Resveratrol and Puerarin loaded polymeric nanoparticles to enhance the chemotherapeutic efficacy in spinal cord injury

Spinal cord injury includes inflammation and apoptosis of neurons, which is difficult to cure by systemic drug administration. Administration of natural active compounds (resveratrol and also Puerarin) by advance drug delivery technology improves the patient’s conditions. Oil-in-water emulsion method was utilized to prepare resveratrol as well as puerarin loaded PLGA nanoparticles. The nanoparticles were subjected to mean zeta potential, mean particle size, encapsulation efficiency as well as in vitro drug release studies. The biochemical parameters i.e. malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), advanced oxidation products (AOPP), catalase (CAT) and nitrite/nitrate levels were tested for the loaded nanoparticles. Reperfusion injury induced rats treated with 10 mg/kg resveratrol and puerarin loaded nanoparticles protects spine from ischemia injury and supports biological parameters. The mean particle size varies from 238 nm to 274 nm and also particle size distribution was mono-dispersed (0.239 to 0.318). Zeta potential value of nanoparticles was observed to be −12.6 ± 2.1 mV. Optimized nanoparticles reveals 72% -79% of drug release over 36 h by diffusion mechanism. Significantly, lowers the levels of plasma nitrite/nitrate level as well as phosphorylation of p38MAPK pathways in reperfusion injury induced rats. The resveratrol and puerarin loaded nanoparticles decreases free radicals produced by reperfusion injury induced rats, as well as decrease of oxidative stress because of IRI. Resveratrol and puerarin loaded nanoparticles decreases GSH, SOD and CAT antioxidant level, which helps in overall health improvement of patients.


Introduction
Spinal cord connected with brain by peripheral tissue and nervous system (Baumann et al. 2010). Spinal cord primarily comprise of neurons, glial cells, astrocytes and oligodendrocytes cells. Spinal neurons, poses cutaneous sensory input are mainly located in the dorsal spinal cord and interconnected in circuits; while neurons responsible for motor control are located in ventral spinal cord. The loss of neurons and axonal degeneration occurs at the lesion site due to spinal cord injury. In some cases severe functional impairment, paraplegia, or tetraplegia may occurs due to motor deficits, loss of sensory inputs and chronic pain (Baumann et al. 2010;Bremner et al. 2009). Secondary spinal cord injury is a degenerative response that includes inflammation, ionic imbalance, excitotoxicity, neurotransmitter accumulation and apoptosis (Conti et al. 2007;Crowley-Weber et al. 2003). The severity of the secondary injury response slowly to systemically administered drug within therapeutic window.
Pain relief, spasticity treatment, rehabilitation and prevention of complications are most focus area in long term therapy for spinal cord injury. The glial scars are developed in the trauma region, which limits the regenerative capacity of central nervous system. (Chou 2010, Mohanty et al. 2010a, Bhaumik et al. 1999. Special methods are required to reconstruct grey and white matter of CNS, which helps in promote axonal growth and reduce scar formation. (Terada et al. 2001). The nanoparticles facilitate passage through the blood-spinal cord barrier and extend therapeutic delivery and/or imaging in the brain and spinal cord to protect it from toxins.
Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenol has antioxidant and anti-inflammatory activity (Frémont 2000;Song et al. 2013). Resveratrol helps in free radical scavenging, increases the antioxidant enzymes, promote neuroprotection and neutralizes oxidative stress. Puerarin (Daidzein-8-C-glucoside) is the major bioactive ingredient in kudzuroot (Terada et al. 2001). Many researchers show that Puerarin has anti-apoptotic, anti-oxidant (Misra and Fridovich 1972), anti-inflammatory (Bremner et al. 2009), anti-arrhythmic (Baumann et al. 2010), and anti-fibrosis (Pekarkova et al. 2001) activity. These therapeutic effects of Resveratrol and Puerarin have much useful for treatment of spinal cord injury (Takahara et al. 1960). The major constrains for oral administration of Resveratrol and Puerarin orally is its short half-life, in which it is rapidly metabolized by first pass metabolism in liver. The nanocarriers useful for significant prolong residence time of the dosage; improve the efficacy of drugs as potential treatment of spinal cord. The main objective of this work was to evaluate the capability of Resveratrol and Puerarin encapsulated in Poly (DL-lactic-co-glycolic acid) nanoparticles as treatment to reduce the oxidative stress in spinal cord injury (Mohanty and Mohanta 2014). PLGA was used due to its biocompatible, biodegradable and optimum mechanical strength for controlled drug release system.

