Long circulating PEGylated-chitosan nanoparticles of rosuvastatin calcium: Development and in vitro and in vivo evaluations

Accepted Manuscript Title: Long Circulating PEGylated-Chitosan Nanoparticles of Rosuvastatin Calcium: Development and in vitro and in vivo evaluations Authors: Mukund Hirpara, Jyothsna Manikkath, K. Sivakumar, Renuka S. Managuli, Karthik Gourishetti, Nandakumar Krishnadas, Rekha R. Shenoy, Jayaprakash Belle, Chamallamudi Mallikarjuna Rao, Srinivas Mutalik PII: DOI: Reference:

S0141-8130(17)32267-5 https://doi.org/10.1016/j.ijbiomac.2017.10.086 BIOMAC 8381

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

22-6-2017 8-10-2017 14-10-2017

Please cite this article as: Mukund Hirpara, Jyothsna Manikkath, K.Sivakumar, Renuka S.Managuli, Karthik Gourishetti, Nandakumar Krishnadas, Rekha R.Shenoy, Jayaprakash Belle, Chamallamudi Mallikarjuna Rao, Srinivas Mutalik, Long Circulating PEGylated-Chitosan Nanoparticles of Rosuvastatin Calcium: Development and in vitro and in vivo evaluations, International Journal of Biological Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.10.086 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Long Circulating PEGylated-Chitosan Nanoparticles of Rosuvastatin Calcium: Development and in vitro and in vivo evaluations

Mukund Hirpara1, Jyothsna Manikkath1, K Sivakumar1, Renuka S Managuli1, Karthik Gourishetti2, Nandakumar Krishnadas2, Rekha R. Shenoy2, Jayaprakash Belle3, Chamallamudi Mallikarjuna Rao2, Srinivas Mutalik1,*

1

Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal

University, Manipal 576104, Karnataka State, India. 2

Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal

University, Manipal 576104, Karnataka State, India. 3

Department of Medicine, Kasturba Medical College, Manipal University, Manipal 576104,

Karnataka State, India

* For correspondence: Dr Srinivas Mutalik Professor, Department of Pharmaceutics Manipal College of Pharmaceutical Sciences Manipal University, Manipal 576104 Karnataka State, India Email: [email protected] ; [email protected] Abstract: The aim of this study was to improve the pharmacokinetics and pharmacodynamics profile of rosuvastatin calcium by formulating long-circulating PEGylated chitosan nanoparticles (NPs). Chitosan was PEGylated by a carbodiimide mediated reaction, using a carboxylic acid derivative of PEG (polyethylene glycol). The NPs were optimised for particle size, polydispersity index, zeta potential and drug entrapment efficiency. In vitro drug release, pharmacokinetic and pharmacodynamics studies of the optimized nanoparticles were performed. PEGylation of chitosan was confirmed by FTIR analysis. Drug-excipient

1

compatibility was studied by differential scanning calorimetry and FTIR analyses. Two batches of nanoparticles were optimized with particle size of <200 nm and entrapment efficiency of ≈14%. In vitro drug release studies revealed cumulative release of 14.07±0.57% and 22.02±0.81% of rosuvastatin over the period of 120 h, indicating appreciable sustained release of drug. TEM analysis showed the spherical structure of nanoparticles. Pharmacokinetic studies indicated that optimized NPs showed prolonged drug release over a period of 72 h. Pharmacodynamics studies in hyperlipidemic rat model demonstrated greater lipid-lowering capability of rosuvastatin nanoparticles in comparison with plain rosuvastatin. The nanoparticles demonstrated substantial prolonged delivery of the drug in vivo along with better therapeutic action, which could be potential drug delivery modality for ‘statins’.

Keywords: Rosuvastatin; Chitosan; Nanoparticles; PEGylation; Atherosclerosis

1. Introduction: Rosuvstatin calcium, is a widely used antihyperlipidemic drug for the treatment of atherosclerosis [1]. Atherosclerosis is a chronic inflammatory condition characterised by deposition of lipids and fibrous elements in the large arteries [2]. This condition is the prime cause of heart disease and stroke and is triggered by a mixture of genetic and environmental factors. The key initiator in the etiology of this disease is the accumulation of low density lipoprotein (LDL) in the matrix of the subendothelium. Rosuvastatin competitively inhibits the enzyme HMG CoA reductase, preventing the conversion of 3-hydroxy-3-methylglutaryl CoA to mevalonate and the production of cholesterol and its circulating blood derivatives, including LDL [3]. The oral bioavailability of this agent is around 20% due to extensive first pass metabolism and higher doses are associated with increased incidence of rhabdomyolysis, proteinuria, hematuria and increased serum creatinine. Additionally, it has low aqueous 2

