Accepted Manuscript CREKA peptide-conjugated dendrimer nanoparticles for glioblastoma multiforme delivery Jingjing Zhao, Bo Zhang, Shun Shen, Jun Chen, Qizhi Zhang, Xinguo Jiang, Zhiqing Pang PII: DOI: Reference:

S0021-9797(15)00278-7 http://dx.doi.org/10.1016/j.jcis.2015.03.019 YJCIS 20329

To appear in:

Journal of Colloid and Interface Science

Received Date: Accepted Date:

13 December 2014 9 March 2015

Please cite this article as: J. Zhao, B. Zhang, S. Shen, J. Chen, Q. Zhang, X. Jiang, Z. Pang, CREKA peptideconjugated dendrimer nanoparticles for glioblastoma multiforme delivery, Journal of Colloid and Interface Science (2015), doi: http://dx.doi.org/10.1016/j.jcis.2015.03.019

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CREKA peptide-conjugated dendrimer nanoparticles for glioblastoma multiforme delivery Jingjing Zhao1, Bo Zhang1 , Shun Shen1, Jun Chen1, Qizhi Zhang1, Xinguo Jiang1, Zhiqing Pang1* 1

Key Laboratory of Smart Drug Delivery, Ministry of Education; Department of Pharmaceutics,

School of Pharmacy, Fudan University; 826 Zhangheng Road, Shanghai, 201203, China

*Corresponding author.

Zhiqing Pang; Tel.: +86-21-5198-0069; Fax: +86-21-5198-0069.

E-mail address: [email protected] Postal address: 826 Zhangheng Road, Shanghai 201203, P.R. China

Abstract Glioblastoma multiforme (GBM) is the most aggressive central nervous system (CNS) tumor because of its fast development, poor prognosis, difficult control and terrible mortality. Poor penetration and retention in the glioblastoma parenchyma were crucial challenges in GBM nanomedicine therapy. Nanoparticles diameter can significantly influence the delivery efficiency in tumor tissue. Decreasing nanoparticles size can improve the nanoparticle penetration in tumor tissue but decrease the nanoparticle retention effect. Therefore, small nanoparticles with high retention effect in tumor are urgently needed for effective GBM drug delivery. In present study, a small nanoparticle drug delivery system was developed by conjugating fibrin-binding peptide CREKA to Polyamidoamine (PAMAM) dendrimer, where PEGylated PAMAM is used as drug carrier due to its small size and good penetration in tumor and CREKA is used to target the abundant fibrin in GBM for enhanced retention in tumor. In vitro binding ability tests demonstrated that CREKA can significantly enhanced nanoparticles binding with fibrin. In vivo fluorescence imaging of GBM bearing nude mice, ex vivo brain imaging and frozen slices fluorescence imaging further revealed that the CREKA-modified PAMAM achieved higher accumulation and deeper penetration in GBM tissue than unmodified one. These results indicated that the CREKA-modified PAMAM could penetrate the GBM tissue deeply and enhance the retention effect, which was a promising strategy for brain tumor therapy.

KEYWORDS: Penetration, retention effect, CREKA peptide, Polyamidoamine dendrimer, glioblastoma

1. Introduction Glioblastoma multiforme (GBM) is the most aggressive central nervous system (CNS) tumor due to its fast development, poor prognosis and terrible mortality[1]. Mean survival time of GMB is approximates 9–12 months and the 5-year survival rate is 10%–35% despite of positive treatment[2, 3]. Radiotherapy or chemotherapy following surgical resection is essential[4]. Among many chemotherapy strategies, nanoparticle drug delivery system has displayed promising future and much work focusing on nanoparticle delivery to brain tumor has been performed[1, 5, 6]. In order to increase drug delivery efficiency to GBM, trans-blood brain barrier (BBB) drug delivery system[7-10], Trans-BBB plus brain tumor cell dual targeting drug delivery system[11], blood tumor barrier (BTB) targeting drug delivery system[12] , glioma extracellular matrix targeting drug delivery system[6] and other strategies were developed for brain tumor targeting therapy. However, poor nanoparticle penetration and retention in the glioblastoma parenchyma were still crucial challenges in GBM therapy. Little work has been down on small nanoparticles to improve drug delivery to GBM tissue by increasing enhanced permeability and retention (EPR) effect and tissue penetration. EPR effect was a vital factor for nanomedicine therapy of tumor[13, 14], and the nanoparticle permeability and retention in brain tumor was relative weak than peripheral tumors owing to special glioma microenvironment[15]. Hobbs’ et al have quantified the microvascular pore size of the U87 glioma[16], which was 7-100 nm, significantly smaller than that of non-cerebral tumors (380-780 nm). Sarin et al[15] pointed out that glycocalyx coating the luminal surface of endothelial cells[17] made the true microvascular physiologic pore size smaller than its anatomic pore size. Given that, proper small particle size is the basic precondition of good nanoparticle permeation in glioma tissue.

