Magnetic Resveratrol Liposomes as a New Theranostic Platform for

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Magnetic resveratrol liposomes as a new theranostic platform for magnetic resonance imaging guided Parkinson’s disease targeting therapy Meili Wang, Lei Li, Xuwu Zhang, Yanping Liu, Ruiyan Zhu, Lanxiang Liu, Yuan Fang, Zhengrong Gao, and Dawei Gao ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b04507 • Publication Date (Web): 23 Oct 2018 Downloaded from http://pubs.acs.org on October 27, 2018

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ACS Sustainable Chemistry & Engineering

Magnetic resveratrol liposomes as a new theranostic platform for magnetic resonance imaging guided Parkinson’s disease targeting therapy Meili Wang1, Lei Li1, Xuwu Zhang1, Yanping Liu1,2, Ruiyan Zhu1,2, Lanxiang Liu3, Yuan Fang3, Zhengrong Gao4, Dawei Gao*1,2 1Applying Chemistry Key Lab of Hebei

Province, Yanshan University, No.438

Hebei Street, Qinhuangdao, PR China 2Hebei Province Asparagus Industry Technology Research Institute, No.12

Donghaidao Street, Qinhuangdao, PR China 3Department of Pathology, the

First Hospital of Qinhuangdao, No. 258 Cultural

Street, Qinhuangdao, PR China 4Department

of Surgery, Beijing Ditan Hospital, Beijing, PR China

Correspondence author: Dawei Gao TEL: +86 335 8387553; FAX: +86 335 8387553; Email: [email protected] Address:

Applying Chemistry Key Lab of Hebei

Province, Yanshan University,

No.438 Hebei Street, Qinhuangdao, PR China

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Abstract Resveratrol (3,5,4’-trihydroxy-trans-stilbene, Res), as a natural product and neuroprotective drug plays important roles in the treatment of Parkinson's disease (PD). However, Res is seriously hindered by the blood-brain barrier (BBB), which limits the access of Res to central nervous system. In addition, the differential diagnosis of PD has been considered one of the most challenging in neurology. Herein, a magnetic targeting drug nanocarrier, Fe3O4 modified Res liposomes (Res-lips@Fe3O4), was designed and fabricated. The factional anisotropy (FA) values and T2 relaxation time of the Res-lips@Fe3O4 treated rats were obtained by Magnetic Resonance Imaging. The Res-lips@Fe3O4 possessed high drug capability and stability, which exhibited strong magnetic response properties. Res-lips@Fe3O4 displayed

sustained and slow drug

release in vitro. In vivo experiments showed that the Res-lips@Fe3O4 could effectively across the BBB and increase the drug content at the target site under the external magnetic field. FA values and T2 relaxation time also demonstrated the best therapeutic effect of the magnetic Res liposomes. Therefore, the Res-lips@Fe3O4 combining with an external magnetic field will provide a potential platform to cross the BBB and treat effectively cerebral disease. Key words: Magnetic targeting liposome; Parkinson's disease; Resveratrol; Magnetic response; Factional anisotropy


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Introduction Parkinson's disease (PD) is one of the chronic progressive disorder that characterized by cellular loss of dopaminergic neurons of substantia nigra (SN) and subsequent deprivation of dopamine in the basal ganglia1. Patients with PD exhibit unique symptoms including bradykinesia, rest tremor, muscle stiffness, stooped posture, and in some cases dementia1-2. However, 60% of dopaminergic neurons and 80% of putamen dopamine have been disappeared when PD related clinical symptoms are recognized clinically3. Due to the progressive property of PD, early evaluation of substantia nigra lesions is important for early diagnosis, disease progression monitoring and long-term drug impact assessment4. Diffusion tensor imaging (DTI) has been demonstrated as an advanced non-invasive magnetic resonance imaging (MRI) parameter to evaluate microstructural changes in PD5-7. FA is one of the most common parameters in DTI analysis. It estimates the degree of anisotropy, i.e., restricts the random motion of water molecules through the normal structure of glial tissue and fiber bundles6, and reduced FA values have been reported in the SN area of some PD patients8-10. Although the mechanisms leading to the selective neuronal death in PD have not been fully explained, convincing experimental evidence suggests that endoplasmic reticulum (ER) stress, oxidative damage and inflammation are crucial factors in the pathogenesis of PD3, 11-12. There is increasing evidence that oxidative stress is one of the major risk factors for substantia nigra degeneration13-14. Convincing evidence from postmortem brain tissue, cell culture and PD animal models suggested that oxidative



