of the enhanced permeability and retention effect (EPR).24 The EPR effect relies on the fact that tumors show abnormal vasculature, which means that nano-size drugs are accumulated in the tumors and show differential accumulation and therefore higher concentrations when compared to the plasma or other organs with proper vasculature. In our work, both polymers exhibited an ability to form nanoparticles, with an average size of 66 nm for the control polymer and 72 nm in the case of the Akt-in ELR, with low PDIs in both cases. This difference in size between the two nanoparticles is statistically relevant and bigger than for previous nanoparticles (55 nm) under the same conditions.34 The size and morphology were corroborated by TEM, cryo-TEM and fluorescence microscopy images (Figure 1). As these previous nanocarriers consisted only of the amphiphilic ELR backbone, this difference with respect to our new nanoparticles is mainly due to the additional functional peptides. To a lesser extent, the presence of the three bioactive sequences also results in a slight increase in the size of the Akt-in nanoparticles (72 nm). We can therefore conclude that both nanoparticles (control and those carrying the Akt inhibitor) meet all the size requirements for reaching the tumor in a controlled manner, as explained above. Furthermore, both control and Akt-in nanoparticles showed the same size when incubated with BSA (Table S2). This could mean that nanoparticles remain stable in systemic circulation and are not affected by plasmatic protein, such as albumin. The critical micellar concentration (CMC) was also studied in order to determine the concentration above which ELRs self-assemble into nanoparticles. The pendant drop method showed that both polymers have their CMC in PBS buffer between 0.21 mg/mL (4.21 µM) for nanoparticles carrying the Akt inhibitor and 0.25 mg/mL (4.49 µM) for control 18 nanoparticles, as shown in Supplementary figure S7. This difference again highlights the fact that the presence of bioactive sequences does not affect the association and self-assembling ability of ELR-based nanoparticles. Experimental measurements are shown in Supplementary material (figure S4-7). Figure 1. Characterization of ELR nanoparticles. TEM images of self-assembled nanoparticles stained with 1% uranyl acetate. A: TEM images of self-assembled nanoparticles stained with 1% uranyl acetate. B: cryo-TEM images of self-assembled nanoparticles stained with 1% uranyl acetate. C: Fluorescence images of selfassembled nanoparticles with conjugated fluorescein. D: Higher magnification of fluorescence images of selfassembled nanoparticles with conjugated fluorescein. Cell nuclei were stained with DAPI. Scale bars: 100 nm for A and B, 50 µm for C and 2 µm for D. The surface charge of nanoparticles is of marked importance as regards the electrostatic interactions between nanoparticles and the cellular membrane and evaluating nanoparticle Intensity (Percent) 19 stability. Due to the negative component of the cellular membrane, cationic particles are typically preferred in order to nonspecifically enter into cells by generating holes and inducing local disorders in the membrane,48 while anionic particles strongly influence membrane structures.49 However, anionic and neutral nanoparticles are thought to enter cells via endocytic pathways. Moreover, positively charged nanoparticles result in membrane depolarization, which reduces the viability of normal cells, thus having a stronger disruptive ability on the lipid bilayer of the cellular membrane.50 Furthermore, neutral and negatively charged nanoparticles are able to enter into the lymphatic system better than cationic carriers, which are more likely to form aggregates with interacting proteins, whereas neutral and anionic particles are thought to avoid renal clearance more efficiently.51 The zeta potential, which determines the surface charge of the nanoparticles, was found to be clearly negative (- 27 mV) due to the presence of glutamic acid residues at the nanoparticle surface. Despite the presence of three lysine residues in the corona, the zeta potential was not altered when compared to previous nanoparticles containing the same amphiphilic backbone reported by García-Arévalo et al.34 Thus, these three lysine residues did not affect the nonspecific internalization of nanoparticles by electrostatic interactions with the cellular membrane. Moreover, the bioactive sequences of nanoparticles carrying the Akt inhibitor did not alter the zeta potential compared to control nanoparticles containing the ELR amphiphilic backbone, thus suggesting that bioactive domains are located at the nanoparticle core. Interestingly, both ELR-based nanoparticles showed stable zeta potential when incubated with BSA (Table S2). This could mean that nanoparticles did not interact with albumin, which is the main protein in the plasma, and remain stable in systemic circulation. 