Materials
Resveratrol (RES) and Puerarin (PUE) purchased from Sigma Aldrich (St. Louis, MO, 454 USA). Poly(lactic-co-glycolic acid) with 50:50 ratio was purchased from Sigma-Aldrich. Polyvinyl alcohol, 6 kg/mol and 80%mol hydrolyzed, was purchased from Polysciences Inc. (Warrington,USA). Acetonitrile (HPLC grade) was obtained from BDH Laboratory Supplies (Pools, UK). All chemicals were of analytical grade and were used as received. DMSO, ethylene diamine tetraacetic acid (EDTA) were purchased from Merck co. (Darmstadt, Germany). All other chemical regents were of analytical grade. All buffers were made with ultrapure water collected from Millipore at 18 MΩ resistances (Millipore, Bedford, USA).

Animals
Male wistar albino rats weighing 210-250 g were purchased and acclimatised to laboratory environment by exposed them to free access of food and water for 24 h. The rats were treated with resveratrol & Puerarin, RES-PUE loaded nanoparticles through intravenous route. Prior to run the animal experimentation, all the procedures were taken to account for consideration and supervision by the Animal ethical clearance committee.

Preparation of RES-PUE-PLGA nanoparticles
Oil-in-water emulsion techniques were used for the drug loading in PLGA nanoparticles and PVA used as co-solvent to stabilized the emulsion. RES-PUE loaded nanoparticles were prepared with aqueous outer phase (PVA and NaCl) covers the inner organic phase of PLGA (0.25 dL/g), Resveratrol (RES) and Puerarin (PUE) in DCM. Micrio-tip probe sonicator at 39 W (VCX750, Sonics and materials Inc., USA) was use as energy source for emulsion preparation over ice bath for 20 s. The nanoparticles formed in emulsion were stabilized by addition of 40 mL of PVA solution (25 mg/mL) and stirred for 18 h at room temperature. The RES-PUE nanoparticles were collected by ultracentrifugation at 115058×g for 20 min at 4°C (Sorvall-WX, ThermoFisher Scientific, USA), washed thrice with ultrapure water to remove free drugs and unbound PVA, lyophilized for 2 days (−80°C and < 10 μm mercury pressure, Lab-conco Lyophilizer, USA) to get the powdered NPs.
To prepare an optimum dosage form, with surplus entrapment, the impact of drug to polymer ratio was studied. Herein, all the variables were found to be constant except that the drug to PLGA ratio showed variations by 20, 30, 40, 50 and60% (w/w) on dry weight basis.

Characterization of resveratrol and Puerarin loaded nanoparticles (RES-PUE nanoparticles)
RES-PUE loaded nanoparticles synthesis was optimized for Drug-polymer ratio, and thus encapsulation and thus encapsulation efficiency varied. Yield of nanoparticle was calculated from the weight of PLGA nanoparticles recovered, divided by the initial weight of PLGA taken for formulation (Cruise et al. 1998;Ohkawa et al. 1979).

Drug encapsulation efficiency (DE)
Encapsulation efficiency was calculated from the ratio of theoretical drug content to fraction of drug detected per unit mass of particle. RES-PUE loaded nanoparticles sample (100 mg) was dissolved in 1 mL of HPLC grade methanol in a microcentrifuge tubes. The solution was mix for 5 min by vortexing (MX-S, Vortex mixer, Biobase) followed by centrifugation (BKC-TH20RL, Biobase) at 115058×g for 20 min at 4°C to collect the supernatant. The collected supernatant was filtered through 0.2 μm syringe filter and 20 μl was injected to RP-HPLC (e2695 separation module, Water's Alliance, Milford, MA, USA). The area under the curve (AUC) and retention time (tR) of the peaks were recorded by extracting the chromatogram at 270 nm and 304 nm wavelength for Resveratrol and Puerarin respectively, using operating software Empower3 pro. Resveratrol and Puerarin encapsulation efficiency and loading capacity was calculated using regression equation obtained from concentration vs AUC of standard Resveratrol and Puerarin respectively.
2.6 Size of particles and zeta potential of RES-PUE loaded nanoparticles The particle size analyzer (Zetasizer, Malvern Panalytical, UK) based on dynamic light scattering technique used for average particle size determination. Zeta potential of nanoparticles shows surface properties of nanoparticles and it was measured by electrostatic or charge repulsion/attraction between particles (Nano ZS analyzer, Malvern Panalytical, UK). Briefly RES-PUE loaded nanoparticles sample solution was prepared with water and solution was placed in separate polystyrene cuvettes meant for particle size and zeta potential measurement.