Furthermore, it has been reported that for poorly permeable tumors only small size nanoparticles (not more than 30 nm) can reach deeper section and achieve a broader tumor tissue distribution [18, 19] and more intracellular accumulation than conventional size (about 100 nm) nanoparticles[20]. However, as we known that the non-edge region of brain tumor has a higher pressure[21], and small nanoparticles are easier to flush back into the blood, resulting in a decreased retention in brain tumor tissue. Therefore, small nanoparticles with high retention effect in tumor are still urgently needed for effective GBM drug delivery. Polyamidoamine (PAMAM) dendrimer was firstly developed by Tomalia in 1985[22], and now it is the most widely researched and applied dendrimer. The character of small size, regular molecular structure and single size dispersion make it a preference choice for tumor imaging[23, 24] and drug delivery[25]. Drug can be either physically embedded in the inner cavity or linked on the surface of PAMAM dendrimer through static absorption or covalent binding[26-28]. Unabridged generation PAMAM dendrimer is amino terminated and is easy to be positively charged at physiological pH making it hemolysis toxic[29, 30] and easy to bind to serum protein and then be excreted[31]. PEGylation of PAMAM can significantly decrease the toxicity of PAMAM[32, 33] and prolong in vivo circulation time[34]. However, PAMAM retention in tumor was weak due to its small size. Tumor-homing peptide CREKA is a type of linear pentapeptide achieved by in vivo phage display[35, 36]. Owing to clotted plasma proteins binding ability, CREKA has been used for the molecular imaging of myocardial ischemia-reperfusion[36]. It has been reported that abundant fibrin deposition occurred in tumor extracellular matrix[37, 38], so CREKA was also be used for tumor diagnosis[39] [24]and tumor targeting drug delivery system[35, 40].

In this research, a small nanoparticle drug delivery system was developed by conjugating peptide CREKA to Polyamidoamine (PAMAM) dendrimer, where PEGylated PAMAM is used as drug carrier due to its small size and good penetration in tumor and CREKA acts as an anchor immobilizing nanoparticles after deep penetration for enhanced retention in tumor. Using fluorescent dye FITC and IR783 as the probes, In vitro binding tests were performed to demonstrate the capability of conjugated nanoparticles binding with fibrin. In vivo fluorescence imaging of GBM bearing nude mice, ex vivo brain imaging were further investigated by a in vivo IVIS spectrum imaging system to show the accumulation of conjugated nanoparticles in GBM. At last, the frozen slices of glioma tissues were observed by fluorescence microscope to analyze the nanoparticle penetration in tumor tissue.

2. Materials and animals The PAMAM G5 (28826 Da, size 5.4 nm) was purchased from Dendritech. CREKA peptide was synthesized by the Chinese Peptide Company (Hangzhou, China). IR783-NHS, a near-infrared dye was kindly gifted by Dr. Cong Li, Fudan University. Fluorescein isothiocyanate isomer I (FITC-I) was purchased from Aladdin (Shanghai, China). Maleimide-poly(ethylene glycol)-NHS (Mal-PEG-NHS, Mw 3400 Da) was obtained from Laysan Bio. Inc (Alabama, USA). Fresh frozen human plasma (FFP) was from Shanghai Blood Center (China) and thrombin was from Siemens Healthcare (Marburg, German). The U87-RFP cells line was bought from the Shanghai SBO Medical biotechnology (Shanghai, China). The bEnd.3 cells line is from ATCC (USA). Dulbecco’s Modified Eagle Medium (high glucose) cell culture medium (DMEM), plastic cell culture dishes, and plates were purchased from Corning Incorporation (Corning, USA). Fetal

bovine serum (FBS)

was purchased from Gibco (CA,

USA).