stress facilitate the cascade leading to dopaminergic cell degeneration, and is closely related to other neurodegenerative processes, such as inflammation and cell death3, 15. Therefore, antioxidants have been recognized as potential neuroprotective agents for the treatment of PD. Resveratrol (3,5,4’-trihydroxy-trans-stilbene, Res) is a polyphenol phytoalexin mainly found in grapes, red wine, peanuts, and other plants. Preclinical studies have demonstrated that Res has many biological properties that are potentially protective of health, its antioxidative16 and neuroprotective effects17-18 have received extensive attention. However, due to the low bioavailability in brain and unfavorable pharmacokinetic characteristics, the biological efficiency of Res in central nervous system diseases is often limited. In addition, delivery drugs to exert superior therapeutic efficacy to the brain tissue is also a challenge because of their inability to cross the highly selective BBB. Therefore, use of nano-delivery systems to deliver drugs across the BBB to improve the limitations seems to be effective in the treatment of PD19. Among the various nano-drug systems, liposomes have attracted much attention as an effective vector for central nervous system (CNS) delivery because of their lipophilic, biodegradability, biocompatibility and nanometer range size (10-200 nm), which allows them easily pass through the tight endothelial cells of BBB20-21. Liposomes provide an established basis for the sustainable treatment diseases by the smart delivery of drugs, which may serve as a sustained drug release system by acting as a long circulating micro-reservoir or localized drug depot22. In addition, some researchers have found that


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chitosan (CTS) has high affinity for the cellular membranes since protonated amino groups, which can interact with carboxylic groups of biomembrane molecular components23. Fe3O4 nanoparticles (Fe3O4 NPs) have been considered as a kind of safe nanomaterials in clinical applications, and are widely used in MRI, cell separation, magnetically guided drug delivery, hyperthermia treatment and so forth24. In particular, Fe3O4 NPs can be magnetized and become physically sensitive to external magnetic fields. Magnetic targeting (MT) can then be used to actively enhance drug deposition at target site, which may increase the therapeutic effects. In this study, a magneto responsive drug delivery system was successfully prepared, which contained Res as a neuroprotective drug. Our synthesis is facile and green, which conform to the principles of green chemistry and sustainable development. At the same time, the drug delivery system good biocompatibility and good biodegradability properties in vivo19, 25. The system could cross BBB effectively via external magnetic field and release Res slowly, and then effect sustainably at the target site. In vitro experiments demonstrated that Res-lips@Fe3O4 possessed high drug capability, stability, and exhibited strong magnetic response properties. The ability of transportation across BBB and therapeutic efficacy of the Res-lips@Fe3O4 were studied by PD rat model, which demonstrated the magnetic liposomes possessed superior therapeutic effect. Experimental HPLC analytical method The quantifications of Res were measured by high-performance liquid



chromatography (HPLC, Agilent 1200, USA). An Agilent ZORBA × 300SB-C18 (4.6 mm × 250 mm, 5 μm) was performed for analysis at 25 °C. The mobile phase was consisted of acetonitrile and 0.1% phosphate buffer (30: 70, v/v). The injection volume was 10 μL and flow rate of mobile phase was set at 1.0 mL/min. The detection wavelength was kept at 306 nm. Standard curves were drawn in the range of 10-100 μg/mL and validated for linearity, precision, accuracy, limit of quantification (LOQ), and limit of detection (LOD). Preparation of drug delivery system Synthesis of Fe3O4 NPs Magnetite Fe3O4 NPs were synthesized with a chemical co-precipitation technique according to previous work19,

26-28.

Typically, FeSO4·7H2O (0.834 g) and FeCl3·6H2O

(1.21635 g) were dissolved in 12.5 mL of deionized water at 50 °C followed by ultrasonic bath (Kunshan ultrasonic instrument co., Ltd; 40 kHz, 250W) for 30 min. Then, 1 mol/L aqueous NaOH (50 mL) was added slowly into the solution under vigorous magnetic stirring (500 rpm) and a black precipitate was immediately formed. The solution was agitated at 85 °C for 20 min, and then washed 4~5 times with deionized water and anhydrous ethanol, respectively. The powders were dried overnight in vacuum oven at 70 °C to obtain Fe3O4 NPs. Synthesis of Res-lips@Fe3O4 57μL acetic acid was dissolved in 9943μL deionized water to get acetic acid solution (0.1 M). The CTS solution (0.1 %, w/v) was prepared by dissolving CTS powder in 0.1