Scanning Electron microscopy
Surface morphological evaluation of RES-PUE loaded nanoparticles werecarried out by scanning electron microscope (EVO, ZEISS Germany) for which lyophilized powder samples were spread on the carbon adhesive sample holder and coated with gold and used in scanning electron microscope (Mohanty and Mohanta 2014).

Atomic force microscopy
Atomic force microscope (AFM) (Axio Observer Z1, Carl Zeiss Microscopy GmbH) was used for visualization of individual as well as groups of particles. It offers visualization in three dimensions, unlike other microscopy techniques. The samples were dispersed in optimum quantity of deionized water by sonication. Small amount of the dilution were put on a cleaned mica plate and incubated to dryness by a stream of nitrogen. This method was followed for all samples.

Fourier transmission infrared spectroscopy (FTIR) analysis
The chemical integrity of the polymer and drug was analysed via Fourier Transmission Infrared Spectroscopic technique (SPECTRUM RX I, Perkin Elmer, USA). Approximately 2 mg of native Resveratrol, Puerarin and RES-PUE loaded nanoparticles samples were mixed separately in 300-400 mg of anhydrous KBr and ground properly in a mortar pestle. The sample mixture was compressed by applying hydraulic pressure of 2000 Kg/ cm 2 (Jasco MP2 mini press) for 2 min. The FTIR spectrum was obtained by scanning all samples under 2 cm −1 resolution power in 4000-400 cm −1 spectral (Mohanty et al. 2010b).

Differential scanning Calorimetry
The physico-chemical properties and drug polymer compatibility of the drugs in the polymer were analysed via Differential Scanning Calorimetric technique (DSC-60, Shimadzu, Japan). The samples RES-PUE loaded nanoparticles, native Resveratrol native Puerarin (2-4 mg) were sealed separately in standard aluminium pans and scanned 10°C/min rate of heat supply at a temperature ranging from 50 to 350°C with continuous nitrogen gas flow of 65 mL/ min (Mohanty et al. 2010a(Mohanty et al. , 2010b.

2.11
In-vitro drug release study RES-PUE loaded nanoparticles, native Resveratrol and Puerarin (each containing 5 mg of Resveratrol and Puerarin) were kept in two separate hermetically sealed dialysis bags (12000-14,000 Da, Sigma-Aldrich). The dialysis bags were hanged in a beaker that was filled with saline buffer of phosphate, maintained at pH = 7.4 (100 mL) as dissolution medium maintained at 37 ± 2°C with continues stirring at 0.93×g. The entire assembly was covered with aluminium foil to prevent loss of dissolution media. 1 mL of samples was collected at specified time intervals. The release media was replenished with 1 mL of fresh dissolution media after every sample withdrawal. The collected samples were subjected to centrifugation at the rate of 13,800 rotations per minute for 30 min and the supernatant was lyophilized (Lab-conco Lyophilizer, USA) for a day. Dissolution was affected by dissolving the lyophilised, dried powder in methyl alcohol and this was analysed via RP-HPLC technique. The entire analysis was repeated thrice (Muthukkumar et al. 1995).

Instigation of ischemia-reperfusion injury in spinal cord
Induction of ischemia in spinal cord according previously published article. Mixture of ketamine (50 mg/kg) and xylazine (5 mg/kg) used as interperitoneal injection to anesthetize rats. Small aneurysm clip was placed between left kidney arteries and cross clamping used to cause ischemia. After surgical treatment animals were preyed on a common diet plan as well as water ad libitum. All animals were anesthetized and give up after 48 h of ischemia-reperfusion treatment. The 3, 4, 5 lumbar segments of spinal cord and blood samples were collected.