Trypsin-EDTA and

penicillin-streptomycin solution were provided by Invitrogen (Merelbeke, Belgium). Double distilled water was purified using a Millipore Simplicity System (Millipore, Bedford, USA). DAPI (4',6-diamidino-2-phenylindole) was from Beyotime Biotechnology Co., Ltd. (Nantong, China). All the other chemical reagents were of analytical grade or chromatographic pure grade, and were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). BALB/c nude mice (male, 4-5 weeks, 18-22 g) were purchased from the Shanghai Slac Laboratory Animal Co., Ltd (Shanghai, China) and maintained under standard housing conditions. All animal experiments were carried out in accordance with the protocols evaluated and approved by the ethics committee of Fudan University.

3. Method 3.1

Preparation

of

CREKA-conjugated

PAMAM

dendrimer

nanoparticle

(CREKA-PEG-PAMAM) As there were 128 primary amino groups on the surface of each PAMAM dendrimer G5 nanoparticle, it is very easy to be modified with a lot of functional groups. As shown in Figure 1, dye-labeled CREKA-PEG-PAMAM was prepared though multi-step chemical reaction. First, FITC was labeled on PAMAM (PAMAM-FITC). Briefly, 32 mg of PAMAM (1.1 µmol) was dissolved in 1 mL of 0.2 M NaHCO3 buffer (pH 9.0) followed by adding 100 µL of FITC in dimethyl sulfoxide (9.5 mg/mL, 2.5 uM). The mixture was reacted for 1 h at room temperature and then the PAMAM-FITC was separated from unconjugated FITC by passing through HiTrap™ desalting column (GE, USA) using saline as eluent. For IR783 labelling (PAMAM-FITC/IR783),

PAMAM-FITC was added into 1 mL of 0.1 M NaHCO3 buffer (pH 8.33), and 1.64 mg of IR783-NHS (2.1µmol) in 40 µL of DMSO was added. The mixture was reacted for 2 h at room temperature. PAMAM-FITC/IR783 was harvested by passing through HiTrap™ desalting column using saline as eluent. For PEGylation of PAMAM (PEG-PAMAM-FITC/IR783), 4.6 mg of Mal-PEG-NHS (1.35 µmol) in 40 µL of DMSO was added to 0.11 µmol of PAMAM-FITC/IR783 in 0.01 M PBS buffer (pH 8.0) and reacted for 1 h. PEG-PAMAM-FITC/IR783 was collected by passing through HiTrap™ desalting column. Finally, for peptide CREKA modification (CREKA-PEG-PAMAM-FITC/IR783), 0.11 µmol of PEG-PAMAM-FITC/IR783 was mixed with 0.80 mg of peptide CREKA (1.32 µmol) in 0.01 M PBS buffer (pH 7.4) and reacted for 2 h at room temperature. CREKA-PEG-PAMAM-FITC/IR783 was collected by passing through HiTrap™ desalting column.

Figure 1 Scheme of preparation of CREKA-conjugated PAMAM dendrimer nanoparticle

3.2 Characterization of dendrimer nanoparticle 3.2.1 1H NMR, particle size and zeta potential Proton nuclear magnetic resonance ( 1H-NMR) spectra of PAMAM, Mal-PEG-PAMAM and CREKA-PEG-PAMAM were recorded in D2O with a Varian Mercury Plus-400 MHz (Agilent Technologies, Santa Clara, CA, USA) apparatus operating at 400 MHz at 25 °C. D2O signals at 4.65 ppm acted as a reference to determine chemical shifts. The integrals of the peaks

corresponding to PAMAM methylene protons and the PEG methylene protons (δ 3.5 ppm) were used to determine the mole ratio of Mal-PEG conjugated to the surface of PAMAM nanoparticles. The disappearance of the peak at 6.70 ppm for the maleimide protons was used to validate CREKA conjugation

with

Mal-PEG-PAMAM.