3.4 Effect of nanoparticles on cell viability 20 The main objective of this work was to develop a novel smart drug delivery system in order to achieve an accurate release of an Akt inhibitor, which was designed to trigger apoptosismediated death of cancerous cells. Thus, based on different expression levels of Akt protein between cancerous and normal cells, the ELR- based nanoparticles were expected to show enhanced effect on cell viability of cancerous cells, compared to normal ones. Once the nanoparticles had been physically characterized, their biological effect on three human cancer cell lines (Caco-2 epithelial colon carcinoma, and two breast cancer lines: MCF-7 and MDA-MB-231) and three normal human primary cell lines (HFF-1 fibroblasts, hMSCs (mesenchymal stem cells) and HUVEC endothelial cells) was examined. Human cancer cell lines were used for this purpose because of their higher expression of Akt protein than normal human cell lines. Furthermore, cancer cells are known to show higher internalization rates due to their faster metabolic state. We used three different concentrations of nanoparticles ranging from the critical micellar concentration (CMC) of 0.25 mg/mL to 1 mg/mL. As shown in the figures 2 and S8, the viability of cells exposed to three different concentrations of both types of particles was studied at increasing incubation times. First of all, we determined the cytotoxic effect of control nanoparticles (Figure S8). Although this type of nanoparticle did not carry any bioactive sequence, internalization could affect cellular viability by destabilizing the membrane. Incubation with control nanoparticles did not significantly affect the viability of any of the six cell lines studied. Indeed, the results showed no difference between either the three different concentrations studied or between the different time points (from 30 to 120 min). Thus, we can conclude that, under the experimental conditions used, the control system does not cause a decrease in the viability of either cancerous or non-cancerous cells. This lack of effect could happen either because control nanoparticles are not internalized or because they do not affect cell viability. As both 21 types of nanoparticles have the same surface components, we expected the same internalization rates, thus meaning that we can conclude that control nanoparticles do not compromise cell viability as they are completely innocuous. Akt-in nanoparticles also showed no effect on the viability of non-cancerous human cells at lower concentrations (Figures 2 and 3). Thus, endothelial cells, mesenchymal cells and fibroblasts were only slightly affected when incubated for 120 minutes with nanoparticles at the highest concentration (1 mg/mL), with cell viability decreasing to 71%, 83% and 77%, respectively. Furthermore, there were no significant differences between non-cancerous cells treated with 0.25 and 0.5 mg/mL nanoparticles carrying the Akt inhibitor at any time. Of the three normal cell lines used, HUVEC cells were the most affected. These results are in accordance with the literature as vascular cells are the most sensitive healthy cells due to their contact with nanoparticles during systemic administration.52 In light of the above, we have provided evidence that the effect of nanoparticles on cell viability is both time- and concentration-dependent. Indeed, there were no significant differences between the viability of normal cells with a concentration of 0.25 and 0.5 mg/mL at any time point. 22 Figure 2. Percentage viability for HFF-1, hMSCs and HUVEC (panel A) and MDA-MB-231, Caco-2 and MCF- 7 (panel B) with respect to untreated cells. Cells were incubated with Akt-in nanoparticles at three concentrations and times and viability was measured using the LIVE/DEAD assay kit. n = 3 independent experiments, mean ± SD. **p < 0.01; ***p < 0.001. In contrast, when cancer cell lines were incubated with nanoparticles carrying the detachable Akt inhibitor, cell viability was strongly affected (Fig. 2). Thus, the viability of cancer cells decreased to less than 20% and 40% after incubation with 0.5 and 1 mg/mL for only 30 minutes, respectively, thus indicating the rapid internalization of these Akt-in NPs. After incubation for 120 minutes, the minimal dose tested, which also corresponds to the CMC of the nanoparticles (Figure 2B), resulted in the death of 55-65% of cancerous cells. Similarly, when the nanoparticle concentration was increased to 0.5 and 1 mg/mL, the effect on cancer 23 cells was markedly higher (cell viability of 4% and 8% respectively). Thus, an increase in the concentration of Akt-in nanoparticles results in a marked reduction in cell survival. This result suggests that this concentration is the minimal dose able to affect 50% cell viability. These findings also show that, of the three cancer cell lines studied, Caco-2 cells are more resistant to treatment with nanoparticles. These differences could be due to the fact that these cell lines have different internalization rates. Interestingly, the 0.5mg/mL concentration strongly affected the viability of cancer cells without affecting normal cells, therefore