Glutathione (GSH) Peptide Conjugated Magnetic Nanoparticles As

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/abseba

Glutathione (GSH) Peptide Conjugated Magnetic Nanoparticles As Blood−Brain Barrier Shuttle for MRI-Monitored Brain Delivery of Paclitaxel Hamed Nosrati,†,‡ Mahsa Tarantash,§ Shayesteh Bochani,∥ Jalil Charmi,†,⊥ Zahra Bagheri,† Mohammadjavad Fridoni,# Mohammad-Amin Abdollahifar,¶ Soodabeh Davaran,□ Hossein Danafar,*,†,∥,△ and Hamidreza Kheiri Manjili*,†,∥ †

Zanjan Pharmaceutical Nanotechnology Research Center, ‡Department of Pharmaceutical Biomaterials, School of Pharmacy, Department of Pharmaceutical Nanotechnology, School of Pharmacy, #Department of Anatomy, Medical School, and △ Department of Medicinal Chemistry, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan 45139-56111, Iran § Chemical Engineering Faculty, Sahand University of Technology, Tabriz 94171-71946, Iran ⊥ Department of Physics, Faculty of Science, University of Zanjan, Zanjan 45371-38791, Iran ¶ Department of Anatomical Sciences and Biology, Medical School, Shahid Beheshti University of Medical Sciences, Tehran 19839-63113, Iran □ Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51656-65811, Iran

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S Supporting Information *

ABSTRACT: In drug delivery science, brain delivery is one of the most important challenges because of the low efficiency of the available treatments. Nowadays, shuttle peptides have attracted more attention because of lower price, reduced immunity, and increased chemical capability. Glutathione (GSH) is one of the blood−brain barrier (BBB) shuttle peptides that has reached the most progressive steps in the path toward clinical application. This project discovered the possibility of GSH-conjugated IONPs as an MRI-monitored paclitaxel (PTX) delivery vehicle across the BBB using BALB/c mouse model. Synthesized shuttle peptide-conjugated nanoparticles were tracked over a certain time by MRI. A one-pot method was used for preparation of IONPs@Asp to form functionalized nanoparticles with two functional groups for linkage of PTX, PEG, and then GSH on the surface of nanoparticles. Afterward, they were analyzed by XRD, TGA, FTIR, TEM, VSM, and DLS techniques. In addition, histological study were performed on the key organs. Here, we exhibit that (1) IONPs@Asp are stable and nontoxic to different cells; (2) conjugation of GSH to nanoparticles promotes their internalization to brain in vivo; (3) final formulation (IONPs@Asp-PTX-PEG-GSH) are effective in MRI visualization; (4) IONPs@Asp-PTX-PEG-GSH begins to eliminate shortly afterward by the kidneys subsequently administration; (5) they were absorbed by liver, spleen, and especially by brain and simultaneously enhancing MRI contrast. Thus, IONPs@Asp-PTX-PEG-GSH are promising MRI-monitored paclitaxel (PTX) delivery vehicle across the BBB. KEYWORDS: shuttle peptides, magnetic nanoparticle, MRI monitored, brain delivery, biodegradability

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melanotransferrin (p97),9 and leptin10 have been used for brain target delivery. Glutathione (GSH) is the BBB shuttle peptide that has extended the most progressive stages in the path toward clinical application. PEGylating was used for protection of GSH from degradation and clearance. Paclitaxel has a wellknown therapeutic drug in a cancers by cooperating with

INTRODUCTION

Therapeutic or diagnostic agent transfer to the brain is the most important subject in drug development because of the growing prevalence of brain cancers.1−3 Though many approaches to bypass the blood brain barrier (BBB) have been suggested.4−6 Over the last two decades between the noninvasive methods, BBB shuttles have verified their potential in preclinical research.7 BBB shuttles can be transport a wide range of cargoes, and carriers across the BBB. Hence, a diversity of protein shuttles such as lactotransferrin, 8 © XXXX American Chemical Society

Received: November 13, 2018 Accepted: February 28, 2019

A

DOI: 10.1021/acsbiomaterials.8b01420 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