Particle

size

and

zeta

potential

of

Mal-PEG-PAMAM and CREKA-PEG-PAMAM were determined by Malvern 3600 Nano-ZS zeta sizer (Malvern Instruments, UK) (n=3). 3.2.2 Determination of CREKA conjugation efficiency and CREKA density on the dendrimer nanoparticle surface The conjugation efficiency of CREKA was determined by the measurement of collected free CREKA solution after separation using desalination column. Concentration of CREKA was determined using an HPLC system (Agilent 1200 series, USA) with an analytical column (250 mm × 4.6 mm; pore size 5 µm; ZORBAX 300SB-C18; Agilent) at room temperature. The elution phase including solvent A (0.1% trifluoroacetic acid in water) and solvent B (80% acetonitrile aqueous solution containing 0.09% trifluoroacetic acid) (A: B = 92: 8, v/v) at the flow rate of 1.0 mL/min. The UV detection wavelength was set at 220 nm, and sample injection volume was 20 µL. Conjugation efficiency of CREKA was calculated as the formula: Conjugation efficiency=(total amount-free CREKA)/total amount×100%. The peptide density on the dendrimer nanoparticle surface was calculated as follows: n=(m1/M1)/(m2/M2),where m1 and m2 were the weight of conjugated CREKA peptide and PAMAM, respectively. M1 and M2 were the molar mass of CREKA peptide and PAMAM, respectively. 3.2.4 Ultraviolet (UV) and fluorescence spectrum scanning To determine whether FITC and IR783-NHS were conjugated to the PAMAM dendrimer

nanoparticles

successfully,

PAMAM-FITC/IR783

was

scanned

by

ultraviolet-visible

spectrophotometer (SHIMADZU UV-2401PC, Japan) and fluorescence spectrophotometer (Agilent, USA) respectively. Then the obtained spectrums were compared with that of free FITC and IR783-NHS. FITC and IR783 density on PAMAM were calculated by the fluorescence intensity using free FITC and IR783 as standard. 3.2.5 In vitro cytotoxicity As an immortalized mouse brain endothelial cell line, bEnd.3 cells were used to evaluate the in vitro cytotoxicity of these dendrimer nanoparticles. Briefly, the bEnd.3 cells were seeded onto a 96-well plate at a density of 104 cells per well and incubated for 24 h. Afterward the cells were incubated with Mal-PEG-PAMAM and CREKA-PEG-PAMAM samples in DMEM with varying concentrations for 4 h at 37 ◦C. The cells treated only with DMEM were used as controls. After exposure to these dendrimer nanoparticles, the cytotoxicity of these formulations was assayed by the MTT method[33]. 3.3 In vitro CREKA-PEG-PAMAM binding with thrombosis Firstly, the in vitro thrombosis clots were prepared as previously described[36]. In brief, FFP clots were formed in 96-well plate. 90 µL of FFP, 5 µL of 0.4 M CaCl2 and 25 µL of thrombin (0.1 U/ml) were transferred into each well followed by a slight shake for 1 min and then the plate was incubated at 37 °C for 90 min. Secondly, 50 µL of PEG-PAMAM-FITC/IR783 solution or CREKA-PEG-PAMAM-FITC/IR783 solution (0.05 µM) were added to each well, and incubated in dark at 37 °C for 30 min. After incubation, the clots were then washed thrice using PBS buffer (0.01M pH7.4) and imaged by the In vivo IVIS spectrum imaging system (PerkinElmer, USA) in the near-infrared channel of IR783.