this intermediate concentration was used for subsequent experiments because it seemed to be the largest therapeutic window in which significant differences in the viability of normal cells when compared to cancerous cells were observed. This is of particular importance as it could allow control by modulating the concentration. The enhanced action of nanoparticles on cancer cell lines when compared to normal cell lines could also be due to the fact that cancer cells are better able to internalize nanoparticles, as demonstrated by Villanueva et al.53 24 Figure 3. Representative fluorescence microscopy images for HFF-1, hMSCs, HUVEC, MDA-MB-231, Caco- 2 and MCF-7 cells after incubation with control nanoparticles or Akt-in nanoparticles. Cells were incubated with Akt-in nanoparticles 0.5 mg/mL for 120 minutes and viability was measured using the LIVE/DEAD assay kit. Scale bars are 100 µm. 3.5 Inhibition of Akt phosphorylation Akt kinase is activated, in response to multiple stimuli, such as growth factors, by phosphatidyl inositol triphosphate products generated by PI3K. These lipid products bind to 25 Akt and induce a conformational change in Akt, thus allowing PDK1 to phosphorylate threonine 308. Moreover, phosphorylation of serine 473 and membrane anchoring are required after threonine 308 phosphorylation for final activation of Akt kinase.28 As we explained above, the mechanism of action of the small peptide inhibitor involved attachment to the Akt kinase, thereby avoiding the phosphorylation of Ser473.30 In order to confirm the specific effect of the peptide inhibitor, immunoblotting assays were performed in Caco-2 and MDA-MB-231 cells after treatment with Akt-in nanoparticles for 2 hours at 37°C. As can be seen from figure 4, Akt phosphorylation was not altered when cancer cell lines were incubated with control nanoparticles. However, when both cell lines were treated with nanoparticles carrying the inhibitor, phosphorylation of Akt protein at Ser473 was prevented. Consequently, we can conclude that the effect of Akt-in nanoparticles on cell viability is due to the accurate inhibitor delivery and its consequent anti-phosphorylation activity, as expected. Treatment Caco-2 MDAMB-231 Control NP - + - - + - Akt-in NP - - + - - + 32 escape from the lysosome, thus meaning that Akt protein is not reached and continues to enhance cell proliferation and block apoptotic cell death. Figure 8. Lysosomal trafficking of nanoparticles carrying Akt inhibitor in MDA-MB-231 and Caco-2 cell lines. Cells were incubated with Pepstatin A (negative control), Akt-in nanoparticles or both, and viability was measured using the LIVE/DEAD assay kit. n = 3 independent experiments, mean ± SD. ***p < 0.001. 4. Conclusion One of the disadvantages of current chemotherapeutic treatments against cancer is the lack of specificity of the drugs, which therefore results in significant damage to healthy tissues.18 In order to improve the selectivity of new drugs, nanotechnological approaches with incorporated targeting systems appear to be the best strategy.2 Furthermore, different types of solid tumor offer multiples obstacles to the drug-delivery systems tested to date. Akt kinase is one of the most interesting of the multiple proteins that can be targeted due to its higher expression in cancerous cells and its role as a key factor controlling multiple signalling pathways and processes, such as cell growth, proliferation and survival.26 The small peptide 33 inhibitor Akt-in was therefore designed in order to block Akt protein, thereby inhibiting both its essential activity and tumor cell growth.30 Thus, we have developed a new smart nanodevice specifically designed including different bioactive peptides so as to achieve the proper delivery of therapeutic agents in targeted cells and tissues. Genetic engineering technics used for the design of these ELR polymers allows us to be able to create different advanced drug delivery systems with diverse applications as therapeutic approach for multiple diseases, taking advantage of its smart stimuli-responsive behavior. In this work, we have developed a new ELR-based nanoparticle carrying a small peptide inhibitor against Akt protein in order to create an advanced approach for application thereof in the therapeutic treatment of cancer. It should be noted that a complex design of the smart nanoparticles, with different actors, was needed in order to successfully release the inhibitor into the cell cytoplasm, thereby avoiding degradative proteases which would not allow the peptide to enter otherwise. We tested the specificity of this novel ELR nanoparticle in 6 different cell lines. Thus, our drug delivery system showed no effect in three primary non-cancerous cell lines, while the same treatment showed lethal effects in breast and colorectal cancerous cells. Moreover, in vitro experiments confirmed that each block included in the polymer was absolutely required for the proper release of the inhibitor in the cellular cytoplasm. Thus, inactivation of lysosomal proteases