Nitrite/nitrate levels in blood plasma
The The UV-Vis spectrophotometer is used for determination of enzymatic conversion of nitrate to nitrite by nitrate reductase at 540 nm. This shows nitrite/nitrate levels based on Griess reaction.

Western blot analysis
Total protein was extracted from tissues of treated and controlled group by utilizing a lysis buffer consisting of 1 M Tris-HCl (pH 7.5), 1 ml 0.5 M EDTA, NP-40 (1% v/v), Trizma base with protease inhibitor. The protein concentration of the supernatant was identified making use of BCA as well as kept at −80 C. The equivalent quantity (50 lg/ well) of proteins were divided in 10% SDS-PAGE, moved to a nitrocellulose membrane layer. The membrane layers were nurtured overnight at 4°C with key antibodies of iNOS, p-p38, overall p38 and β-actin. The equivalent amount of IgG antibodies in alkaline phosphatase were used for incubation for 2 h at RT. The membrane layers were established utilizing ECL reagent and protein signals evaluated by ChemiDoc after being normalizd to cognate β-actin signals.

Quantification of advanced oxidation products (AOPP) and malondialdehyde (MDA)
AOPP and MDA in lipid peroxidation were determined by following the reported method of Ohkawa. Briefly, Thiobarbituric acid (TBA) and trichloroacetic acid (TCA) was used for preparation of homogenates and absorbance was recorded at 532 nm. Addition of MgCl 2 and phosphor tungstate to tissue homogenates helps in determining AOPP in the supernatant at 340 nm absorbance read in acidic conditions. The level of AOPP and MDA was shared as nM of MDA or AOPP reactants/100 g of wet tissues.

Quantification of reduced glutathione (GSH)
The elevated levels of glutathione were determined by previously reported method of Ellman. The tissue homogenates were combined with 5% TCA and 0.3 M disodium hydrogen phosphate. Add 1.0 ml of Ellman's reagent to the homogenates mixture. Record the absorbance of sample against blank containing TCA at 420 nm. The quantity of glutathione is expressed as mg/g of protein.

Quantification of superoxide dismutase (SOD) activity
The SOD of samples was determined following by reported method by Misra and Fridovich. Briefly, 1.8 mM epinephrine was used for initiation of the response as well as absorbance read at 480 nm. Non-polar solvents were added in tissue homogenates followed by 0.6 mM EDTA solution as well as 0.1 M carbonate buffer solution. The superoxide dismutase activity revealed as 50% inhibition of epinephrine in one minute/mg protein.

Quantification of catalase (CAT) activity
The CAT activity was performed by previously reported method by Takahara. Briefly, the tissue homogenate was mixed with 50 mM phosphate buffer and absorbance was recorded by the addition of 30 mM H 2 O 2 solution at 240 nm. The enzyme activity was revealed as l moles of H 2 O 2 decomposed/min/mg protein.

Statistical analysis
Analyses were performed in triplicate (n = 3) and the outcomes were expressed as mean ± SD. For the statistical analysis the experimental data was tested by on-way analysis of variance (ANOVA). The values of p < 0.05 were characteristic of statistical significance.

Loading capacity and drug entrapment efficiency
The drug loading and entrapment efficiency of PLGA nanoparticles is depends on the solvent affinity of drug, polymer and surfactant. It's previously reported that PLGA have encapsulation efficiency of~82%w/w. We have prepared five different RES-PUE loaded nanoparticles formulations taking different ratio of drug, PLGA and keeping same PVA content (Chou 2010;Mohanty et al. 2010a). The RES-PUE loaded nanoparticles fabricated with a ratio of 1:3 drug-polymer shows, higher entrapment efficiency of 74.85% and loading capacity of 15.50% (Table 1).

Particle size and zeta potential of RES-PUE loaded nanoparticles
The photon correlation spectroscopy is one of the easy and rapid method used for measurement of particle size. In RES-PUE loaded nanoparticles the mean particle size was ranges from 238 nm to 274 nm and particle size distribution was mono-dispersed (0.239 to 0.318) (Fig. 1a). The stability of RES-PUE loaded nanoparticles in water can be attributed from single peak at particle size distribution plot (Chou 2010;Mohanty et al. 2010b). Polymers and additives used in formulation influence the zeta potential of particle surface charge. Zeta potential of RES-PUE loaded nanoparticles was found to be −12.6 ± 2.1 mV (Fig. 1b).