3.4 In vivo imaging of glioma-bearing nude mice and biodistribution study U87 orthotopic glioma bearing model mice were established by injecting U87 cells (106 cells in 5 µL of PBS) into the right brain of each nude mouse (2 mm lateral to the bregma and 5.0 mm deep from the dura) at a flow rate of 3.0 ml/min using a stereotaxic apparatus. Three weeks after U87MG implantation, PEG-PAMAM-FITC/IR783 or CREKA-PEG-PAMAM-FITC/IR783 was injected into the glioma-bearing nude mice through tail vein at a dose of 0.6 µmol/kg. After injection, the in vivo fluorescence imaging was performed at 2 h, 4 h, 12 h, and 24 h by In Vivo IVIS spectrum imaging system (PerkinElmer, USA), respectively. For ex vivo imaging, 3 mice in each group were sacrificed at 12 h and 24 h post-injection followed by heart perfusion with PBS (0.01 M, pH 7.4) and 4% paraformaldehyde. The main organs including brain, heart, liver, spleen, lung and kidney, were collected and imaged using In Vivo IVIS spectrum imaging system. To quantify the biodistribtution of nanoparticle, three weeks after U87MG implantation, the glioma-bearing

nude

mice

were

injected

with

PEG-PAMAM-FITC/IR783

or

CREKA-PEG-PAMAM-FITC/IR783 at a dose of 0.6 µmol/kg. Twelve hours and twenty four hours later, 6 mice in each group were sacrificed and the heart, liver, spleen, kidneys, lung, left brain and blood were collected and homogenized. The fluorescence of the homogenate at 790 nm with an excitation wavelength of 760 nm was read using a Tecan Infinite M200 Pro Multiplate Reader (Switzerland). The resulting signal was then multiplied by the corresponding organ weight to obtain the total organ fluorescence (n = 6) and the relative distribution of the nanoparticles in each organ was calculated as ID% (Percentage of injected dose). 3.5 Distribution of CREKA-PAMAM-FITC/IR783 in glioma tissue Three

weeks

after

U87MG

inoculation,

PEG-PAMAM-FITC/IR783

or

CREKA

-PAMAM-FITC/IR783 were administrated to the mice by i.v. injection at a dose of 0.6 umol/kg. The mice were sacrificed 24 h later after injection followed by heart perfusion with PBS (0.01 M, pH 7.4) and 4% paraformaldehyde. Then the brains were collected and dehydrated with 15% sucrose until subsidence and in 30% sucrose until subsidence in turn. After that, the brains were embedded in OTC (Sakura, Torrance, CA, USA), frozen at 80 ℃ and were sliced into 20-µm brain slices. Subsequently, these slices were stained with 1 mg/ml DAPI for 10 min at room temperature and then the distribution of fluorescence signal was analyzed with the fluorescence microscope (Leica DMI4000B, Germany). The overlay of green nanoparticles with red glioma cells was analyzed by Image J 1.48v software. 3.6 Statistical analysis Results are given as means ±standard deviation. Statistical significance between groups was tested by one-way analysis of variance, after which post-hoc tests with the Bonferroni correction were used for comparison between individual groups. Statistical significance was set at P < 0.05. 4. Result and discussion 4.1 Characterization of dendrimer nanoparticles

Figure 2 1HNMR spectra of PAMAM, Mal-PEG-PAMAM and CREKA-PEG-PAMAM in D2O. As shown in Figure 2, PAMAM has multiple peaks between 2.2 and 3.2 ppm which were from the methylene protons of its branching units. The typical methylene protons of PEG at 3.5 ppm and a characteristic peak of the Mal group in PEG at 6.7 ppm showed the successful conjugation of Mal-PEG to PAMAM. The disappearance of the Mal peak in the NMR spectrum of CREKA-PEG-PAMAM indicated that the Mal group had reacted with the thiol group of CREKA and CREKA was successfully conjugated to PEG-PAMAM. Furthermore, the integrals of the peaks corresponding to the PEG methylene protons and PAMAM methylene protons were used to quantify the number of PEG chains per PAMAM with the assumption of 280 methylene protons per PEG and 2032 per PAMAM. It was shown that Mal-PEG-PAMAM had a PEG/PAMAM proton ratio of 1.64, implying an average of 11.9 PEG chains per PAMAM.

Figure 3 The particle-size distribution and surface zeta potential of Mal-PEG-PAMAM (green line) and CREKA-PEG-PAMAM (red line).

