Peptide Nanosponges Designed for the Delivery of Perillyl Alcohol to

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Peptide Nanosponges Designed for the Delivery of Perillyl Alcohol to Glioma Cells Asanka S. Yapa, Tej B. Shrestha, Sebastian O. Wendel, Madumali Kalubowilage, Jing Yu, Hongwang Wang, Marla Pyle, Matthew T. Basel, Yubisela Toledo, Raquel Ortega, Aruni P. Malalasekera, Prem S. Thapa, Deryl L Troyer, and Stefan H. Bossmann ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00305 • Publication Date (Web): 11 Dec 2018 Downloaded from http://pubs.acs.org on December 11, 2018

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Scheme 1: Perillyl alcohol inhibits glioma cell invasion, migration, and proliferation by interfering with Rasprenylation.37 The majority of Ras family proteins (Ras superfamily of small guanosine triphosphatases (GTPases) terminate with a C terminal CAAX (C=Cys, A=aliphatic, X=any amino acid) tetrapeptide sequence.44 196x126mm (300 x 300 DPI)


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Scheme 3: Functions of the different regions of each peptide branch in (D-POH)10K20 and (D-POH)10R20 nanosponges. Red: trigonal linker, green: oligopeptide for enhanced uptake (K20 >> R20), black: biotin is added to enhanced water-solubility and to further enhance uptake. 178x144mm (300 x 300 DPI)



Figure 7: Cell viabilities of neural progenitor cells (NPC) and murine glioma cells (GL26) as a function of the concentration of type (D-POH)10K20 (green) and (D-POH)10R20 (blue) nanosponge in serum-containing medium (10% FBS, 5% horse serum) after 48h of exposure. The red line shows the results of the control experiment (only PBS was added). NPC: The calculated p-values for type D-POH)10K20 vs. PBS control are p = 6.1 x 10-6 (highly significant) and for (D-POH)10R20 vs. PBS control are p = 0.30 (not significant). GL26: The calculated p-values for type (D-POH)10K20 vs. PBS control are p = 1.6 x 10-6 (highly significant) and for (D-POH)10R20 vs. PBS control are p = 0.76 (not significant). 106x130mm (600 x 600 DPI)


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Figure 9: Uptake of rhodamine B-labeled (D-POH)10K20 nanosponges by murine glioma cells (Gl26) using a Zeiss Aciovert 40 CF microscope (40X). A: bright field microscopy, B: fluorescence microscopy, total intensity, C: blue and red filter combined. Individual bright red spots are discernible, indicating endocytosis. 148x330mm (300 x 300 DPI)



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Peptide Nanosponges Designed for the Delivery of Perillyl Alcohol to Glioma Cells Asanka S. Yapaa‡, Tej B. Shresthab‡, Sebastian O. Wendelb‡, Madumali Kalubowilagea, Jing Yua, Hongwang Wanga, Marla Pyleb, Matthew T. Baselb, Yubiselaa Toledo, Raquel Ortegaa, Aruni P. Malalasekeraa, Prem S. Thapac, Deryl L. Troyerb*, Stefan H. Bossmanna*

‡

a

b

c

These authors have contributed equally

Department of Chemistry, Kansas State University, Manhattan, KS, USA

Department of Anatomy & Physiology, Kansas State University, Manhattan, KS, USA

Microscopy and Analytical Imaging Laboratory, University of Kansas, Lawrence, KS, USA Present addresses: HWW: 101 BIVAP Innovation Center, Kimball Ave., Manhattan, KS, USA SOW: Department of Biology, Kansas State University, KS, USA APM: Department of Chemistry, Southwestern College, Winfield, KS, USA RO: National Institute of Health, Bethesda, MD


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KEYWORDS Peptide Nanosponges, AFM, Caspase-mediated Activation, Cell Uptake, Neural Progenitor Cells, Murine Glioma, Perillyl alcohol

ABSTRACT

Peptide nanosponges of low polydispersity are spontaneously formed from trigonal supramolecular building blocks in aqueous buffers, which feature cationic and/or anionic oligopeptides (n=5-20) and a hydrophobic unit.

In contrast to classical liposomes/vesicles,

nanosponges feature interwoven hydrophilic and hydrophobic nanodomains, and are readily taken up by mammalian cells. Perillyl alcohol is known to be a simple, but effective small molecule drug against glioma multiforme. However, its efficacy is limited by poor bioavailability. In order to make perillyl alcohol bioavailable, two nanosponges consisting of 10 aspartates, to which perillyl alcohol is attached by means of an ester bond, and 20 lysines or arginines (type (D-POH)10K20 and (D-POH)10R20) were synthesized, purified, and characterized by Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). These nanosponges were then tested in cell cultures of murine glioma cells (GL26) and murine neural progenitor cells (NPC), because the latter was previously utilized in cell-based cancer therapy. The two nanosponges exhibited significantly different biophysical properties (size distribution and zeta potentials). Consequently, different efficacies in killing GL26 and NPC were observed in serum containing culture media. The results from these experiments confirmed that the type (D-POH)10K20 nanosponge is a promising candidate for the (cell-mediated) cytotherapy of glioblastoma.


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INTRODUCTION

Nanosponges were originally developed as topical delivery systems. 1-3 Since 19904 their applications have been expanded to pulmonary5, intravenous6 and oral delivery of drugs1, 3, slowrelease polymers7, decoys for multi-resistant infections8, and systems designed for the capture of toxins or pathogens for proteomics studies.9 Classic nanosponges for medicinal applications are comprised of hyper-crosslinked (co)polymers, such as cyclodextrin-derivatives polystyrenes, and other polymers that are usually insoluble in aqueous buffers. They are chemically and physically stable and increase the stability and bioavailability of encapsulated drugs. Inorganic and hybrid nanosponges that are known as well.10-13 Inspired by Zhang et al., we have prepared self-assembled nanosponges from rationally designed peptides. 14 Like in cyclodextrin-derived nanosponges, which were the most versatile type to date3, 6, 15, 16, our novel nanosponges feature hydrophobic domains, hydrophilic domains, and water-filled nanocavities, as TEM studies and Coarse-Grained Molecular Dynamics simulations indicated.17,

18

The presence of both, hydrophilic and

hydrophobic domains in peptide nanosponges and the absence of alpha-helices or beta-sheets are significant differences to peptide hydrogels.19