Figure 1. Schematic image of synthesis of IONPs@Asp-PTX-PEG-GSH.

microtubules.11 PTX was used as a model drug and conjugated to IONPs@Asp through a ester linkage for examine the ability of transport the therapeutic agents across BBB via GSH shuttle peptide assistant. Because of the extensive applications of iron oxide magnetic nanoparticles (IONPs), they have received wide consideration. Magnevist (Gd-DTPA) was commercialized in 1988 as the first MRI contrast agent. At this time, commercial MRI contrast agents are gadolinium-based chelates as positive contrast agents.12 Recently, researchers confirmed that gadolinium-based chelates can increase nephrotoxicity.13−15 In this case, an overall warning has been reported by the FDA for gadolinium-based contrast agents.16 Past recent decades, IONPs-based contrast agents have received further and further consideration for MRI because of their biocompatibility and safety profiles.17 For this reason, IONPs have been recently widely studied as MRI contrast agents.18−22 As a result, some marketed MRI contrast agents based on IONPs are currently accessible. But, the in vivo half-life time of reported IONPs is normally short. Hence, promotion the in vivo abilities of new IONPs is an interesting scientific challenge. Previously, our research group reported the amino acid functionalized IONPs for any uses especially in biomedical scope.11 The amino acids due to cheap, and biocompatible structures is one of the best candidate for surface coating of nanoparticles such as IONPs.23 Additionally, PEG can be prolonged the blood circulation time of nanoparticles.24,25 In the current study, we used a simple one-pot technique for fabricating L -aspartic acid (Asp)-coated functionalized (IONPs@Asp). In next step, PTX was conjugated to IONPs@Asp through a biocleavable ester linkage to study the ability of synthesized nanoparticles to cross the BBB, noted as IONPs@Asp-PTX. Next we conjugated activated PEG on the surfaces of IONPs@Asp-PTX (IONPs@Asp-PTX-PEG) for improving the water solubility and prolonging the circulation time of the nanoparticles. Finally, we functionalized IONPs@Asp-PTX-PEG by GSH (IONPs@Asp-PTX-PEGGSH) as BBB shuttle peptide for enhancing the brain delivery of the PTX. The physicochemical characteristics of the particles were analyzed by different techniques. After determine biocompatibility, cytotoxicity and hemocompatibility of final formulation the transportation across the BBB was monitored with MRI. Also, MRI technique was used for the general biodegradability and the blood clearance of the IONPs@Asp-PTX-PEG-GSH. The MRI signal intensity was tracked for 24 h after injection. The biocompatibility and stereological study was investigated for more than 15 days after the nanoparticles administration using hystomorphometric analysis. The histology results for liver, kidney, heart, spleen, and brain specimens also determined the pathological changes induced by IONPs.