Table 1 Number size and Zeta potential of dendrimer nanoparticles Mal-PEG-PAMAM

a

CREKA-PEG-PAMAM

Number Size (nm)

6.53 ± 0.07

7.52 ± 0.35

Polydispersity index

0.19 ± 0.03

0.21 ± 0.03

Zeta Potential (mv) a

3.26 ± 0.14

3.39 ± 0.37

Measured in NaCl solution (1 mM). Data are presented as the means ± standard deviation; n = 3. As shown in Figure 3 and Table 1, after PEGylation and CREKA modification, the mean

particle size of Mal-PEG-PAMAM and CREKA-PEG-PAMAM were slightly increased to 6.53 ± 0.07 nm and 7.52 ± 0.35 nm, respectively. The zeta potential of Mal-PEG-PAMAM and CREKA-PEG-PAMAM were 3.26 ± 0.14 nm and 3.39 ± 0.37 nm, respectively, indicating that

CREKA conjugation slightly increased the zeta potential of Mal-PEG-PAMAM, which may relate to the weak positive charge of CREKA[35]. Many previous reports have demonstrated that nearly neutral particles (ξ-10—10 mV) is easier to avoid opsonized and Kupffer cells clearance[41, 42] and distribute deeply and homogenously in tumor[43-45]. HPLC analysis revealed that the CREKA conjugation efficiency was approximately 95%, and the density of CREKA on the surface of dendrimer nanoparticles was 11.4±0.3, which is reasonable for active targeting drug delivery system. In addition, previous reports have proved that more CREKA on the surface of nanoparticle or nanoworm did not induce better targeting efficacy. Instead, excessive peptides might significantly reduce blood circulation half-life of nano-preparation[46]. As shown in Figure 4A, the peaks in PAMAM-FITC/IR783 UV absorption spectrum (499 nm, 693 nm and 769 nm) were basically consistent with the peaks in free FITC (481 nm) and free IR783 (688 nm and 763 nm) UV absorption spectrums. The peak of PAMAM-FITC/IR783 fluorescence emission spectrum (Figure 4 B&C), 531.94 nm and 796.98 nm, were close to the fluorescence emission peaks of free FITC (530.00 nm) and free IR783 (799.04 nm). Compared with UV spectrum of free FITC and IR783, the corresponding peaks of FITC and IR783 in PAMAM-FITC/IR783 slightly shifted right, which might due to the conjugation of FITC and IR783 with terminal amino-groups in PAMAM. According to the fluorescence intensity of PAMAM-FITC/IR783, there were 1.8 FITC and 1.5 IR783 on the surface of PAMAM nanoparticles.

Figure

4

UV

absorption

spectrum

and

fluorescence

emission

spectrum

of

CREKA-PEG-PAMAM-FITC/IR783. (A) The UV spectrum scanning of IR783 (red line), FITC (green line) and PAMAM-FITC-IR783 (blue line). (B) Fluorescence emission spectrum of free FITC (green line) and PAMAM-FITC/IR783 (blue line) with the excitation wavelength of 430 nm. (C) Fluorescence emission spectrum of free IR783 (red line) and PAMAM-FITC/IR783 (blue line) with the excitation wavelength of 760 nm.

Figure 5 In vitro cytotoxicity of dendrimer nanoparticles against bEnd.3 cells with varying concentrations from 1to 100 µM (n = 4).

As shown in Figure 5, Mal-PEG-PAMAM and CREKA-PEG-PAMAM displayed little toxicity against bEnd.3 cells. Even at the highest concentration (100µM), the cellular viability was always above 90%. There were no significant differences in cellular viability between Mal-PEG-PAMAM and CREKA-PEG-PAMAM groups. These results indicate that these dendrimer nanoparticles have good safety.

4.2 In vitro fibrin clot binding

Figure 6 In vitro binding of dendrimer nanoparticles to FFP clots. (A) IVIS spectrum imaging and (B) corresponding semi-quantitative fluorescence intensity of FFP clots after incubation with PBS

(Control), CREKA-PEG-PAMAM-FITC/IR783 (Targeted) or Mal-PEG-PAMAM-FITC/IR783 (Untargeted). Values were means±SD, n=3.