In contrast to most classic nanosponges, which can only bind relatively small drug molecules, the novel peptide nanosponges can incorporate virtually any therapeutic (macromolecule), including peptides, either by association with the hydrophilic or hydrophobic nanosponge components, or by means of using a weak chemical bond to tether the drug during transport. Like classic nanosponges, peptide nanosponges are chemically stable in a wide pH-range and stable up to 300 °C.1, 7 They form clear dispersions and can be synthesized by means of very simple procedures, such as shaking or ultrasound-mediated dispersion in aqueous buffers. 17, 18


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To date, more than 130 types of brain20 and central nervous system (CNS) tumors have been discovered. This study focusses on one type of brain cancer; Glioblastoma multiforme (GBM) or Glioblastomas, which are highly malignant. Other than histology, the 2016 CNS WHO accounts molecular parameters for the CNS tumor classification.21 According to the latest WHO tumor classification, glioblastoma is a grade IV astrocytoma21 and it is the most common and most aggressive/fatal primary glioma found in humans. 22-24 Glioblastomas are found in both cerebral hemispheres of the brain, as well as the spinal cord.23 Glioblastomas can be difficult to treat because of their histopathologically heterogeneous nature25 and finger-like tentacles.26 This is why the treatment plans for glioblastoma often combine surgery, radiation, and chemotherapy.27 However, many chemotherapeutics are unable to effectively cross the blood–brain barrier28, leading to poor treatment outcomes. Consequently, the mortality of glioblastoma is very high.29 Maximally 5 percent of glioblastoma patients survive more than five years after diagnosis.29 According to Fonseca et al., the molecular genetics of malignant gliomas provides new targets for antineoplastic agents. Altered activation of the Ras/MAPK and PI3K/Akt pathways in gliomas30, 31 are promising therapy targets.23, 32, 33 These signaling pathways play a critical role in regulating diverse cellular functions including cell survival, cell cycle progression and cellular growth34 in healthy cells, as well as in cancer cells. Overexpression of the oncogenes EGFR and PDGFR22-24, 35 and “mutations and deletions of tumor suppressor genes TP53 and PTEN” 23 are the roots of those overactive signaling pathways. 23 The Ras protein family, belonging to the class of small, membrane-associated GTPases, plays a vital role in cellular signal transduction.36 Elevated levels of Ras proteins are observed in glioblastoma patients. In order to become functionally active, Ras proteins must be attached to the


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inner cell membrane.36 This membrane anchoring is facilitated by a posttranslational modification on Ras proteins. A farnesyl group is covalently attached to the cysteine on C-terminal of the CAAX motif of Ras protein. This process is catalyzed by the enzyme farnesyl transferase. 36 Unanchored Ras is unable to fulfill its signaling functions, resulting in inhibited cellular proliferation.23 Hence, farnesyl transferase inhibitors can be considered as a new class of antineoplastic drugs, which act by altering cell signal transduction and thereby inhibiting proliferation and survival of malignant cells (Scheme 1).23 Recent studies have revealed that the naturally occurring monoterpene perillyl alcohol (POH) (IUPAC: [4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]methanol) is a potent pharmacological inhibitor of the Ras-mediated signaling pathway.23, 37-39 In recent phase I clinical trials, Azzoli et al. showed that the maximum tolerance dose of POH is 8400 mg/m2 per day when delivered orally.40 According to the phase I and phase II human clinical trial results, oral administration of POH does not exert hepatic, renal or neurobiological toxicity, but it does cause gastrointestinal tract disturbances41, such as nausea, vomiting and diarrhea.40, 42, 43 To successfully use POH clinically, it is necessary to find effective POH delivery strategies, limiting side effects. The novel PeptideNanosponges17,

18

that were developed earlier by us are ideal candidates for biomedical

applications, because they are virtually non-toxic, highly biocompatible, and biodegradable.17, 18


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OH

Acetyl-CoA

Farnesyl-diphosphate

Perillyl alcohol

FTase

H-Ras-CAAX

H-Ras-F Farnesylated proteins

Scheme 1: Perillyl alcohol inhibits glioma cell invasion, migration, and proliferation by interfering with Ras-prenylation.37 The majority of Ras family proteins (Ras superfamily of small guanosine triphosphatases (GTPases) terminate with a C terminal CAAX (C=Cys, A=aliphatic, X=any amino acid) tetrapeptide sequence.44

The novel nanosponges were designed by utilizing the self-assembling properties of (cholesterol-(K)nDEVDGC)3-trimaleimide and (cholesterol-(D)nDEVDGC)3-trimaleimide units that both feature a trigonal linker, a cleavable sequence designed for caspases-3, 6, and 7 (DEVDGC45) and either an oligo-lysine or oligo-aspartic acid sequence (n = 20).17 The C-terminus of each oligopeptide is attached it to the trigonal linker via Michael addition to maleimide46, whereas the N-terminus is tethered to a hydrophobic cholesterol anchor. A sponge-like dynamic structure is spontaneously assembled, due to the formation of ion pairs, intense hydrogen bonding, and the occurrence of hydrophobic regions and water-filled nanocavities, as Coarse-Grained Molecular Dynamics simulations suggest.17 Spontaneous molecular self-assembly is a free energy driven spontaneous process, which offers many advantages when synthesizing tunable nanoscale structures, including adjustable size, shape and surface chemistries. 47-49


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We demonstrated with different characterization methods, such as dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) that the obtained nanosponges are of low polydispersity and their sizes range from 100 to 150 nm in diameter.17, 18 These nanosponges are essentially not toxic and are rapidly taken up by mammalian cells.17 Therefore, they have the potential of becoming well-suited materials for cell-based drugdelivery applications in cancer therapy and theranostic applications.

Nanosponge-based Delivery Platforms for Perillyl Alcohol (POH) We have developed two nanosponges for POH delivery by designing peptide chains containing segments of D10K20, and D10R20, thus combining oligoaspartic acid units with oligolysine or oligoarginine units (Scheme 2). These peptides were again coupled to trimaleimide via the facile maleimide-thiol coupling reaction.46 To the N-terminus of the peptides biotin was attached to enable targeted delivery by the nanosponge. Then POH was covalently bound to the peptide via the carboxyl groups of the aspartic acid side chain. This ester bond is designed to survive the uptake by neural progenitor cells50 and subsequent transport to gliomas through the blood brain barrier following cytokine gradients.50 Slow hydrolysis in the presence of esterases51 will eventually result in apoptosis of the delivery cell when the tumor site is reached, and the payload is released. The novel drug-loaded carriers are biotin-((D-perillyl alcohol)10 K20DEVDGC)3trimaleimide (type (D-POH)10K20) and biotin-((D-perillyl alcohol)10R20DEVDGC)3-trimaleimide (type (D-POH)10R20).