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MATERIALS AND METHODS

Entire procedures were done permitting to the appropriate strategies, regulations and the Ethics Committee of the Zanjan University of Medical Sciences. Materials. All materials that was used for synthesis on nanoparticles and other analysis were obtained from Sigma-Aldrich Chemicals, USA. PTX was gifted from Sobhan Oncology Company (Rasht, Iran). Synthesis of IONPs@Asp-PTX-PEG-GSH. The synthesis of IONPs@Asp-PTX-PEG-GSH involved a five-step procedure. Schematic delineation of synthesis of IONPs@Asp-PTX-PEG-GSH was outlined in Figure 1. Step 1, Synthesis of IONPs@Asp. Under consistent blending with a magnetic stirrer, 0.55 g of FeCl3·6H2O, 0.2 g of FeCl2·4H2O, 0.845 g of Asp, and 100 mL of deionized water in N2 were mixed. Fifteen milliliters of NH4OH, 25% was added dropwise to reaction medium under consistent mighty stirring. Next, it was continued for 6 h, and then chilled to r.t. The IONPs@Asp were separated magnetically (1.2 T), washed multiple times, and then parched in a vacuum oven. Step 2, Synthesis of IONPs@Asp-PTX. One hundred milligrams of IONPs@Asp, 7 mL of ethanol, 35 mg of free PTX, 31.4 mg of EDC, and 18.8 mg of NHS were mixed at room temperature in the dark and pH 8.2 condition. The PTX-modified particles (IONPs@Asp-PTX) were separated magnetically and washed with ethanol before dried. Step 3, Synthesis of Carboxyl-Terminus PEG (HOOC-PEG-COOH). Previously, Mathiyalagan et al. described the synthesis of carboxylterminus PEG (HOOC-PEG-COOH) with succinic anhydride.26 Briefly, 1 g of PEG (MW 4000 g/mol; 0.25 mmol), 0.6 g o succinic anhydride (0.6 mmol), 0.06 g of DMAP (0.5 mmol), 10 mL of anhydrous dioxane, and 0.05 g of TEA (0.5 mmol) were mixed at rt. Afterward, the resultant HOOC-PEG-COOH was collected with cold diethyl ether. Step 4, Synthesis of IONPs@Asp-PTX-PEG. To a solution the HOOC-PEG-COOH (0.088 g, 0.04 mmol), EDC (0.24 g, 0.12 mmol), and DMAP were added. Next, the IONPs@Asp-PTX was added to the reaction, and stirred for 24 h. The synthesized IONPs@ Asp-PTX-PEG were separated magnetically, were washed three times with water, and then dried. Step 5, Synthesis of IONPs@Asp-PTX-PEG-GSH. Finally, the GSH as targeting agent conjugated to the activated carboxyl-terminus of PEG on the surface of IONPs@Asp-PTX-PEG. The suspension of 100 mg IONPs@Asp-PTX-PEG were mixed with 40 mg GSH. This suspension was stirred overnight at RT. Next GSH conjugation, the IONPs@Asp-PTX-PEG-GSH were separated magneticaly, and the resulting supernatant was discarded. Deionized water washing the separated IONPs@Asp-PTX-PEG three times with using magnetic separation, and then dried in a vacuum. In Vitro Drug Loading and Release. The loaded drug was computed the change in PTX concentration in the solution beforehand and later conjugation reaction. Equation 1 was used for determine of percentage of drug loading: B


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Figure 2. (a) TEM images of IONPs@Asp-PTX-PEG-GSH; (b) VSM curves of IONPs, IONPs@Asp-PTX, and IONPs@Asp-PTX-PEG-GSH, (c) cytotoxicity of pure IONPs and IONPs@Asp-PEG on HFF-2 and HEK-293 cell lines; (d) hemolysis assay of RBCs incubated with IONPs, IONPs@Asp, IONPs@Asp-PTX, IONPs@Asp-PTX-PEG, and IONPs@Asp-PTX-PEG-GSH. water, 0.5 mL of PBS, and 0.5 mL of sample with concentration of 10 mg/mL. The samples were incubated and centrifuged at 13000 rpm for 15 min, and the supernatant absorbance was identified by Eppendorf Bio Photometer at 540 nm. Equation 2 used for calculation of hemolysis:

%drug loading initial PTX concentration − final PTX concentration = × 100 initial PTX concentration (1) For investigation of release profile, briefly, 1 mL of the final formulation solution was located into a dialysis bag (12 kDa). Then was immersed in 20 mL of release medium (PBS, pH 7.4 and pH 5.8, 2% w/v tween 80) at 37 °C with continuous shaking. The released amount of PTX was determined by UV−Vis absorbance at 227 nm. Characterization. XRD, TGA, FTIR, TEM, VSM, and DLS techniques were applied for characterization of synthesized nanoparticles. In Vitro Study. Cell Culture. HFF2 and HEK293 cell lines which achieved from Pasteur Institute (Tehran, Iran) were used in cell toxicity test. The cells cultured in DMEM, accompanied by 10% fetal bovine serum, 100 mg/mL penicillin G and 100 mg/mL streptomycin (Gibco, Germany) at 37 °C. Cytotoxicity. The cell culture medium containing IONPs and IONPs@Asp-PEG adding in a dilution runs (0, 0.06, 0.09, 0.15, 0.19, 0.30, and 0.40 mg/mL NPs) to each well in 96-well plate. After that 72 h of incubation, 20 μL of MTT (5 mg/mL) adding to wells. After 4 h, the medium was replaced with 100 μL of DMSO to dissolve the formazan crystals. The absorbance of each well was read at 570 nm by a microplate reader. Hemocompatibility. The in vitro hemolysis assay was performed by previously reported procedure.27 Briefly, HRBCs were achieved via centrifugation of blood at 3000 rpm for 10 min the. After remove of supernatant, the HRBCs washed four times with PBS solution and dispersed in PBS. 0.5 mL of diluted HRBCs was mixed with 0.5 mL of