To assess in vitro fibrin clots binding ability of different agents, FFP clots were formed in 96-well plates, incubated with PBS or different dendrimer nano-agents, and imaged by In Vivo IVIS spectrum imaging system in the IR783 channel. As shown in Figure 6A, the fibrin clots incubated with fibrin-targeted agent CREKA-PEG-PAMAM-FITC/IR783 showed obviously stronger

fluorescence

intensity

than

fibrin

clots

incubated

with

untargeted

PEG-PAMAM-FITC/IR783, and there was little fluorescence on the surface of fibrin clots incubated with PBS. The semi-quantitative results also revealed that the clot fluorescence intensity in CREKA-PEG-PAMAM-FITC/IR783 group was 3.4-fold than that of unmodified agent (Figure 6B) (P < 0.001), which were quite consistent with previous report[36]. These results indicated that the CREKA modification could significantly enhance fibrin binding capacity of these dendrimer nanoparticles. 4.3 In vivo imaging In order to investigate the glioma targeting ability of CREKA-PEG-PAMAM-FITC/IR783, in vivo imaging of GBM-bearing mice administrated with PBS, CREKA-PEG-PAMAM-FITC/IR783 or Mal-PEG-PAMAM-FITC/IR783 was performed using In Vivo IVIS spectrum imaging system. As

shown

in

Figure

7A,

CREKA-PEG-PAMAM-FITC/IR783-treated

the mice

brain was

fluorescence higher

intensity than

that

of of

Mal-PEG-PAMAM-FITC/IR783-treated mice at each time point. Ex vivo images of brains (Figure 7 B) further demonstrated that the glioma targeted group had higher fluorescence intensity at glioma site than that of control group and untargeted group at 12 h and 24 h. Similarly, the

corresponding brain semi-quantitative results displayed that there was a significant difference between the fluorescence intensity of targeted group and untargeted group at 12 h and 24 h. The higher accumulation in glioma of targeted nanoparticles might due to three reasons. Firstly, small targeted dendrimer nanoparticles penetrated deeply in tumor tissue. Secondly, targeted dendrimer nanoparticles in tumor tissue interacted with fibrins in the ECM to keep nanoparticles immobilized in the tumor which greatly enhanced the nanoparticle retention effect. Thirdly, some targeted nanoparticles interacted with fibrins to form coaggregates, which could bind to integrin on the surface of GBM cells, and ultimately became internalized. The internalization of these nanoparticles further enhanced the nanoparticle retention effect.

Figure 7 (A) In vivo fluorescence imaging of GBM-bearing nude mice administrated with PBS (left), Mal- PEG-PAMAM-FITC/IR783 (middle), and CREKA-PEG-PAMAM-FITC/IR783 (right) at different time points (2 h, 4 h, 12 h, 24 h). (B) Ex vivo fluorescence imaging and the corresponding semi-quantitative fluorescence intensity of brains from GBM-bearing nude mice administrated with PBS (Control), Mal-PEG-PAMAM-FITC/IR783 (Untargeted), and CREKA-

PEG-PAMAM-FITC/IR783 (Targeted) at 12 h and 24 h. (C) the corresponding semi-quantitative radiant efficiency results of brains.

Figure 8 Ex vivo fluorescence imaging of major organs and biodistribution of dendrimer nanoparticles. (A) Ex vivo fluorescence imaging of major organs and blood of glioma bearing nude mice administrated with PBS (left, Control), Mal-PEG-PAMAM-FITC/IR783 (middle, Untargeted), and CREKA-PEG-PAMAM-FITC/IR783 (right, Targeted) at 12 h and 24 h. (B) the corresponding semi-quantitative results of major blood and organs. (C) Relative fluorescence intensity per gram of tissue (n = 6). (D) Relative nanoparticles uptake per organ (n = 6). As shown in Figure 8, ex vivo fluorescence imaging and corresponding semi-quantitative results of major organs showed that both targeted and untargeted dendrimer nanoparticles were mainly distributed in kidney, liver and spleen (Figure 8A&B). Further biodistribution study revealed similar results. Figure 8C showed the relative nanoparticle content per gram of tissue. It

demonstrated that the biodistribution of targeted and untargeted dendrimer nanoparticles was similar, and kidney, liver contained the highest amount of nanoparticles. To better understand the overall particle distribution, the fluorescence signals were multiplied by the measured weight of the corresponding organs, with the weight of the blood being estimated as 6% of the total body weight. Figure 8D showed the relative signal in each organ normalized to the total injected dose. After accounting for the tissue mass, it could be observed that the nanoparticles were distributed mainly in the kidney and the liver. In previous reports, conventional nanoparticles (size about 100 nm) accumulated mainly in mononuclear phagocyte system (MPS)-related organs, liver and spleen [47, 48], while in the present study the nanoparticles distributed most in kidney and liver (Figure 8 C&D), which might due to the small particle size and positive surface zeta potential of dendrimer nanoparticles[49].