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Scheme 2: Chemical structures of nanosponge types D(POH) 10K20 and D(POH)10R20: G: glycine, D: aspartic acid, V: valine, E: glutamic acid, K: lysine, R: arginine. The chemical composition of the peptide components has been verified by MALDI-TOF. The data is included in the SI section. The “critical aggregation concentrations (cac)19”, e.g. the concentrations at which nanospongeformation spontaneously occur, have been determined for the nanosponge types D(POH)10K20 and D(POH)10R20, as well as for their precursors featuring no attached perillyl alcohol units following a methodology developed earlier.17,

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The resulting structures and the effects of caspase-6

digestion on the morphology of D(POH)10K20 nanosponges have been investigated by Atomic Force Microscopy (tapping mode) as well. Nanosponges for cytotherapy of gliomas using neural progenitor cells50 as drug carriers are only viable if it can be proven that a) the nanosponges are toxic to glioma cell cultures and b) that they


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are less toxic to neural progenitor cells. Therefore, we have determined cell viabilities in serum containing media.

RESULTS AND DISCUSSION DLS Characterization of the Nanosponges The effective diameters and the polydispersity index (PDI) values of the nanosponges obtained by dynamic light scattering measurements (DLS) are summarized in Table 1. Continuous monitoring by DLS for 12 hours at 298 K revealed that type (D-POH)10K20 and (D-POH)10R20 nanosponges are very stable in aqueous solution (PBS). The corresponding correlation curves and number-averaged size distributions are shown in Figure 1. As summarized in Table 1, the “cac”19 of the (Biotin-D10(Perillyl alcohol10)K20DEVDGC)3trimaleimide is about 0.0011 mM. At that concentration, nanosponges of the order of 2 micrometers in diameters are formed with relatively low polydispersity (PD = 0.134 to 0.282). In distinct contrast, supramolecular aggregation of (Biotin-D10(Perillyl alcohol10)R20DEVDGC)3trimaleimide does not occur until the concentration of the supramolecular building blocks is about 45 times greater. Interestingly, the resulting type (D-POH)10R20 nanosponges are smaller in diameter (approx. 360 nm) (PD = 0.307 to 0.113).


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Table 1: Diameter and CAC of Type (D-POH)10K20 and (D-POH)10R20 Nanosponges Concentration cac19

Nanosponge

Diameter

Type

/ nm (DLS17, / mM 18 )

/ mM

(D-POH)10K20

1955 ± 258

0.0011

0.0011

(D-POH)10K20

1880 ± 205

0.00165

(D-POH)10R20

360 ± 52

0.0488

(D-POH)10R20

421± 32

0.0676

Polydispersity

/ mV52, 53 0.134 0.282

0.0488

Zeta-Potential

20 ± 1

0.307 0.113

-2±3

It is of mechanistic interest that the cac of type (D-POH)10K20 is approx. 5 times lower than the cac of the binary nanosponge (cholesterol-(D)20DEVDGC)3-trimaleimide and (cholesterol(K)20DEVDGC)3-trimaleimide, (type DK20) of 0.0050 mM (total concentration, 0.0025 mM (D) and 0.0025 mM (K)).17 In type DK20 each peptide sequence is attached to a terminal hydrophobic cholesterol unit (log P 40 = 7.39) and charge-attraction, as well as hydrogen-bonding can occur in aqueous buffers.41 In contrast, perillyl alcohol has a log P = 1.95, which is not hydrophobic. The differences between type (D-POH)10K20 and type K20 are even more pronounced. For (cholesterol(K)20DEVDGC)3-trimaleimide a cac of 0.080 mM was determined by means of DLS, which is 70 times larger than the cac of (Biotin-D10(Perillyl alcohol10)R20DEVDGC)3-trimaleimide. The zeta potentials of type (D-POH)10K20 nanosponges in PBS were positive, which was expected considering that they contain 20 lysine segments. In sharp contrast, the nanosponges featuring R 20 segments possess zeta potentials that are very close to 0 mV. This was quite surprising and a clear indication that the structures of type (D-POH)10K20 and (D-POH)10R20 nanosponges are very different! Whereas in (D-POH)10K20 we observe oligo-lysine chains at the surface, in (DPOH)10R20 the surface is characterized by either the presence of aspartate-perillyl units, and/or the


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nanosponge is efficiently attracting chloride and/or ((di)hydrogen)phosphate anions from PBS. This is evidenced by the observed difference in zeta-potential of (D-POH)10K20 (𝜁 = 21.9 ± 0.52𝑚𝑉) and (D-POH)10R20 (𝜁 = −1.65 ± 1.02𝑚𝑉) (Figure S5).


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Figure 1: Correlation curves (C()) of dynamic light scattering measurements of (BiotinD10(Perillyl alcohol10)K20DEVDGC)3-trimaleimide nanosponges (type (D-POH)10K20) and (Biotin-D10(Perillyl alcohol10)R20DEVDGC)3-trimaleimide nanosponges (type (D-POH)10R20) in deoxygenated PBS buffer below and above their critical aggregation concentration”.19 In Figure 2, the concentration dependence of type (D-POH)10K20 and (D-POH)10R20 nanosponges is shown. Both curves begin at their respective (estimated) cac’s. Apparently, the sizes of both types of nanosponges can be adjusted by selecting their concentration. Rapid changes of the observed nanosponge diameters have been measured during the first 10 min. After that time, the observed diameters did not change within the experimental errors for 24h.

Figure 2: Average hydrodynamic diameters, as measured by DLS, as a function of type (DPOH)10K20 and type (D-POH)10R20 nanosponge concentrations. In each curve, the typical experimental error is indicated.