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M acetic acid25, 29. Then, 5 mL of Res-liposomes were added drop wise into 5 mL CTS solution, the resulting mixture was stirred 2 h to obtain Res-lips@CTS. 1 mg Fe3O4 NPs were dispersed in 4 mL deionized water and sonicated for 2.5 h. Then, the Fe3O4 solution and above Res-lips@CTS were mixed with a volume ratio of 1: 3, and the mixtures were incubated at 25 °C for 3 h. Entrapment efficiency The quantities of Res encapsulated in liposomes were detected by HPLC. Briefly, 1 mL Res-lips@Fe3O4 was dissolved in the same amount methanol for disruption of liposomal structure. Then mixtures were centrifuged for 30 min at 10,000 rpm to separate the inorganic and organic components. The supernatant was filtered by organic membrane (0.45 μm) before being measured via HPLC. The encapsulation efficiency (EE) was calculated with the following formula: weight of drug loaded

EE (%) = total weight of drug added in the preparation × 100%

(1)

“weight of drug loaded” was the amount of encapsulated drug. Firstly, resveratrol standard solutions with different concentrations (10–100 μg/mL) were prepared, and then the corresponding peak area was obtained by HPLC analysis. According to the concentrations and corresponding peak area, a standard curve (y=-151.3+140x, R2=0.999) of resveratrol was received. Next, the samples were determined by HPLC analysis to get the peak area, and the resveratrol concentrations can be calculated by standard curve. “weight of drug loaded”=concentrations×dilution ratio×sample volume. In vitro drug release characteristics


(2)


To measure the release properties of Res from different liposomes, the samples of free Res, Res-liposomes, Res-lips@CTS or Res-lips@Fe3O4 (1 mL) were put into dialysis bags (MWCO: 12,000-14,000), separately, then dialysis bags were immersed in PBS (200 mL, pH=7.4). The experimental apparatus was placed in an oscillating water bath at 37 °C and shaken horizontally with 100 rpm. At scheduled intervals (1, 2, 4, 8, 12, 16, 20, 24, 32, 40, 48, 60 and 72 h), 1 mL aliquots of the samples were withdrawn, and the contents of Res were measured by HPLC. Stability study In order to detect the dispersion of nanocomposites in various physiological solutions, the Res-lips@Fe3O4 were added in PBS, cell culture medium and fetal bovine serum (10 % in cell culture medium) with a volume ratio of 1:1, respectively, and then the observation was conducted to check if there was agglomeration or sediment in the solution. In addition, the solutions of Res-lips@Fe3O4 were stored in PBS at 4, 25, and 37 °C for one month. At scheduled intervals, the particle diameters and EE for liposomes were measured. In vivo experiments Animals and experimental design Male Sprague-Dawley rats (3-4 months, 180-200 g) were fed under constant temperature (22 °C, 50-60 % humidity) and relative humidity with alternating 12 h cycles of light and dark. In the whole experiment, the rats were fed in standard laboratory cages and had free access to food and water. The changes in the hair color and body weight,


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food consumption were used to evaluate the status of animals. All the animal experiments were permitted by the institutional animal use committee in Yanshan University and conducted in ethically and humanely. Thirty-six animals were randomly divided into six groups containing 6 animals each (Table 1): saline+saline (Sham group), 6-OHDA+saline (PD group), 6-OHDA+Free Res, 6-OHDA+Res-lips@CTS,

6-OHDA+Res-lips@Fe3O4

and

6-OHDA+Res-lips@Fe3O4+magnetic field (MF). For 6-OHDA+Res-lips@Fe3O4+MF group, a piece of magnet (D10 × 5 mm, N35) was fixed on each rat’s head to simulate external magnetic fields. Table 1 Groups of animals in the experiment Groups

Number

Treatment

Sham group

6

saline+saline

PD group

6

6-OHDA+saline

Free Res group

6

6-OHDA+Free Res

Res-lips@CTS group

6

6-OHDA+CTS@Res-lips

Res-lips@Fe3O4 group

6

6-OHDA+Fe3O4@CTS@Res-lips

Res-lips@Fe3O4+MF group

6

6-OHDA+Fe3O4@CTS@Res-lips +MF

Different Res formulations (0.5 mg/kg/day) were administered by intraperitoneal (ip.) route once a day during a period of 35 consecutive days. One-week treatment later,