4.4 In vivo distribution of nanoparticles in glioma

Figure

9

In

vivo

distribution

of

Mal-PEG-PAMAM-FITC/IR783

(upper)

and

CREKA-PEG-PAMAM-FITC/IR783 (lower) at 24 h after administration. Blue: cell nuclei stained by DAPI. Green: FITC-labeled dendrimer nanoparticles. Red: glioma cells expressing red fluorescence protein. White dash lines: border of the glioma. Image D was merged by image A, B

and C, and image H was merged by image E, F and G.

In order to evaluate the penetration of CREKA-PEG-PAMAM-FITC/IR783 in brain tumor tissues, in vivo distribution of CREKA-PEG-PAMAM-FITC/IR783 in glioma slices were imaged using fluorescence microscope (Leica DMI 4000B, Germany). As shown in Figure 9, Mal-PEG-PAMAM-FITC/IR783 distributed sparsely at the edge of tumor and in the deep while CREKA-PEG-PAMAM-FITC/IR783 distributed extensively and reached the deep section of the tumor. These results suggested that PEG-PAMAM-FITC/IR783 could penetrate deeply into the tissue but with little retention while CREKA conjugation greatly improved nanoparticles retention in

tumor

tissue.

Estimated

by

Image

J

software,

about

31

%

of

CREKA-PEG-PAMAM-FITC/IR783 distributed in the glioma cells expressing red fluorescence protein

(Figure

9

H).

The

extensive

and

abundant

distribution

of

CREKA-PEG-PAMAM-FITC/IR783 in glioma tissue depends on suitable small particle size and effective specific ligand. Firstly, the suitable particle size helps dendrimer nanoparticles pass through small physiological pores on blood tumor barrier (BTB) smoothly, and move to the distant tissue outside vascular. Secondly, the fibrin binding peptide CREKA keep dendrimer nanoparticles immobilized in the tumor greatly enhanced nanoparticles retention effect, which contributes to the next cellular internalization of nanoparticles. The good penetration and retention of CREKA-PEG-PAMAM in tumor make it a potential candidate for GBM targeting drug delivery system, and many anti-glioma drugs especially enzyme sensitive or gene drugs can be delivery to glioma through the novel delivery system. Moreover, PAMAM was widely used in diagnostic imaging, so the delivery also could be used for brain tumor diagnosis. Since the vasculature of

new growing low-grade gliomas extremely resembles that of normal brain[50] and the small particle size allows dendrimer nanoparticles cross BBB [51], the delivery system is also a preferred choice of early brain tumor detection. Conclusion An effective GBM targeting drug delivery system was developed in the present study, which is expected to be a potential strategy for the treatment of malignant brain tumor. In vitro binding with fibrin displayed higher fibrin binding ability of CREKA modified dendrimer nanoparticles than other agents. In vivo imaging and distribution in glioma indicted that modified dendrimer nanoparticles can penetrate into deep area of glioma and achieve better retention than unmodified nanoparticles. In summary, these promising results indicated that the CREKA-modified dendrimer nanoparticles might be a potential drug delivery system for the clinical therapy of glioma. Acknowledgement The work was supported by the National Basic Research Program of China (973 Program, 2013CB932502), National Natural Science Foundation of China (81302714), the State Scholarship Fund, and “Zhuoxue” program of Fudan University.

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CREKA peptide-conjugated dendrimer nanoparticles for glioblastoma multiforme delivery.

Glioblastoma multiforme (GBM) is the most aggressive central nervous system (CNS) tumor because of its fast development, poor prognosis, difficult con...
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