Scanning Electron microscopy
Scanning electron microscopic studies were performed for surface morphology and particle size measurement of nanoparticles. The SEM images of particles shown in Fig. 1c, obtained directly by visualization through scanning electron microscope, so it appears smaller than the hydrodynamic layer obtained by Zetasizer.

Atomic force microscopy
Three dimensional surface morphology studies of the RES-PUE loaded nanoparticles were carried out by AFM on cleaned Si substrates (Fig. 1d). The lateral sizes of the nanoparticles varies from image to image because of the variation in the tip shape (Dunne et al. 2000). The height of nanoparticle were varies from 9 to 11 nm.

Differential scanning calorimetry (DSC) studies
The physical state of drug incorporated in PLGA nanoparticles was determined by DSC studies. The DSC curve of native Resveratrol, Puerarin, PLGA and RES-PUE loaded nanoparticles were shown in Fig. 2a. The sharp endothermic peak at 262°C and 189°C in native Resveratrol and Puerarin respectively shows the melting point of native drug. The formulation mixture shows the endothermic peak at 183°C, that of PLGA. In addition it shows less intense peak of Resveratrol and  Puerarin, which may be due to surface bound drugs (Mohanty et al. 2010a;Sahoo et al. 2004).

FTIR spectroscopy
The FTIR spectrum of Resveratrol, Puerarin, PLGA and RES-PUE loaded nanoparticles were presented in Fig. 2b.
In addition peaks at 2441 cm-1 and 3406 cm-1 in both PLGA and RES-PUE loaded nanoparticles conforms the presence PLGA in the formulation. The presence of hydrogen bonding between hydroxyl groups of Resveratrol and carbonyl group of PLGA shifts O-H stretching band to higher wave number (Song et al. 2013;Mohanty et al. 2010b;Sahoo et al. 2004).

In vitro drug release studies
In the present study cumulative percentage release data of drug from native Resveratrol, Puerarin and RES-PUE loaded nanoparticles divulges sustained release action of formulation (Fig. 2c). The native Resveratrol and Puerarin shows 96% and 98% of drug release respectively in just 6 h of study, whereas the optimized formulation of RES-PUE loaded nanoparticles shows only 72% -79% of drug release over 36 h by diffusion mechanism.
3.2 In vivo study 3.2.1 Effect of RES-PUE loaded nanoparticles on plasma nitrite/nitrate levels RES-PUE loaded nanoparticles alter the circulating plasma nitrite/nitrate levels induced by reperfusion injury. The plasma nitrite/nitrate level in control group was less than the reperfusion injury induced group (Lafci et al. 2013). Administration of RES-PUE loaded nanoparticles by i.v. route decreases the nitrite/nitrate level in plasma of reperfusion injury induced rats (Fig. 3a). However, the control group rats did not show any change in nitrite/nitrate levels in plasma even after treated with RES-PUE loaded nanoparticles.

Effect of RES-PUE loaded nanoparticles on protein expressions
Effect of RES-PUE loaded nanoparticles on IRI-induced, iNOS protein expression was measured by western blot. The protein (Fig. 3b) expression was not altered in control, RES-PUE loaded nanoparticles and surgery groups (Lemmon et al. 2014). The increased level of iNOS protein expression was normalizes after treatment with RES-PUE loaded nanoparticles in reperfusion injury induced rat groups.

Effect of RES-PUE loaded nanoparticles on p38MAPK pathway
Effect of RES-PUE loaded nanoparticles on reperfusion injury induced group was studied by activation of p38MAPK pathway indicating p38MAPK phosphorylation. As shown in Fig.  4, the p38MAPK phosphorylation did not alter in control (Ohkawa et al. 1979). But, p38MAPK was distinctly phosphorylated in reperfusion injury induced group treated with RES-PUE loaded nanoparticles.

Effect of RES-PUE loaded nanoparticles on malondialdehyde and AOPP
The level of Malondialdehyde (Fig. 5a) and AOPP (Fig. 5b) formed from lipid peroxidation/ oxidation was used to investigate the effect of RES-PUE loaded nanoparticles (Ellman 1959). The reperfusion injury induced groups treated with RES-PUE loaded nanoparticles shows decreased level of Malondialdehyde and AOPP as compared to control group.