6-OHDA was administered according to the following procedure. Briefly, the rats were narcotized with chloral hydrate for stereotaxic surgery, then 6-OHDA was dissolved in 0.02 % ascorbate saline (5 μg/μL) to be injected into the right nigrostriatal: antero-posterior: -4.8, medio-lateral: +1.9, dorso-ventral: -8.0 (mm from Bregma). The injection rate was 1 μL/min and the needle was kept in place for an additional 10 min for 6-OHDA diffusion30. Meanwhile, the sham-operated rats were received 0.02 % ascorbate saline (1μL/min) in the right medial forebrain bundle of the nigrostriatal, which was used the control (Sham group). Penicillin was injected once two days to prevent infections. The photo of operation was displayed in Figure S1 (Supporting Information). Assessment of Blood-brain Barrier Integrity Evans Blue’s (EB) penetration was considered as a label of Blood-brain Barrier integrity according to the previous study19, 31-32. Briefly, the Sham, PD model rats were injected intraperitoneally with 2% (w/v) EB solution at 40 mg/kg and allowed the dye-albumin complex to circulate in the whole body for 3 h. Then the rats were deeply anaesthetized and perfused with normal saline to remove the Evans Blue-Albumin complex. Finally, brain tissues were removed, weighed, and EB was extracted by formamide at 37 °C for 72 h. The concentration of EB was detected by UV spectrophotometer at 630 nm. Behavioral testing After administrated with 6-OHDA, the rats were detected at the 7th, 14th, 21st and 28th days by the apomorphine hydrochloride-induced rotational behavior test. Briefly, the


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animals were allowed to habituate for 15 min, then apomorphine hydrochloride was injected intraperitoneally (0.5 mg/kg), and their total numbers of turns towards the direction ipsilateral of the lesion were counted for 90 min with an automatised rotameter. The results are expressed as the number of ipsilateral turns per minute. Measurement of FA value FA values in SN and corpus striatum (CS) regions were acquired by MRI scanner (Siemens Verio 3.0 T, Germany) at the 7th, 14th, 21st and 28th days after the rats were injected 6-OHDA. Briefly, the rats were narcotized with chloral hydrate, and then were placed in a four-channel special coil. The coronal diffusion tensor images were collected by plane spin echo diffusion-weighted imaging sequences with the following parameters: Repetition time (TR) = 1800 ms, Echo time (TE) = 110 ms, slice thickness = 2.0 mm, resolution = 1.5×1.0×2.00 mm3 and Field of view (FOV) = 65×65 mm2. The diffusion tensor imaging (DTI) was carried out using the Siemens Verio 3.0 T MR Leonardo 3682 workstation, and the image processing was completed using the diffusion tools in the Neuro 3D software package. Then FA values were obtained from the target regions on the slice of the maximum level of SN and CS respectively. CS was divided into three equal zones, and then averaged the values. The interested regions were drawn by two experienced researchers and averaged the results. Measurement of T2 relaxometry T2 relaxation times in right SN were acquired by MRI scanner at 7th, 14th, 21st and 28th days after 6-OHDA injection. Briefly, the rats were narcotized with chloral hydrate,



and then were placed in a four-channel special coil. The lesion was detected by T2 mapping of coronal slices acquired with the following parameters: Repetition time (TR) = 3000 ms, Echo time (TE) =225 ms, slice thickness = 2.0 mm and Field of view (FOV) =74 mm2, 33. The T2 image was carried out using the Siemens Verio 3.0 T MR Leonardo 3682 workstation, and the image processing was completed using the diffusion tools in the Neuro 3D software package. Then T2 relaxation times were obtained from the target regions on the slice of the maximum level of SN, which was divided into two equal zones, and then averaged the values. The interested regions were drawn by two experienced researchers and averaged the results. Pharmacokinetic study To determine the blood circulation time of drug, the blood samples were collected at 1, 2, 4, 8, 12 and 24 h after last injection (the 35 needles) of different Res formulations. The Res content in the plasma was determinated according to reported methods34-36. Briefly, each blood sample was first collected in a test tube, centrifuged at 3000 rpm for 15 min at 4 °C to isolate plasma, then 500 μL of plasma was mixed with equivalent acetonitrile, and the sample was incubated at 20 °C in the dark overnight. Finally, the resulting mixture was centrifuged at 10000 rpm for 10 min, and the supernatant was detected via HPLC. The Res content of rat plasma was measured by comparing with the calibration curve. Parameters including area under the plasma concentration time curve from zero to time infinity (AUC), elimination half-life (t1/2), and maximum plasma concentrations (Cmax) were evaluated by using the Origin 8.5 software.