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TEM Analysis of Type (D-POH)10K20 and type (D-POH)10R20 Nanosponges

The morphology of Biotin-(D10(Perillyl alcohol10)K20DEVDGC)3-trimaleimide (A) and Biotin(D10(Perillyl alcohol10) R20DEVDGC)3-trimaleimide (B) on graphite is shown in Figure 3. Whereas type (D-POH)10R20 nanosponges appear to be spherical, type (D-POH)10K20 nanosponges form rod-like structures. The main axis of the nanorods formed by type (D-POH)10K20 is about 100nm, whereas a highly polydisperse size distribution (diameters ranging from 40 nm to 400 nm) is found for the spheres from type (D-POH)10R20. In agreement with earlier research on peptide nanosponges featuring cholesterol labels17, 18, the structures of type (D-POH)10K20 and type (DPOH)10R20 nanosponges feature water-filled cavities of less than one to three nanometers in diameter.18 This water evaporates in high vacuum, thus leaving bright voids within the structure. It is noteworthy that the TEM-derived sizes of type (D-POH)10K20 and type (D-POH)10R20 nanosponges do not correlate with their DLS-derived sizes (Table 1). This was anticipated since in previous experiments, a similar behavior for cholesterol-labeled nanosponges was found.

17, 18

The use of HOPG (Highly Ordered Pyrolytic Graphite) as surface in TEM, the use of uranyl acetate as contrast agent, and the desiccation of the nanostructures in high vacuum during the TEM characterization have been identified as major factors for the observed differences. To date, type (D-POH)10K20 are the only examples of trigonal nanosponges that feature rod-like structures. This anomaly will be further investigated in the future. However, it should be considered that the high aspect ratio observed in TEM may arise from the measurement conditions.


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Figure 3: Bright-field TEM of Biotin-(D10(Perillyl alcohol10)K20DEVDGC)3-trimaleimide (A) and Biotin-(D10(Perillyl alcohol10) R20DEVDGC)3-trimaleimide (B) on graphite (10 µL of 0.050 mM in PBS). Uranyl acetate (0.005 mM in PBS) was used as staining agent. A FEI Technai G2 transmission electron microscope at an electron acceleration voltage of 200 kV was used.54

Atomic Force Microscopy of Type (D-POH)10K20 and type (D-POH)10R20 Nanosponges Figure 4 shows the AFM images (tapping mode) type (D-POH)10K20 and (D-POH)10R20 nanosponges. Both types form polydisperse supramolecular (mainly two-dimensional) aggregates on MICA surfaces. Whereas (D-POH)10K20 nanosponges show a maximum in diameter of 40  10


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nm (Figure 5), type (D-POH)10R20 nanosponges form aggregates of 100  30 nm in diameter. The resulting structures are approx. 3 nm in height for both nanosponge types.

Figure 4: AFM images (tapping mode) and height profiles of (D-POH)10K20 (A) and (D-POH)10R20 (B) nanosponges (0.050 mM of each nanosponge in PBS) on MICA. The effect of caspase-6 digestion on (D-POH)10K20 nanosponge morphology on MICA was studied by means of a series of AFM experiments. The AFM images and height profiles obtained at 0h, 1h, 2h, and 3h are shown in the SI section. Figure 5 summarized the size distributions of the supramolecular aggregates of (D-POH)10K20 on MICA in the absence and presence of caspase-6.


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Figure 5: Size distribution (relative percentage) of supramolecular aggregates of 0.10 mM Biotin(D10(Perillyl alcohol10)K20DEVDGC)3-trimaleimide nanosponges on MICA in the absence and presence of 10-8 M caspase-6 (Enzo Lifesciences). 1.0ml of the freshly prepared nanosponge stock solution in PBS was deposited onto a freshly peeled MICA sheet, followed by removing of the solvent by using a gentle nitrogen stream (2 min). 0h: no caspase added, 1-3h, incubation of 1 to 3 hours at 37oC in the presence of 10-8 M caspase-6 prior to deposition on MICA. Quantitative analysis of the size distribution of the nanosponges during caspase-6 digestion was performed using the program NIH IMAGE.55 As shown in Figure 5, the main diameter of type (D-POH)10K20 nanosponge on MICA is 40  10 nm. Incubation with caspase-6 leads to a subsequent increase in the diameters of the supramolecular aggregates, indicating enzymatic cleavage and reorganization of the aggregates. These findings can be regarded as proof that the consensus sequence DEVDGC56 is cleaved by caspase -6.


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Cell Experiments and MTT Assays

Cell viability was measured by using the MTT assay, which is sensitive to cell proliferation.57 The murine glioma cell line (GL2658) was selected, because it is considered one of the best cell lines for immunocompetent mouse models for glioblastoma. 58 Neural progenitor cells (NPC) have been successfully used by us for cell-mediated delivery of nanotherapeutics to tumors.50, 59 LC50 values for all experiments performed were calculated using the Graphpad Prism software60 and are summarized in Table 2.

Statistical analysis Data will be presented as means +/- standard error. Means of the groups will be first evaluated for normality, then analyzed by ANOVA followed by the Newman-Keuls Post Hoc procedure, or where indicated, Student’s t test.61 Statistical significance will be set at p < 0.05.

Cell Toxicity Assays Before performing cell toxicity experiments with nanosponges containing perillyl alcohol, the toxicity of perillyl alcohol itself was tested. The results shown in Figure S10 prove that perillyl alcohol is virtually not toxic to neural progenitor cells and glioma cells in the chosen concentration range. The same concentrations of perillyl alcohol were used, either as free perillyl alcohol in the control experiments, or chemically bound to both nanosponges. We have also tested the toxicity of the nanosponges without attached perillyl alcohol. Both, type D 10K20 and type D10R20 nanosponges were essentially not toxic to both GL26 and NPC cells. After establishing this, we proceeded to testing nanosponges with chemically attached perillyl units.


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The cell experiments reported here comprise measuring the cell viabilities of neural progenitor cells and murine glioma cells after adding type (D-POH)10K20 and (D-POH)10R20 nanosponges for 24h and 48h. The concentrations of both nanosponges that were added to the cell cultures ranged from 0 to 0.16 𝜇g/mL. The experiments conducted in the presence of 10% FBS exhibited promising results for type (DPOH)10K20 nanosponges. They were found to be very effective against GL26 cells after both 24 and 48 hours of incubation. It is noteworthy that type (D-POH)10K20 nanosponges show also a modest activity against neural progenitor cells, albeit only at the highest tested concentration. This finding does not rule NPCs out as transport cells for (D-POH)10K20 in future animal models testing cell-mediated glioma therapy, because the payload can be adjusted to safe concentrations during cell-mediated transport. Type (D-POH)10R20 did not exhibit any activity, neither against GL26, nor against NPC cells in serum-containing medium. Potential reasons for the observed differences in the activities of (D-POH)10K20 and (D-POH)10R20 nanosponges will be discussed below.