Effect of RES-PUE loaded nanoparticles on GSH, SOD and CAT antioxidant activities
The GSH non-enzymatic antioxidant activity level (Fig. 6a) alters in RES-PUE loaded nanoparticles treated reperfusion injury induced group (Takahara et al. 1960). Similarly, the SOD (Fig. 6b) and CAT (Fig. 6c) enzymatic level didn't alter in control group, but significantly increases in RES-PUE loaded nanoparticles treated reperfusion injury induced group (Misra and Fridovich 1972).

Discussion
The increase in Drug to polymer ratio increases the encapsulation efficiency. It was observed that increase in the drug to PLGA ratio from 20% to 40% increases the drug encapsulation efficiency of nanoparticles. The increases in drug loading saturate the surface matrix if the PLGA nanoparticles, which favour the leaching, process of drug. So, the formulation with 1:3 drug to PLGA ratio was selected and used for all further studies.
The higher zeta potential value of the particles shows higher stability of nanoparticle formulations. Negative charge can be attributed tothe presence of free carboxyl groups of PLGA on the nanoparticle surface (Chou 2010;Mohanty et al. 2010a). The projected PEG chains of PVA prevent aggregation of the particle despite of lower zeta potential. The SEM and AFM image of RES-PUE loaded nanoparticles was found to be spherical in shape with smoother surface (Mohanty et al. 2010b;Mohanty and Mohanta 2014). The DSC and FTIR studies also reveal that Resveratrol and Puerarin were present as molecular dispersion form in the PLGA matrix.
In vitro drug release studies shows that the formulation shows a burst release of drug at initial stage (0.25 h and 0.5 h), attributes the presence of surface bound drugs. Low melting point and hydrophobic nature of PLGA provides better sustained release action on solidification (Mohanty et al. 2010a;Sahoo et al. 2004). In vivo studies shows no change in nitrite/nitrate levels and increased level of iNOS protein expression in plasma after treated with RES-PUE loaded nanoparticles. The reperfusion injury induced group treated with RES-PUE loaded nanoparticles shows decrease in p38 phosphorylation, Malondialdehyde and AOPP level as compared to control group.
Cytokines production and neutrophils activation causes loss of membrane lipid functions (Sa and Das 2008), which generates free radicals and results in cell death (Mackay et al. 2006). The free radical causes reoxygenation and changes the pathology of neurologic dysfunction in reperfusion injury induced group. The group treated with 10 mg/kg of RES-PUE loaded nanoparticles protects spinal cord from ischemia.
Oxidative stress induced in injured spinal cord tissues due to over production of nitric oxide. Similarly, several studies have been focused on inhibition of p38MAPK pathway by suppressing oxidative stress in reperfusion injury induced group (Conti et al. 2007;Maggio et al. 2012). Significant decrease in p38MAPK expression and plasma nitrite/nitrate levels were observed in RES-PUE loaded nanoparticles treated reperfusion injury induced group (Sgaravatti et al. 2008). The Malondialdehyde and AOPP levels were in creased in reperfusion injury induced group and it correlated with cytokines levels (Pekarkova et al. 2001).
The administration of RES-PUE loaded nanoparticles to reperfusion injury induced group significantly increases the antioxidant level i.e. GSH, SOD and CAT, which are acts as defence.

Conclusion
The multifunctional resveratrol and puerarin loaded nanoparticles were developed to enhance the oral bioavailability of resveratrol and puerarin. RES-PUE loaded nanoparticles protect spinal cord injured rat models. The RES-PUE loaded nanoparticle blocks the p38MAPK signalling pathway and expressed antioxidant effects. The further investigation may be acceptable to determine the efficacy of RES-PUE loaded nanoparticles on human spinal cord reperfusion injuries. Further, the work has to take for larger population which will allow us to get clearer picture in the data analysis.
Reperfusion injury induced free radicals were reduced by RES-PUE loaded nanoparticles treatment by increasing enzymatic and non-enzymatic antioxidant levels.
obtained prior to initiation of the research work on animals, and the experiments were performed in accordance with the animal ethical committee guidelines and regulations of Republic of China.