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Biodistribution study To determine the sustainability of Res-lips@Fe3O4, the brain samples were collected at 1, 2, 4, 8, 12 and 24 h after last injection (the 35 needles) of different Res formulations. The Res content in brain was determined according to HPLC. In addition, after rats were treated with different Res formulations for 4 h, all of them were sacrificed under anesthesia, and primary organs including brain, heart, liver, spleen, lung and kidney were excised. Tissue specimens were washed with PBS and weighed after removing excess liquid. The Res contents were determined by the HPLC analysis. Briefly, tissue samples (200 mg) were first suspended in 1 mL of saline and homogenized with tissue homogenizer. Then, transfer 0.5 mL homogenate into 1.5 mL micro tube, and the same volume of acetonitrile was added and mixed, the resulting sample was centrifuged for 10 min at 10000 rpm and the supernatant was detected via HPLC. Results and discussion HPLC analytical method The HPLC calibration curve of Res were designed over the concentration range of 10–100 µg/mL (r2=0.999) and standard curve of was found to be linear. The accuracy of 5, 50 and 100 μg/mL was 85.6%, 92.4%, and 89.3%, respectively. The coefficients of variation (CV) for intra and inter day precision were less than 5%. LOD and LOQ were 20 ng/mL and 26 ng/mL, respectively. Characterization The synthesis strategy of Res-lips@Fe3O4 was as follows. Briefly, Res-lips were



fabricated using ethanol injection method (Figure 1A), and CTS was combined through the electrostatic interactions between NH3+ groups of CTS and negatively charged phosphates (Res-lips@CTS). Next, the synthesized Fe3O4 NPs (about 10 nm, Figure S2, Supporting Information) were incubated with the Res-lips@CTS for 2 h using the coprecipitation method, and then the architectures of Res-lips@Fe3O4 were formed by self-assembly processes. The Res-lips@Fe3O4 were characterized by transmission electron microscope (TEM, HT7700, Japan), showing smooth, monodisperse and almost spherical morphology structures (Figure 1B), and the average size of Res-lips was approximately 124 nm, which increased to approximately 163 nm when modified with Fe3O4 NPs. The outer layer has approximately 39 nm, which attribute to the CTS polymer reacts with iron oxide nanoparticles through glycosidic bonds37.

Figure 1. TEM images of A) Res-lips, B) Res-lips@Fe3O4 The elemental maps of EDX (Figure 2A) provide effective evidence that Fe elements have been combined with the Res-lips@CTS. Figure 2B shows the XRD


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images of the Fe3O4 NPs and Res-lips@Fe3O4. The diffraction peak positions at 30.22°, 35.60°, 43.05°, 53.64°, 57.10°, and 62.85° could be attributed to the (220), (311), (400), (422), (511), and (440) planes of Fe3O4, respectively38-39, and also confirmed to be the spinel structure of Fe3O4 (according to the JCPDS file of Fe3O4 (PDF No. 65-3107))40. Besides, XRD peaks for Res-lips@Fe3O4 were similar to those of Fe3O4 nanoparticles (Figure 2B a), revealing that Fe3O4 nanoparticles were successfully coated on the liposomes. Meanwhile, the homogeneously dispersed Res-lips@Fe3O4 showed a fast response to an externally magnetic field and the separation process can be completed in 2 min (Figure S3 Supporting Information).

Figure 2. A) EDS analysis of Res-lips@Fe3O4; B) XRD spectra of a) Fe3O4 NPs and b) Res-lips@Fe3O4 In vitro drug release characteristics The in vivo circulation time of liposomes is an important factor affecting the potential pharmaceutical efficacy, and release model in vitro can partly reflect the characteristics of drug residence time41. The in vitro drug release characteristics of



different Res formulations were studied in PBS and the results shown in Figure 3. Free Res displayed faster diffusion property and almost all drugs were released within 12 h, which was basically consistent with the expected diffusion rate of low molecular weight molecules across the dialysis membrane41. In contrast, the release rate turns slower (12 h, 30%) in the form of liposomes under the same conditions, which means the bilayer membrane of liposomes can achieve the encapsulated Res sustained-release. A further decrease in the release rate of Res was observed for the Res-lips@Fe3O4, only 23.7 % release within 12 h (retained 76.3%), which may be due to the external CTS-Fe3O4 NPs layer. After incubation for 72 h, the Res-lips still retained 53.6% Res and Res-lips@Fe3O4 contained 65.6% Res. The results showed that Res-lips@Fe3O4 can release drugs slowly, continuously and steadily.