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Figure 6: Cell viabilities of neural progenitor cells (NPC) and murine glioma cells (GL26) as a function of the concentration of type (D-POH)10K20 (green) and (D-POH)10R20 (blue) nanosponge in serum-containing medium (10% FBS, 5% horse serum) after 24h of exposure. The red line shows the results of the control experiment (only PBS was added). The black line is a second control that was introduced by adding 5 x 10-9 M of active recombinant caspase-6 (Enzo Lifesciences) to (D-POH)10R20. NPC: The calculated p-values for type D-POH)10K20 vs. PBS control are p = 3.1 x 10-6 (highly significant) and for (D-POH)10R20 vs. PBS control are p = 0.29 (not significant). After addition of caspase-6, p = 3.57 x 10-6 was calculated, indicating statistical significance. GL26: The calculated p-values for type D-POH)10K20 vs. PBS control are p = 4.2 x


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10-6 (highly significant) and for (D-POH)10R20 vs. PBS control are p = 0.36 (not significant). After addition of caspase-6, p = 0.42 was calculated (not significant).

One further control experiment has been added to the MTT experiments shown in Figure 6: 2 𝜇l of 5 x 10-9 moles L-1 of active recombinant caspase-6 (purchased from Enzo Lifesciences) was added to each well before the cells were incubated for 24h in the presence of (D-POH)10R20. Caspase-6 will cleave the consensus sequence DEVDGC, which leads to a partial release of the payload (see Figures 7 and 8).44 However, this measure was unable to enhance the cell toxicity of (D-POH)10R20, mainly because perillyl alcohol is released outside of the cells.


20


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Figure 7: Cell viabilities of neural progenitor cells (NPC) and murine glioma cells (GL26) as a function of the concentration of type (D-POH)10K20 (green) and (D-POH)10R20 (blue) nanosponge in serum-containing medium (10% FBS, 5% horse serum) after 48h of exposure. The red line shows the results of the control experiment (only PBS was added). NPC: The calculated p-values for type D-POH)10K20 vs. PBS control are p = 6.1 x 10-6 (highly significant) and for (D-POH)10R20 vs. PBS control are p = 0.30 (not significant). GL26: The calculated p-values for type (DPOH)10K20 vs. PBS control are p = 1.6 x 10-6 (highly significant) and for (D-POH)10R20 vs. PBS control are p = 0.76 (not significant).


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Table 2: LC50 values (in 𝜇g/ml and nmol/l) of type (D-POH)10K20 (m = 17466.05 g mol-1) and (D-POH)10R20 nanosponges (m = 19134.9 g mol-1) for GL26 and NPC cell lines. Experiments were conducted in serum containing media. (D-POH)10K20

(D-POH)10R20

LC50

LC50

LC50

LC50

μg/ml nmol/L μg/ml nmol/L 24h GL26 0.075

4.29

>1

> 52.3

NPC

> 57.3

>1

> 52.3

GL26 0.068

4.14

>1

> 52.3

NPC

32.10

>1

> 52.3

>1 48h

0.614

Caspase-Mediated Cleavage Two types of nanosponges, (D-POH)10K20 and (D-POH)10R20 were developed as nanoshuttles to transport the anticancer agent, perillyl alcohol into glioma tumor cells, and for uptake by neural progenitor cells, which can potentially serve as transport cells in future cytotherapies of glioblastoma. The peptide nanosponges contain a trigonal maleimide linker and three branches. Their major difference is the presence of either a K20 or a R20 block in (D-POH)10K20 and (DPOH)10R20. Both contain the DEVDGC consensus sequence for caspase-3, -6, and -7 cleavage56 and a terminal D10 unit to which 10 perillyl alcohol molecules are bound via ester functions. The latter can be hydrolyzed by numerous esterases and carbonic anhydrase, which are overexpressed in many gliomas51, 62, 63. Mammalian esterases are known to tolerate a broad variety of substrates. 51,


22


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Furthermore, caspase-6 and potentially other proteases can release 7 ± 2% of perillyl alcohol

after 24h of incubation (Figure 8).

Figure 8: High performance liquid chromatograms of D10K20 nanosponges (blue) (concentration: 0.10 mmol L-1 in HEPES buffer) and D10K20 nanosponges (0.10 mmol L-1 in HEPES buffer) after incubation with caspase-6 (Enzo Lifesciences, 2.5 x 10 -9 moles L-1, 60 min (orange) and 24h (red) at 298K). It is clearly discernible that the consensus sequence DEVDG is cleaved by caspase-656, as indicated by the appearance of a new peak at 3.45 min (A). This peak is virtually consistent with the injection of monomeric (D-POH)10K20DEVDGC (not shown). Furthermore, a fraction of perillyl alcohol (approx. 7 ± 2%) is released in the presence of caspase-6 after 24 of incubation (B). The experiments summarized in Figure 8 demonstrate that activation of the nanosponges with caspase-6, as well as other effector caspases56, is principally possible. Apparently, the added caspase-6 decreases in activity during incubation, resulting in incomplete cleavage of the “branches” from their trigonal linker.


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Scheme 3 shows the different regions of each branch in the trigonal building blocks for the nanosponges. Whereas D10 is responsible for the reversible binding of perillyl alcohol by means of esterification, K20 (and to a much lesser extent R20) are designed to enhance cellular uptake by endocytosis, following the apoptosis of the transport cell. The consensus sequence can be cleaved by all effector caspases (e.g. caspase -2, -3, -6, -7)56, which are overexpressed in virtually all solid tumors. Evidence for this mechanism was obtained by HPLC analysis (Figure 6). Caspase-6 cleaves the consensus sequences of nanosponges that have been taken up by transport cells, thus triggering their release by means of apoptotic processes, enhancing the porosity of the transport cells and then dissecting them into apoptotic bodies.66 These processes facilitate nanosponge release from the transport cells, which integrate with tumor tissue and are, therefore, vulnerable to by-stander effects, because effector caspases frequently retain their activity in solid tumors.66

Scheme 3: Functions of the different regions of each peptide branch in (D-POH)10K20 and (DPOH)10R20 nanosponges. Red: trigonal linker, green: oligopeptide for enhanced uptake (K20 >> R20), black: biotin is added to enhanced water-solubility and to further enhance uptake.