%hemolysis =

A sample − A negative A positive − A negative

100 (2)

In Vivo Study. All procedures were done according with the related guidelines, regulations and the Ethics Committee of the Zanjan University of Medical Sciences. MR Imaging. The physiological solution of the IONPs@Asp-PTXPEG-GSH were injected intravenously into the cross tail vein at a dose of 20 mg Fe/kg to BALB/c mice with weight of about 20 g, then followed by ex vivo MRI scanning. The injected mice were arbitrarily divided into 9 groups (n = 4): control, treated with IONPs@Asp-PTX-PEG-GSH in 8 time intervals: 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after injection. The IONPs@Asp-PTXPEG-GSH injected intravenously. Then, the mice were sacrificed at planned time intervals and desired organs were removed. After washing the removed organs with physiological saline, they immersed in 4% paraformaldehyde at 4 °C. The organs were fixed in a culture plates using 1% agarose gel. The fixed organs were scaned by 1.5-T MRI. The imaging parameters: TR/TE = 6440 ms/91 ms, FA = 90°, FOV = 2 cm × 2 cm, slice thickness = 1 mm. Macro Imaging software (version 1.47v) was used for analyze of MRI images. The signal intensity were found at the ROI. The signal intensity of extracted organs were obtained before and after the administration of C


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Figure 3. (a) MRI signal trend and (b) signal change intensity in brain at different interval times. Each bar shows the mean of four measurements ± SD. (c) Photograph of dispersed brain powder in water (6 h after injection) in the presence of the magnetic field.

Figure 4. In vivo MRI studies: MRI signal change intensity in (a) liver, (b) heart, (c) kidneys, and (d) spleen at different interval times. IONPs@Asp-PTX-PEG-GSH in each mice. The formula (eq 3) was applied for the signal variation of organs: %signal change =

Ipre − Ipost Ipre

organs were washed with physiological saline. The formalin 4% was used for immersing of organs for 24 h, and imbedded in paraffin, divided, and stained with H&E protocol. The hystopathological study was done on mice treated with IONPs@Asp-PTX-PEG-GSH that were sacrificed 15 days after injection. Cell Density Estimation. The optical dissector way was applied for computed the numerical density of hepatocytes. Also, for stereological study of brain, the number of neuron and glial cells were calculated by the optical dissector method.

100 (3)

Ipre and Ipost were the signal intensity of the organ before and after the injection of IONPs@Asp-PTX-PEG-GSH at programmed time intervals, respectively. Stereological Study. The same organs, MRI scanned, were used to hystomorphometric analysis. After the imaging was taken up, the D


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ACS Biomaterials Science & Engineering An identical interlude of stirring the phase and regular constant arbitrary sample were applied for choice of the positions of the microscopic fields. Only when nucleus was sited totally or incompletely within the including mount, was counted. Also, if it did not extent the excepting line. Following formula was used for Numerical density (Nv) calculation (eq 4):

v=

ΣQ t ΣPha /f BA

(4)

The ΣQ, h, a/f, ΣP, t, and BA are the amount of the nuclei, the height of the dissector, the frame area, the total number of the unbiased counting frame in all fields, the real section thickness measured in every field using the microcator, and the block advance of the microtome.28 The relative numerical density of the livers hepatocytes were calculated using the following formula (eq 5): %relative numerical density =