Figure 3. In vitro release profiles of free Res, Res-lip and Res-lips@Fe3O4. Data were expressed as mean ± S.E. (n = 3) Stability study The stability of nanocomposites in biologic system is of great significance for their


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biomedical applications. As shown in Figure 4, the Res-lips@Fe3O4 can fast disperse in PBS, cell culture medium and fetal bovine serum (10 % in cell culture medium) respectively. There was no agglomeration or sediment for at least 7 days, indicating Res-lips@Fe3O4 were very stable in physiological conditions. In addition, after being stored one month at 4 °C, the nanocomposites did not show any significant changes on morphology and no detachment of Fe3O4 NPs from the Res-lips@Fe3O4 (Figure S4). On the contrary, the Res-lips@Fe3O4 had significant reduction on EE (%) from 86.94 ± 1.94 to 48.08 ± 2.94, and 25.17 ± 2.04 after one month of storage at 25 and 37 °C, respectively (Figure 5A). The particle sizes also increased in varying degrees (Figure 5B). Therefore, the system of Res-lips@Fe3O4 possesses good stability under low temperature.

Figure 4. Digital images of Res-lips@Fe3O4 after incubated with PBS, DMEM, DMEM with 10% FBS for 7 days



Figure 5. Storage stability Res-lips@Fe3O4. A) EE; B) Size changes after storage at 4, 25 and 37°C for one month. Data were expressed as mean ± S.E. (n = 3). Assessment of Blood-brain Barrier Integrity Evans Blue (EB) dye binds to serum albumin (67 kD) immediately after intravenous injection and was also used to measure BBB integrity31. As shown in Figure 6, EB has been circulated to all organs (heart, liver, spleen, lung and kidney) except the brain, which means that the BBB remains intact. In addition, EB was barely detectable in PD group (0.836 ± 0.05 ng/g) after 6-OHDA injury, and no significant difference in total EB content between sham (0.83± 0.06 ng/g) and PD groups. These results indicated that the BBB of rats was integrated after the injection of 6-OHDA.


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Figure 6. Integrity of BBB after injection of 6-OHDA. A) Sham group; B) PD group Behavioral testing The examination of rotational behavior for the 6-OHDA treated rat is a validated means of evaluating the anti-parkinsonian therapeutic potential of molecules for PD30. Apomorphine hydrochloride is believed to directly act on the hypersensitive dopaminergic receptor at the damaged site and induce the contralateral rotations in PD rats42. In this study, the numbers of apomorphine hydrochloride induced rotations were measured with an automatised rotameter at the 7th, 14th, 21st and 28th days after 6-OHDA lesions. As illustrated in Figure 7, all the PD rats displayed a dramatic increase in ipsilateral rotations as compared sham group, which means that the successful construction of PD model. The rotation numbers of the free Res group (14.7 ± 3.85 rpm) were fewer than that of PD group (16.3 ± 1.45 rpm) at the 28th day, confirming the potential neuroprotective effects of Res to PD rats. Res-lips@Fe3O4+MF administration resulted in the lowest levels of ipsilateral rotations among all Res groups at the 28th day (7.7 ± 2.33). Apparently, an equimolar doses of Res exhibited different neuroprotective effects, which were depended on the physicochemical properties of the administered Res formulations and the extra magnetic field. The Res-lips@Fe3O4 can effectively cross the BBB under the magnetic field to delay PD progression, and display obvious therapeutic effect.



Figure 7. Effects of apomorphine hydrochloride treatment on contralateral rotation in different Res-treated PD rats. Data were expressed as mean ± S.E. (n = 3) Measurement of FA value It has been demonstrated that conventional brain MRI has the highest value in the diagnosis of central nervous system diseases6. DTI is a MRI technique that can reveal the microstructural integrity of brain tissues43. FA is one of the most common parameter in DTI analysis, and the decreased FA value had been found in PD patients using region-of-interest (ROI) analysis8-10. In order to evaluate the drug efficacy, the changes of FA value in right SN and CS were measured by MRI scanner with a four-channel special coil once a week after the rats were injected 6-OHDA. Figure 8A showed the changes of FA in the right SN on the treated rats, and the red dot in Figure 8B was the location of the right SN in MRI scanner. On the 7th day after injury, a maximal decrease of the FA value was observed in SN, which attributed to the mechanical injury. Many clinical studies have demonstrated that neuronal loss in early PD patients was associated with decreased FA values in the SN44-46. FA values in Res