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Nanosponge Uptake by Means of Endocytosis

In Figure 9, bright field and fluorescence microscopy images of rhodamine B-labeled (DPOH)10K20 nanosponges are show that were taken up by glioma cells. The uptake of the same nanosponges by neural progenitor cells is shown in Figure S13. Whereas blue fluorescent Hoechst staining reveals the positions of the nuclei, red fluorescence indicates the location of the rhodamine B-labeled nanosponges. They can be predominantly discerned in the cytoplasm. The presence of intense red dots is consistent with nanosponge uptake via endocytosis.67 Furthermore, Figure S12 shows the same nanosponges after 24h of incubation at 37 oC in RPMI medium in the absence and presence of serum. It is apparent that the peptide nanosponges are stable in serum and that they have diameters of 1 to 3 m in both media, which is consistent with the findings by DLS. Furthermore, these images indicate that the high aspect ratio found in TEM is most likely due to the experimental conditions of this characterization method.


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Figure 9: Uptake of rhodamine B-labeled (D-POH)10K20 nanosponges by murine glioma cells (Gl26) using a Zeiss Aciovert 40 CF microscope (40X). A: bright field microscopy, B: fluorescence microscopy, total intensity, C: blue and red filter combined. Individual bright red spots are discernible, indicating endocytosis.

What Effect is Responsible for the Observed Cytotoxicity?

From the control experiment shown in Figure 6 and summarized in Table 2, it is apparent that the presence of caspase-6 does not enable efficient cell killing by (D-POH)10R20, whereas (DPOH)10K20 is able to kill GL26 glioma cells in low nanomolecular concentrations, even in the


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presence of serum. It is our conclusion that the uptake, not the enzymatic activation of the perillyl alcohol containing nanosponge is the critical step in killing the glioma cells. This is in agreement with the control experiment (see SI section), in which perillyl alcohol in the absence of nanosponges, but in the same concentration as when bound to the nanosponges, was added to both, GL26 and NPC cell cultures. In both cases, perillyl alcohol was virtually not toxic to the cells, because it is not taken up efficiently at those low concentrations. Since the overall metabolism of GL26 cells is higher than of NPC cells, the killing efficacy of (D-POH)10K20 nanosponges is higher in the cancer cells at both 24h and 48h. Based on the observation of significantly higher toxicity against GL26 cells, K20 is far more efficient than R20 in facilitating cellular uptake of the nanosponges. The presence of biotin at the N-terminal end of the peptide branches does not enable fast receptor-mediated uptake of the nanosponges, at least not in the presence of serum, when biotin is not a limiting nutrient.

CONCLUSION Based on the experiments discussed here, one of the two trigonal nanosponges, type (DPOH)10K20 is a promising candidate for delivering perillyl alcohol to glioblastomas. The second candidate, type (D-POH)10R20, fails to kill murine glioma cells (GL26) in the presence of serum. Both, type (D-POH)10K20 and (D-POH)10R20 are taken up by neuronal progenitor cells and show either no or low toxicity, as determined in cell viability experiments. We attribute the different biological effects that are caused by the two types of nanosponges to the observed significant differences in nanosponge sizes (according to DLS and, to a lesser degree, also AFM) and surface charge. Type (D-POH)10K20 nanosponges are positively charged, whereas (D-POH)10R20 nanosponges are negatively charged.


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EXPERIMENTAL SECTION Amino acids, Fmoc-Cys(Trt)-Rink Amide MBHA resin and N,N,N′,N′-Tetramethyl-O-(1Hbenzotriazol-1-yl)uronium hexafluorophosphate (HBTU) were purchased from peptides international Inc, Louisville, KY, USA. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCxHCl) was procured from Oakwood Chemical, West Columbia SC, USA. 4Dimethylaminopyridine (DMAP) was purchased from ACROS Organics, New Jersey, USA. Perillyl alcohol, Piperidine and Triisopropylsilane (TIPS) were purchased from Sigma Aldrich. N,N-Diisopropylethylamine (DIPA), trifluoroacetic acid, ether, methylene chloride and dimethylformamide (DMF) were purchased from Fisher Scientific.

Peptide Synthesis and Biotin Coupling The D10K20DEVDGC and D10R20DEVDGC peptides were synthesized by iterative solid phase peptide synthesis according to standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) protocol.34 Fmoc-Cys(Trt)-Rink Amide MBHA resin was used as solid support. Three equivalents of Fmoc (N-(9-fluorenyl)methoxycarbonyl) protected amino acid and HBTU were dissolved in a DIEA/DMF solution, and added to the MBHA preloaded with 0.20 mmol of amino acid per g. The solution was removed from the resin after 30 min of reaction at RT. This process was repeated once. Then, the next Fmoc group was removed by using 20%(v/v) piperidine in DMF. Stepwise addition of Fmoc-protected amino acids resulted in the desired peptides. Biotin was coupled to the N- terminal aspartic acid (D) applying standard peptide coupling conditions described above. The synthesized peptide was purified by dialysis (MWCO 3500) and then lyophilized.


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D10K20DEVDGC Peptide Synthesis and Rhodamine B Coupling

The D10K20DEVDGC peptide was synthesized by iterative solid phase peptide synthesis according to standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) protocol. H-Cys(Trt)-2-ClTrt resin was used as solid support (0.09 mmoles). A 1/3 of this D10 K20DEVDGC peptide (0.03 mmol) was used to couple Rhodamine B. After removing the Fmoc protecting group of the N- terminal aspartic acid (D), Rhodamine B was added by applying the same peptide coupling conditions except the reaction was done once and it was kept for 24 h to react. The Rhodamine B labeled peptide and the D10K20DEVDGC peptide were cleaved by adding TFA:TIPS:H2O cocktail solution. Peptides were collected into cold ether and washed 6 times with cold ether to get the pure Rhodamine BD10K20DEVDGC (pink) and D10K20DEVDGC peptides (white).

Synthesis of Type D10K20 and D10R20 Nanosponges via Michael Addition of D10K20DEVDGC or D10R20DEVDGC to Trimaleimide

The biotinylated peptide (D10K20DEVDGC or D10R20DEVDGC) was dissolved in degassed, 1X PBS solution, pH 7.4. Trimaleimide24 in degassed DMF was added drop-wise to the peptide solution (peptide: trimaleimide molar ratio; 4:1) while stirring at RT under inert atmosphere. The reaction was carried out for 24 h followed by two dialysis steps (MWCO 3500/10000), followed by freeze-drying. Yield: 85% of (Biotin-D10 K20DEVDGC)3-trimaleimide and 87% of (BiotinD10R20DEVDGC)3-trimaleimide.