and inhibition of vesicles acidification resulted in abolished effect of nanoparticles. Overall, based on our findings, we can conclude that this smart nanodevice could be a novel strategy for the proper release of therapeutic agents at molecular level in targeted cells. This study is the first to report an accurate smart nanodevice against Akt protein after intracellular 34 activation. Interestingly, our system improved the accuracy of the inhibitor in a time- (our system was 12 times faster than the inhibitor alone) and dose-dependent manner (5 times lower amount of inhibitor), compared to previous works with Akt-in.30 This improved action of Akt-in when carried in nanoparticles could be due to the better internalization of nanoparticles compared to nude peptides and their shielding effect, which protects the inhibitor from cellular proteases. It is worthy to mention that our new therapeutic system is not limited to one type of cancer, as it is targeted to Akt kinase protein, which is overexpressed in multiple neoplastic diseases, such as colon, pancreatic, breast, ovarian and lung cancer. Further studies are needed to study the accuracy of these nanoparticles in in vivo models better resembling the tumor environment and its interactions with nanocarriers. In the future, patients overexpressing Akt may be candidates for therapeutic treatment with nanoparticles bearing the inhibitor, which could improve the problems caused by current non-specific chemotherapeutic drugs. Supporting Information Amino acid sequence of polymers (Table S1), SDS-PAGE of ELRs purification (Figure S1), High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MALDITOF/MS) analysis of control polymer (Figure S2) and Akt-in polymer (Figure S3), Differential Scanning Calorimetry (DSC) analysis of ELRs (Figure S4), Dynamic Light Scattering analysis of nanoparticle size (Figure S5), Dynamic Light Scattering analysis of nanoparticle zeta potential (Figure S6), Dynamic Light Scattering analysis of nanoparticle size and zeta potential after incubation with 5% BSA (Table S2), Critical Micellar Concentration (CMC) analysis of ELRs using the pendant drop method (Figure S7) and Live/Dead analysis of HFF-1, hMSCs, HUVEC, MDA-MB-231, Caco-2 and MCF-7 35 incubated with control nanoparticles at three concentrations and times (Figure S8). This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding author *Corresponding author: a[email protected] BIOFORGE (Group for Advanced Materials and Nanobiotechnology), CIBER-BBN, University of Valladolid, 47011 Valladolid, Spain. Conflict of interest The authors declare no competing financial interest. Acknowledgments The authors are grateful for financial support from the European Social Fund (ESF) and the European Regional Development Fund (ERDF), as well as funding from the EU (NMP-2014- 646075), the MINECO (PCIN-2015-010, MAT2015-68901-R, MAT2016-79435-R and MAT2016-78903-R), the JCyL (project VA317P18), the CIBER-BBN, the JCyL and the Instituto de Salud Carlos III under the "Network Center of Regenerative Medicine and Cellular Therapy of Castilla and Leon". The authors would like to thank R. García for her technical assistance. References (1) Saxena, R.; Nanjan, M. J. Elastin-like polypeptides and their applications in anticancer drug delivery systems: a review. Drug Deliv. 2015, 22 (2), 156-67. 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Chem. 2013, 288 (5), 3136-52. 39 TOC Abstract: 40 Supplementary material 3K LAEL E50 I60 CatD H5 Akt-in Control MGKKKP V (LAEL)3 [(VPGVG)2 (VPGEG)10 (VPGVG)2] [VGIPG]60 Akt-in MGKKKP V (LAEL)3 [(VPGVG)2 (VPGEG)10 (VPGVG)2] [VGIPG]60 VQEYVYD LFHAIAHF HIHGGWH GLIHGWY AVTDHP DRLWAW ERF Table S1. Composition of polymers. Amino acid sequence of ELR polymers. Figure S1. Characterization of ELR polymers. The expression vectors containing the selected ELR genes were transformed into Escherichia coli BLR (DE3) strain for production. The ELR was then bioproduced in Escherichia coli in a 15-L bioreactor and purified by several cooling and heating purification cycles (Inverse Transition Cycling) following centrifugation, thereby taking advantage of the ability of these recombinamers to aggregate above their transition temperature. Finally, the polymer was dialyzed against ultrapure water type I and sterilized by filtration (0.22 μm filters). A: Purification of control polymer measured by SDS-PAGE. 1: protein marker; 2: cold supernatant; 3: cold pellet; 4: hot supernatant; 5: hot pellet; 6: cold supernatant; 7: cold pellet; 8: protein marker; 9: pure lyophilized polymer. Purification of Akt-in polymer measured by SDS-PAGE. 1: protein marker; 2: production sample; 3: production sample; 4: cold pellet; 5: cold supernatant; 6: hot pellet; 7: hot supernatant; 8: protein marker; 9: pure lyophilized polymer 41 Figure S2. Characterization of control polymer. A: The amino acid composition was verified by high performance liquid chromatography (HPLC). B: The molecular weight of the recombinamer was determined by mass spectrometry (MALDI-TOF/MS).