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formulation administration groups were higher than that of PD group because of the neuroprotection of Res. In addition, following 28 days administration, the average of FA value in free Res group was 0.24 ± 0.004, whereas FA values in Res-lips@CTS and Res-lips@Fe3O4 groups were 0.31 ± 0.008 and 0.31 ± 0.005, respectively, which indicated that the neuroprotective effects of Res was increased by entrapping in liposomes. Notably, the neuroprotective effects of Res-lips@Fe3O4 plus magnetic field was significantly enhanced (0.33 ± 0.006). Figure 8C showed the changes of FA in the right CS, showing a similar trend with that of right SN. Figure 8D was the typical images illustrating the location of the right CS in MRI scanner. There was the lowest FA value in CS of PD group at the 28th day (0.2 ± 0.004). FA value in Res-lips@Fe3O4+MF group (0.26 ± 0.003) was remarkably higher than that of Res-lips@Fe3O4 group (0.24 ± 0.006), closing to that of sham group (0.276 ± 0.003) on the 28th day. This also means that Res-lips@Fe3O4+MF group possessed the best neuroprotective effect. The main reason was that the Res-lips@Fe3O4 could easily cross BBB under a magnetic field, so more Res was transported to the diseased site in the brain, eventually enhanced the drug treatment efficacy.



Figure 8. FA values of treated rats measured using MRI. A) Changes of FA in the right SN on treated rats; B) Region-of-interest location of right SN in MRI scanner (red dot); C) changes of FA in the right CS on the treated rats; D) Location of the right CS (red/blue/green dot), respectively, Data were expressed as mean ± S.E. (n = 3).*p < 0.01 vs PD group; #p < 0.05 vs Res-lips@Fe3O4. Measurement of T2 relaxometry The changes of T2 relaxation time at the right SN were measured by T2 maping (Figure 9 and Figure 10). At the 7th day after injury, a significant increase in T2 relaxation time was observed in right SN compared with the sham group, and the lesion is


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clearly defined by the increase of the T2 relaxation time. It is believed that, the increase in T2 relaxation time in pathological tissues can be attributed to an increase in interstitial water related to demyelination, oedema, gliosis and neuronal loss47-48. The increased T2 relaxation time in SN after 6-OHDA injection, highlighting the oedematous process caused by dopaminergic neuronal death33. After 28 days administration, the average of T2 relaxation time in free Res group was 71.15 ± 0.54 ms, whereas those in Res-lips@CTS and Res-lips@Fe3O4 groups were 66.87 ± 0.47 ms and 65.46 ± 0.65 ms, respectively, which indicated that the neuroprotective effect of Res was increased by entrapping in liposomes. Notably, the neuroprotective effect of Res-lips@Fe3O4 plus magnetic field was significantly enhanced (59.8 ± 0.55 ms), which mainly attributed to the significant magnetic response properties of nanocomposites under external magnetic field.

Figure 9 T2 images of rats (Res-lips@Fe3O4+MF group) at three different time points: pre-lesion, at the 7th and 28th day after 6-OHDA injection.



Figure 10 Changes of T2 relaxation time in the right SN on treated rats. Data were expressed as mean ± S.E. (n = 3).*p < 0.01 vs PD group; #p < 0.01 vs Res-lips@Fe3O4. Pharmacokinetic profiles of Res Figure 11 presented the plasma concentration-time profiles of Res in PD rats after intraperitoneal administration (0.5 mg/kg). The pharmacokinetic parameters of the different Res formulations are showed in Table 2. The study found that free Res can be rapidly cleared from the blood compartment within 6-8 h of administration. Compared with the pharmacokinetics of free Res, the Res-lips@CTS displayed an approximately 1.5 times higher of t1/2 (7.98±0.67 vs 13.28±0.72 h) and nearly 2 times of AUC (26.25±1.5 vs 49.67 ± 1.77 μg/mL·h), implying the rapid blood clearance of the free drugs at physiological conditions. The application of Res-lips@Fe3O4 under the external magnetic field markedly increased the bioavailability and extended the circulation time of Res, improving AUC (73.48 ±2.04 μg/mL·h) by approximately 3 times and increasing the Cmax 2 times approximately (3.23±0.15 vs 5.75±0.2 μg/mL), in comparison with free Res.


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These results could attribute to three factors, the drug slow release from liposomes, the ideal size of the liposomes (155.7 ± 1.6 nm) and the impact of extra magnetic field. The drug slow release property of the liposomes has been demonstrated in vitro. In addition, it has been reported that small liposomes (≤ 200 nm) are likely to accumulate between different cell types in the liver (hepatocytes or Kupffer cells) due to their ability to cross the vascular endothelium and largely to avoid the first phagocytes encountered49,50. Then, the extra magnetic field has also decreased RES effect of Res-lips@Fe3O4 except offering magnetic targeting.