Figure 5. Number of neuron and glial cells of brain.

numerical density hepatocytes total volume of liver (5)

synthesized nanoparticles the ζ-potential was determined by DLS. The result show that the ζ-potential is −17.2 mV. In XRD pattern of IONPs@Asp and IONPs@Asp-PTXPEG-GSH we can be seen the characteristic XRD peaks of with Bragg reflection of Fe3O4 standards from a JCPDS file (JCPDS no 019−0629) (Figure 1S). This matching show that the synthesized IONPs did not changed during the production process. The results of FTIR proved that Asp, PTX, PEG and GSH successfully conjugated to the surface of IONPs (Figure 2S). For IONPs@Asp, the peaks at ∼3400, 2922, 1620, and 1388 cm−1 due to stretching vibration of N−H, stretching of methylene groups C−H, and stretching vibrations of CO and C−O, respectively. Moreover, we can be seen the Fe−O vibration peak at 621.31 and 418.14 cm−1 in IONPs@Asp spectrum. The PTX characteristic peaks were seen at: ∼ 3500 cm−1 because to stretching of O−H groups, ∼2900 cm−1 due to aliphatic C−H stretch, ∼1500 to ∼1735 cm−1 due to aromatic ring (CC) stretching frequency, ∼1050 cm−1 due to aromatic C−H bonds, ∼1630 cm−1 due to CO amide stretching, and 1451 cm−1 due to CH2 scissoring mode of PTX.11 In the FT−IR spectrum of IONPs@Asp-PTX, CO stretching vibration (1620 cm−1) associated with carboxylic acid end group of IONPs@Asp was lifted to upper frequencies (1631 cm−1). This can be show conjugation of the PTX with IONPs@Asp with esterification reaction. Moreover, after reaction of PEG with succinic anhydride, CO stretching vibration (1735 cm−1) was appeared that related to formed esteric bond. In the spectra of IONPs@AspPTX-PEG-GSH the absorption bands looking at 800−900 cm−1 are related to the C−O−C stretching vibration of PEG chains. Additionally, the absorption bands looking at 1532 cm−1 are ascribed to the formed amide bond. Figure 3S shows the TGA curves of the IONPs@Asp, IONPs@Asp-PTX and IONPs@Asp-PTX-PEG-GSH. The result show that the organic phase were increased in step by step of synthesis process. For example in the TGA curve of IONPs@Asp-PTX-PEG-GSH, net IONPs content was 77%. The remaining 23% was from Asp, PTX, PEG, and GSH. Also, the magnetic properties of IONPs, IONPs@Asp-PTX and IONPs@Asp-PTX-PEG-GSH were studied by VSM. As can be seen in Figure 2b the saturation magnetization (Ms) of IONPs, IONPs@Asp-PTX and IONPs@Asp-PTX-PEG-GSH

Volume Estimation. The volumes of kidney proximal and distal convoluted tubules, livers sinusoid, spleens white pulp, red pulp, and hearts muscle fibers were calculated using the Cavalieri method. The volume of the tissue was estimated by the following formula (eq 6): V=

∑P

a t p

(6)

The ΣP, a/p, and t were the total points hitting the tissue sections, was the region related with each point and the space between the sampled segments, respectively.29 The relative volume of the of kidneys proximal and distal convoluted tubules, livers sinusoid, spleens white pulp, red pulp, and hearts muscle fibers were considered applying the following formula (eq 7): %relative volume =

A T

(7)

where A is the volume of kidney proximal and distal convoluted tubules, livers sinusoid, spleens white pulp, red pulp, and hearts muscle fibers for each case and T is total volume of the related organ. For example, for calculating relative volume of kidney proximal tubules, A is the volume of kidney proximal tubules and T is the total volume of liver. Statistical Analysis. The one-way ANOVA tests and multiple t test were used for statistical significance identify.