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Perillyl alcohol loading to (Biotin-D10R20DEVDGC)3 -trimaleimide

(Biotin-D10R20DEVDGC)3-trimaleimide was dissolved in 1X PBS solution, pH 7.4. Perillyl alcohol in DMF was added drop-wise while stirring rapidly to obtain a uniform solution. Then a mixture of EDC and DMAP in 1X PBS solution, pH 7.4, was added and stirred for 24 h (peptide: alcohol: EDC: DMAP molar ratios; 1:50:1.2:1). The product was purified by solvent extraction using dichloromethane. The aqueous phase was freeze dried to obtain final product. Yield: 55% of (Biotin-D10(Perillyl

alcohol10)R20DEVDGC)3-trimaleimide.

The

resulting

product

was

qualitatively analyzed by HPLC (Shimadzu NexeraSR) utilizing a reverse phase (C18) column and H2O / CH3CN + 1% CF3COOH as eluent. The organic phase was increased from 0.5% to 40% within 30min. The corresponding HPLC chromatogram can be found in the SI section.

Perillyl alcohol loading to (Biotin-D10K20DEVDGC)3 -trimaleimide

The synthetic procedure for (Biotin-D10(Perillyl alcohol10)K20DEVDGC)3-trimaleimide was identical with the procedure for (Biotin-D10(Perillyl alcohol10)R20DEVDGC)3-trimaleimide, with the exception of using 1X PBS with an adjusted pH = 5.5 (instead of pH = 7.4) for EDC coupling. Yield: 48% of (Biotin-D10(Perillyl alcohol10)K20DEVDGC)3-trimaleimide. The resulting product was qualitatively analyzed by HPLC (Shimadzu NexeraSR) utilizing a reverse phase (C18) column and H2O / CH3CN + 1% CF3COOH as eluent. The organic phase was increased from 0.5% to 40% within 30min. The corresponding HPLC chromatograms can be found in the SI section.


30


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Synthesis of Rhodamine B Labeled Nanosponges

The Rhodamine B-D10K20DEVDGC (0.03 mmol) and D10K20DEVDGC (0.06 mmol) peptides were dissolved in 2.5 ml of degassed 1X PBS. Trimaleimide (0.0225 mmol) was dissolved in 40 µl of degassed DMF and added into the peptide solution. The reaction was carried out at room temperature under inert atmosphere for 24 h while stirring. Then, the solution was dialyzed in distilled water (MWCO 3500). Solutions were freeze-dried to get the pure Rhodamine B labeled nanosponges.

Nanosponge Formation and DLS Characterization

Separate solutions of (Biotin-D10(Perillyl alcohol10)K20DEVDGC)3-trimaleimide and (BiotinD10(Perillyl alcohol10)R20DEVDGC)3-trimaleimide in deoxygenated PBS buffer were prepared and filtered through 200 µm filters. The prepared stock solutions were 0.55 mM and 2.00 mM. All other stock solutions were prepared by diluting the original solutions with deoxygenated PBS buffer. The hydrodynamic diameters and polydispersity indexes (PDI) of the formed nanosponges were measured by dynamic light scattering (DLS, ZetaPALS, Brookhaven Instruments Corp., Holtsville, NY).52, 53 All measurements were carried out at 298 K, using 658 nm laser wavelength, and 90 degree detection angle. Data was collected from an average of three measurements over 60 seconds. DLS was also used to estimate the critical aggregation concentration (cac)19 of the nanosponges. DLS measurements were performed in the following manner: stock solutions of 0.55 mM were prepared for Biotin-(D10(Perillyl alcohol10)K20DEVDGC)3-trimaleimide, and of 2.0 mM for


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Biotin-(D10(Perillyl alcohol10)R20DEVDGC)3-trimaleimide. 1.0 𝜇L aliquots of the stock solution of (Biotin-D10(Perillyl alcohol10)K20DEVDGC)3-trimaleimide were given stepwise to 1.0 mL of PBS. DLS was measured after 10 min at incubation of 37 oC after addition of each 1.0 𝜇L aliquot. For (Biotin-D10(Perillyl alcohol10)R20DEVDGC)3-trimaleimide, aliquots of 5.0 𝜇L were added. DLS was measured after 10 min at incubation of 37 oC after addition of each 5.0 𝜇L aliquot.

TEM Characterization

Samples for transmission electron microscopy (TEM) were prepared by dropping 10 µL of 0.050 mM Biotin-(D10(Perillyl alcohol10)K20DEVDGC)3-trimaleimide or Biotin-(D10(Perillyl alcohol10) R20DEVDGC)3-trimaleimide solutions in PBS directly on a glow discharged TEM grid. Uranyl acetate (0.005 mM in PBS) was used as staining agent in all TEM experiments. Nanosponge morphology on HOPG was examined by means of bright-field transmission electron microscopy (TEM) using a FEI Technai G2 transmission electron microscope at an electron acceleration voltage of 200 kV. High resolution images were captured using a standardized, normative electron dose and a constant defocus value from the carbon-coated surfaces. All TEM measurements were performed at the Microscopy and Analytical Imaging Laboratory of the University of Kansas. 54

AFM Characterization and Caspase-6 Digestion Experiment

Samples for atomic force microscopy (AFM) were prepared by adding 1.0 ml of nanosponge stock solution (0.10 mM of either Biotin-(D10(Perillyl alcohol10)K20DEVDGC)3-trimaleimide or Biotin-(D10(Perillyl alcohol10)R20DEVDGC)3-trimaleimide in PBS) onto a freshly peeled MICA


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sheet, followed by removing of the solvent by using a gentle nitrogen stream (2 min). AFM images were taken by a Nanoscope AFM image system (Digital Instruments) utilizing TESPA-HAR probes in tapping mode. The spring constant of the tip was 50 N/m and the frequency was 350 kHz. The set point, P gain and I gain were set at 1.2, 0.6 and 0.5, respectively. The images were gathered with 256x256 pixel resolution at a scan rate of 1 Hz. The images were then analyzed by the Nanoscope software (Bruker) and by Adobe Photoshop (contour plots). The caspase-6 digestion experiments were performed using 0.10 mM Biotin-(D10(Perillyl alcohol10)K20DEVDGC)3-trimaleimide in PBS on mica in the presence of 10 -8 M caspase-6 (Enzo Lifesciences). All other experimental parameters remained as described above. Quantitative analysis of the size distribution of the nanosponges during caspase-6 digestion was performed using the program NIH IMAGE.55