Figure 11. Plasma Res concentration-time curves. Data were expressed as mean ±S.E. (n = 3). Table 2 Plasma pharmacokinetic parameters of different Res formulations treated rats Formulations

AUC (μg mL-1h-1)

t1/2 (h)

Cmax (μg mL-1)

Free Res

26.25±1.5

7.98±0.67

3.23±0.15

Res-lips@CTS

49.67±1.77*

13.28±0.72

4.38±0.12*



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Res-lips@Fe3O4

57.46±1.46*

13.55±0.83

4.85±0.16*

Res-lips@Fe3O4+MF

73.48±2.04*#

14.81±0.83*

5.75±0.2*#

Data were expressed as mean ±S.E. (n = 3). *p < 0.01 vs Free Res; #p < 0.01 vs Res-lips@Fe3O4. Tissue biodistribution assay To explore the tissue distribution and site accumulation, the Res concentrations in brain

and

major

organs

were

measured

in

different

Res-treated

rats.

In

Res-lips@Fe3O4+MF group, the organs of the reticuloendothelial system (i.e., liver and spleen) captured large amounts of liposomes (Figure 12), which is consistent with the current research19. Regarding brain accumulation, a higher content of Res (7100 ± 490 ng/g) was found in Res-lips@Fe3O4+MF group than Res-lips@Fe3O4 group (4500 ± 470 ng/g), so the magnetic Res liposomes can effectively traverse the BBB to accumulate in the brain target site and enhance the therapeutic effect of PD under the external magnetic field.

Figure 12. Res concentrations in different tissues after 4 h of Res-treatment. Data were


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expressed as mean ± S.E. (n = 3). The change of brain drug content was studied to evaluate sustainability of magnetic nanocarrier. The Res content in brain was determined at 1, 2, 4, 8, 12 and 24 h after last injection of different Res formulations. In Res-lips@Fe3O4+MF group, besides the higher Res concentration in brain, the Res also lasted a long time in the brain with high concentration (Figure 13). The results showed that after Res encapsulated in magnetic liposomes, the duration of drug effect in brain was also extended, which demonstrated the sustainability of Res-lips@Fe3O4.

Figure 13. Brain Res concentration-time curves. Data were expressed as mean ±S.E. (n = 3). Systemic toxicity investigation Different Res formulations (0.5 mg/kg/day) were injected intraperitoneally (ip.) once a day for 35 consecutive days, and no death and abnormal behaviors were observed in rats. There was no difference in body weight between the treatment groups and sham group (Figure 14a), and also no significant difference in major organ indexes, including



the heart, liver, spleen, lungs, and kidneys (Figure 14b). The results verified that the Res-lips@Fe3O4 had not obvious toxic side effects.

Figure 14. A) Body weight changes of Res- treated rats; B) Organ coefficients of Restreated rats. Data were expressed as mean ± S.E. (n = 3). In summary, a novel efficient drug delivery system for PD therapy was designed and fabricated based on magnetic responsive delivery by mild and green method. The Res-lips@Fe3O4 can traverse rapidly BBB and accumulate at the lesions of brain to achieve the efficient treatment of PD rats. At the same time, the nanocarrier exhibits high drug capability, stability, sustained drug release and strong magnetic targeting. The magnetic Res liposomes can effectively prevent the drug leakage before reached the target site showing a significant slow release property. Their FA and T2 relaxation time measured by MRI demonstrated the Res-lips@Fe3O4 possessed the superior therapeutic effect, meanwhile, body weight and organ indexes also hinted the nontoxicity of system. Therefore, the Res-lips@Fe3O4 provides a meritorious platform focusing PD therapy. Associated Content Supporting Information Materials and instruments, synthesis of Res-liposomes, details of


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characterization experiments. Additional experimental data in Figure S1-S4. Stereotaxic injection, TEM image and magnetic responses properties. Notes The authors declare no competing financial interest. Acknowledgments This work was funded by the National Natural Science Foundation (No. 21476190, 21776238), the Hebei province key basic research Foundation (No.15961301D) and Hebei Province higher education science and technology research Project (No. ZD2017084). Reference 1.

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For Table of Contents Use Only

Synopsis: The preparation process of slow release magnetic targeting drug nanocarriers for Parkinson's disease therapy.


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Magnetic Resveratrol Liposomes as a New Theranostic Platform for

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