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RESULTS AND DISCUSSION Synthesis. The IONPs@Asp were prepared by coprecipitation procedure according to the previously reported method.11 As synthesized IONPs@Asp provide two functional group including amine and carboxylic acid for linkage of PTX (to −COOH), and PEG (to −NH2). The conjugation of IONPs@Asp with PTX was performed by covalent bonding. The carboxyl-terminus PEG (HOOC-PEG-COOH) was synthesized with succinic anhydride assistance. Two sides of HOOC-PEG-COOH were reactivated to its succinimide using EDC and NHS and then reacted with IONPs@Asp-PTX to conjugate activated HOOC-PEG-COOH with the amine (−NH2) group of Asp in which DMAP was used as a catalyst and EDC and NHS was used as a condensation agents. Finally, the activated end side of attached HOOC-PEG-COOH was reacted with GSH as shuttle for penetration to brain. Characterization. The TEM result show that the core diameter of the formulated IONPs were 15.85 ± 3.11 nm (Figure 2a). After measurement of internal diameter of E


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Figure 6. (a) Relative numerical density of hepatocytes (b), the relative volumes of muscle fibers of heart, (c) sinusoid of liver, (d) proximal tubules of kidneys (e), distal tubules of kidneys, (f) white pulp of spleen, and (g) red pulp of spleen. The results show no significant difference between control and treated groups.

Figure 7. Histological study of the brain, liver, spleen, heart, and kidneys after 15 days. N, neuron cells; G, glial cells; MF, muscle fibers; CV, central vein; G (kidney), glomerulus; RP and WP, red pulp and white pulp.

The UV−vis were used to monitor the absorbances IONPs@Asp-PTX-PEG-GSH nanoparticles for the corresponding dispersion solution. If nanoparticles sediment, they can be monitored by a decreased absorbance. Results of 48h shows that nanoparticles have excellent stability. We did not see any significant decrease in intensity of absorbance. Cytotoxicity Assay. For in vivo applications the toxicity of IONPs@Asp-PEG should be studied. The inhibitory effect on cell viability of IONPs and IONPs@Asp-PEG were perfomed on HFF-2 and HEK-293 cell lines by a standard MTT assay at the range of 0.06, 0.09, 0.15, 0.19, 0.30, and 0.40 mg/mL. The obtained results were illustrated in the Figure 2c the cell lines

were 75 emu/g, 47 emu/g and 51 emu/g, respectively. The result show that the Ms of modified nanoparticles are smaller than Ms of IONPs. Also the IONPs@Asp-PTX-PEG-GSH with Ms of 51 emu/g is one of the best candidate for biomedical applications. The total of PTX bonded on the IONPs@Asp-PTX-PEGGSH was computed with a standard linear-fit curve of free PTX in ethanol 3.98%. The PTX release study in Figure 4S show that amount of release at pH 5.8 is more than pH 7.4 within a 96 h. The result show that only 25% of the drug was released. These release behavior confirmed extraordinary strength of the ester bond between PTX and nanoparticle. F