Cell Experiments, MTT Assays and Microscopy

The cytotoxicity of Biotin-(D10(Perillyl alcohol10)K20DEVDGC)3-trimaleimide and Biotin(D10(Perillyl alcohol10)R20DEVDGC)3-trimaleimide nanosponges was assessed by utilizing the MTT assay36 on C17.2 neural progenitor cells (NPCs)50, which were a gift from Dr. V. Ourednik (Iowa State University) to Dr. D. L. Troyer, DVM (Kansas State University, Anatomy & Physiology). NPCs were originally developed by Dr. Evan Snyder. 38 These cells were maintained in DMEM supplemented with 10% FBS (Sigma-Aldrich), 5% horse serum (Invitrogen), 1% glutamine (Invitrogen), and 1% penicillin/streptomycin (Invitrogen). GL26 murine glioma cells39 were cultured in RPMI 1640 medium with 10% FBS, and 5% CO2. The percentage of viable cells was determined after 24 and 48 hours of incubation. Cells were seeded in T-25 flask. After 24 h


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of incubation at 37 °C and 5% CO2, cells were re-plated in a 96 well plate at 8000/cm2 density and further incubated for

24 h at 370C, 5% CO2, to obtain 80 % confluency before the type (D-

POH)10K20 or (D-POH)10R20 nanosponges were added. Concentration series of type (D-POH)10K20 or (D-POH)10R20 nanosponges (0.0, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20, 40, 60, 80, 100 µmoles L -1) were prepared by dissolving the nanosponge components in the same media that were used for culturing the cells. Cells were incubated for 24/48 h at 37 °C. Eight replicates were prepared for each concentration. A portion of 10 µL of MTT reagent (5 mg/ml in DI water) was added to each well, and the plates were incubated for another 4 h at 37 °C. Finally, 100 µL of 10% sodium dodecyl sulfate in 0.010M HCl was added into each well and incubated for 24 h at 37 °C. Their absorbance was recorded by using a plate reader at 550nm and 690nm. PBS solution was used as control for all the experiments. A Zeiss Aciovert 40 CF microscope was used for imaging the uptake of Rh B-D10(POH)K20 Nanosponges. The typical procedure comprised four steps: 1) Plate NPCs at 10,000/cm2 density 24h prior to the procedure; 2) remove old medium and add 300 µl of NPC medium per well, 3) add the rhodamine B labeled, DPOH10K20 nanosponge (stock solution concentration = 10 mg/ml), volumes added (ml) = 5, 10, 15, 20, 25, 35, 50, 75,1 00,1 50 per well (2 repetitions), 4) incubate the plate at 37 °C for 24 h. Protocol for Hoechst Staining (48 well plate): 1) remove cell medium and wash cells with 1X PBS once. 2) fix cells: add 200µl of 10 % normalized formaldehyde solution/well and keep for 15 min, 3) wash cells with 200 µl/well of 1X PBS 3 times, 5 min, 4) stain cells: Add 200 µl/well of Hoechst solution (1:500 dilution in 1X PBS) and keep for 10 min, 5) wash cells with 200 µl/well of 1X PBS 3 times, 30 sec/wash, 6) add 200 µl/well of 1X PBS to keep the cells hydrated.


34


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ASSOCIATED CONTENT Supporting Information. The SI section contains a collection of FTIR spectra, zeta-potential data, and HPLC characterization of the nanosponges, as well as their characterization by means of MALDI-TOF. A general procedure for determining the cac of the nanosponges by means of DLS and AFM images (tapping mode) of D-POH)10K20 nanosponges on MICA in the presence of caspase-6, as well as microscopy images of nanosponges in RPMI medium (with and without serum) and microscopy images of the nanosponge uptake in neural progenitor cells, are also included. AUTHOR INFORMATION Corresponding Authors Prof. Dr. Stefan H. Bossmann, Kansas State University, Department of Chemistry, Manhattan, KS 66506, Phone: 785-532-6817, Fax: 785-532-6666, Email: [email protected] Prof. Dr. Deryl L. Troyer, DVM, Kansas State University, Anatomy & Physiology, Manhattan, KS, Phone: 785-532-6405 Email: [email protected] Present Addresses HWW: 101 BIVAP Innovation Center, Kimball Ave., Manhattan, KS, USA SOW: Department of Biology, Kansas State University, KS, USA APM: Department of Chemistry, Southwestern College, Winfield, KS, USA RO: National Institute of Health, Bethesda, MD


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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡ Asanka S. Yapa, Sebastian O. Wendel and Tej B. Shrestha contributed equally.

Funding Sources This work was funded by NSF (DMR 1242765 and CBET 1656989), and the Johnson Cancer Center at Kansas State University. Notes The authors declare no competing financial interest.

ABBREVIATIONS AFM: Atomic Force Microscopy, CDI (Carbonyl-di-imidazole, CNS: Central Nervous System, DIEA: N,N-Diisopropylethylamine, DLS: Dynamic Light Scattering, EGFR: Epidermal Growth Factor Receptor, GBM: Glioblastoma multiforme, HBTU: (2-(1H-benzotriazol-1-yl)-1,1,3,3tetramethyluronium

hexaﬂuorophosphate,

HEPES:

4-(2-Hydroxyethyl)-1-piperazine-

ethanesulfonic acid, HOPG: Highly Ordered Pyrolytic Graphite, MALDI-TOF: Matrix Assisted Laser Desorption Ionization - Time of Flight, NPC: Neural Progenitor Cells, PBS: phosphatebuffered saline buffer, PDGFR: Platelet-derived growth factor, PI3K/Akt: Phosphatidylinositol-3Kinase and Protein Kinase B, PTEN: Phosphatase and Tensin Homolog, Ras/MAPK: Family of oncogenes from Harvey and Kirsten murine sarcoma viruses /mitogen-activated protein kinase, TEM: Transmission Electron Microscopy, TIPS: Triisopropylsilane


36


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REFERENCES 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11.

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