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physiological solution of the IONPs@Asp-PTX-PEG-GSH, there is no noticeable IONP accumulation in the liver section of the mice (the signal change intensity of this organ in Figure 4a is less than 15). However, the signal change intensity of other organs certified the presence of IONPs@Asp-PTX-PEGGSH in other organs (e.g., the heart and kidneys). The IONPs@Asp-PTX-PEG-GSH were distinguished in the brain, kidneys, heart, spleen and liver (Figure 4). MRI measurements were accomplished for 0−24 h. One of the biggest drawbacks in encountering IONPs through blood circulation is the reticuloendothelial system. This system can recognize unknown materials in our body and catch IONPs administrated through intravenous injection and send up into the spleen and liver.31 Therefore, it is worthwhile to study the escape IONPs@Asp-PTX-PEG-GSH from reticuloendothelial system. This state is shown in Figure 4a for liver signal change intensity after injecting of the IONPs@ Asp-PTX-PEG-GSH. In the first hour accumulation IONPs in the liver is not as high intensity as other times. By passing the time, the signal change intensity in other organs clearly is higher and confirmed that the IONPs@Asp-PTX-PEG-GSH exist in blood circulation. Resovist as a commercial IONP was observed to gather in the liver almost 5 min after injection,31 whereas, our outcomes show that the IONPs@Asp-PTX-PEGGSH has a longer circulation. The IONPs@Asp-PTX-PEGGSH were shown to collect in the liver about 2 h after injection. After 2 h, the liver capture was remarkably darker than in the primary scan and the signal change intensity is larger than the control liver. In Figure 4a, a persistent darkening could be observed 2 to 8 h after injection that implied gathering of particles in liver. Again, in 24 h after injection, the signal change intensity is lower than the signal change intensity at 8 h. It is probable that IONPs are destroyed in this stage but the released iron may be kept in ferritin and/or hemosiderin and can affect the MRI signal in liver. Figure 4b shows the MR imaging signal change intensity in the hearts of injected mice. Our result proves that as long as the IONPs@Asp-PTXPEG-GSH existd in the circulating blood, the darkening and signal change intensity of the heart is larger as well. For instance, the signal change intensity values at 1 and 24 h after injection in heart is approximately are 18 and 1 respectively. It means that IONPs@Asp-PTX-PEG-GSH were expelled from the blood circulation. In precise observations, our results state that approximately all IONPs are almost removed from blood circulation after 24 h. The MRI scans of kidneys and its related signal change intensity proven the clearance by the kidneys after the injection (Figure 4c). The kidneys have a key role for the removal of the IONPs@Asp-PTX-PEG-GSH from blood circulation. Figure 4d indicates spleen MR imaging after injecting of the IONPs@Asp-PTX-PEG-GSH. In the first hour after injection, there is not any notable iron accumulation in the spleen. Whereas, the signal change intensity in the kidneys and heart confirmed that the IONPs@Asp-PTX-PEG-GSH exist in blood circulation. After 30 min, a robust and tenacious blackening until 6 h, conforming to the extreme contrast signal could be observed. Whereas, the signal change intensity decrease after 8 h. It is likely that IONPs degrade in time in spleen. Thus, MRI results related to T2 contrast show that these nanoparticles can be proper choice in diagnostic applications.

were protected with IONPs and IONPs@Asp-PEG for 72 h various concentrations with the range of 0.06, to 0.40 mg/mL. The result confirm the nontoxic effect of IONPs@Asp-PEG on the viability of HFF2 and HEK-293 cell lines. In order to investigate the hemocompatibility for further medical and pharmaceutical applications, the in vitro hemolysis assay was investigated. Figure 2d show the hemolytic activity result of samples. The negligible hemolytic effect of samples was observed in the experiment concentration. For IONPs@ Asp-PTX-PEG-GSH, < 3.52% hemolytic activity was discovered. The in vitro cytotoxicity studies confirmed the biocompatibility and safety of synthesized nanoparticles. MRI-Monitored Brain Delivery. In vivo and ex vivo IONPs biodistribution were done with MRI detection.30 With increasing concentration of IONPs in tissues, signal intensity is increasing too and it is an excellent sensitivity ratio for pre to post contrast administration in MRI. Deposition and existence of IONPs@Asp-PTX-PEG-GSH in the tissue is illustrated by orientation of darkness. In this part is appointed to evaluate evasion reticuloendothelial system and transporting through BBB related to IONPs@Asp-PTX-PEG-GSH sample. As a prevalent procedure, aforementioned sample was injected into the tail vein of mice and the brain was analyzed in various and certain intervals by using MR imaging. Since our goal is to investigate the crossed of IONPs@Asp-PTX-PEG-GSH across the BBB, we monitored the brain uptake of IONPs@Asp-PTX-PEGGSH at different time points By paying more attention to the results, it is divulged that the IONPs@Asp-PTX-PEG-GSH remains more in bloodstream in the compare to standard commercial contrast agents. Astonishingly, the presence of IONPs@Asp-PTX-PEG-GSH were sensed in the brain after a quarter minutes (15 min). The brain scan was significantly darker than the control brain (Figure 3a, b) after 30 min. After injecting of the IONPs@Asp-PTX-PEG-GSH, MR imaging of the brain was shown that the shuttle peptide targeted nanoparticles were appeared in the brain after 15 min and noticeable increase in signal change intensity occurred in 30 min (intensity is higher than 16 as shown Figure 3b). In parallel states, the signal change intensity of heart and kidneys also gave support to presence of the IONPs@Asp-PTX-PEGGSH in blood circulation. After 6 h, the brain of the mice capture showed a durable dark-oriented until 24 h. To achieve the objectives, we monitor the brain uptake of IONPs@AspPTX-PEG-GSH in our experiment and show a notable accumulation of the IONPs@Asp-PTX-PEG-GSH in the brain exist at 30 min to 24 h. Naked Eye Observation Brain Delivery. To evaluating and confirm the passing of synthesized shuttle peptide through the BBB we designed a simple ocular experiment. For this case, the brain tissue 6 h after injection was removed, homogenized, dried, and dispersed in deionized water. The prepared brain suspension was set in the side of the magnetic field. Because IONPs@Asp-PTX-PEG-GSH have high magnetic properties, if these particles exist in brain tissue we can be seen the IONPs with naked eye besides the magnetic field. Figure 3c shows dispersed brain powder (6h after injection) photograph. This test is a strong reason for proven successful delivery of these shuttle type particles to the brain. MRI Monitoring and Biodegradation Studies. In the first sight of Figure 4a, after passing 1 h injecting a G


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ACS Biomaterials Science & Engineering Notes

Consequently, we can see the gradual clearance and biodegradation that due to the decreased signal change intensity at 24 h after injection. Stereological Study. For stereological study of brain, the number of neuron and glial cells were calculated by the optical disector method, respectevely. The stereological study results also showed no significant difference between control and treated group at many time intervals as shown in Figure 5. For stereological study of heart, liver, kidneys, and spleen, the volume of muscle fiber, the number of hepatocytes and sinusoid volume, proximal tubule and distal convoluted tubule volume, and white pulp and red pulp volume, respectively, in the control and treated group with the IONPs@Asp-PTXPEG-GSH were compared. The stereological study results presented no significant difference between control and treated group as shown in Figure 6. Histopathological Study. Histological study of the key organs of mice were performed. The mice were injected intravenously and next, after 15 days, the heart, liver, kidneys, brain, and spleen organs were harvested. As we can see in Figure 7, there is no toxicity detected in harvested key organs. The H&E-stained tissue results demonstrated that IONPs@ Asp-PTX-PEG-GSH were not toxic in vivo experiments. Also, we did not detect any mortality upon intravenous injections of the IONPs@Asp-PTX-PEG-GSH. Consequently, all of current study results propose that these shuttle peptide functionalized IONPs can be improved for further clinical application.

This study was permitted by the Ethics Committee of the Zanjan University of Medical Sciences, and the study participants signed an informed consent. All procedures were approved in accordance with Ethics Committee of the Zanjan University of Medical Sciences guidelines and regulations. All investigational procedures were accepted by Zanjan University of Medical Sciences and licensing committee The authors declare no competing financial interest.

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REFERENCES

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CONCLUSION In calculation, the capability to monitor of IONPs local in vivo study by implementing MRI methods offers a major benefit to noninvasively validate the localization of the drug delivery vehicle at the respective target site. The sequel of experiment showed a noticeable accumulation of the IONPs@Asp-PTX-PEG-GSH at 30 min and maintained even at 24 h in brain. Our result affirms that for early hours there is no significant accumulation in the liver. Experimental results prove more circulation in the bloodstream. Also, to validate biodegradation and clearance with body organs, we performed a qualitative evaluation of heart, kidneys, liver, and spleen by the MRI technique at programmed time intervals. ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b01420.

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ACKNOWLEDGMENTS

This work was supported by the Zanjan University of Medical Sciences (Grant A-12-430-23).

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Additional data and figures such as X-ray diffraction (XRD) patterns, FTIR spectra, TGA curves, and release study figures (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Hamed Nosrati: 0000-0002-7487-8188 Hossein Danafar: 0000-0001-8956-7895 H


Article

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