solubility and its rate of absorption depends on the rate of its dissolution in the gastro-intestinal tract (GIT). Its half-life is around 20 h, necessitating daily administration of this drug [4]. Therefore, this study was aimed at developing long-circulating rosuvastain nanoparticles, which would lower the frequency of administration of this drug. Chitosan is a natural, non-toxic, β-1,4-linked glucosamine polysaccharide, primarily obtained from the exoskeletons of crustaceans. Chitosan has been widely explored as a prospective carrier for the delivery of bio-actives due to its biocompatibility and biodegradability [5]. It has been used to achieve sustained and controlled delivery of therapeutic agents. Additionally, chitosan has been reported to possess the property of reducing the cholesterol levels, which may make chitosan as an attractive polymer in the development of drug delivery systems for the treatment of cardiac diseases [6, 7]. In this study, we sought to formulate nanoparticles of rosuvastatin calcium using PEGylated chitosan as the matrix, for improving the biopharmaceutical profile of this drug. PEGylation refers to the modification of biological molecules including drugs, delivery vehicles, proteins and other macromolecules by their covalent conjugation with polyethylene glycol (PEG) [8]. PEG is a non-toxic and non-immunogenic polymer which alters the hydrophobicity of a molecule and improves its aqueous solubility. Moreover, it increases stability, decreases proteolysis and renal excretion, thereby reducing the dosing frequency and improving the pharmacokinetics. PEGylation also improves the efficacy and safety of the therapeutic agent. PEGylation is especially useful for formulating therapeutic agents that are intended for chronic delivery, where a single dose of formulation can produce its effect till a long duration of time [8, 9]. It has been reported that PEGylation prevented the aggregation of chitosan nanoparticles loaded with methotrexate and insulin; additionally PEGylation reduced the recognition of the nanoparticles by the macrophages thus allowing them to circulate continuously in the blood for a longer period of time as compared to chitosan nanoparticles [9,

3

10]. Hence in this work, PEGylated chitosan nanoparticles loaded with rosuvastain calcium were prepared and evaluated.

2. Materials: Rosuvastain calcium (RC) was a kind gift from Orbicular Pharmaceutical Technologies,

Hyderabad,

India.

Chitosan

Dimethylaminopropyl)-N′-ethylcarbodiimide

(medium

hydrochloride

molecular (EDC)

weight), and

N-(3-

N-hydroxy

succinimide (NHS) were purchased from Sigma Aldrich, St Loius, USA. Polyethylene glycol (PEG) was procured as methoxy poly(ethylene glycol) succinimidyl valerate (mPEG SVA; MW 5000) from Laysan Bio Inc., USA. Methanol and acetonitrile were of HPLC grade and procured from Finar, Mumbai, India. All other reagents and chemicals used were of analytical grade.

3. Methodology: 3.1. Analytical methodology for RC: Analysis and quantification of RC was performed by reverse phase HPLC. The HPLC system (LC-2010 CHT, Shimadzu, Kyoto, Japan) configured with variable wavelength UV detector, quaternary low pressure gradient pumps, column oven, high throughput auto sampler and LC solution software for data analysis. Separation was obtained using an octadecylsilane (C18) analytical column (250 × 4 mm) with particle size 5µm (Genesis, UK), maintained at 25 °C. Acetonitrile and phosphate buffer (pH 3.0±0.05) mixture (50:50) was used as the mobile phase at a flow rate of 1 mL/min and the injection volume was 20 μL. The detection wavelength was 243 nm. The method was validated for linearity, accuracy and precision. The developed analytical method showed a retention time of 6.8 min for RC. The method was found to exhibit

4

linearity in the range of 10-20,000 ng/mL (R2 > 0.999), accuracy (98.5 -101.0%) and precision (RSD < 1.0% for inter- and intraday precision). 3.2. Preparation of PEGylated chitosan: Chitosan was grafted with PEG using a carboxylic acid derivative of PEG (mPEGSVA) by a carbodiimide-mediated reaction [11, 12]. Chitosan (50 mg) was dissolved in 1% v/v acetic acid solution. After obtaining a clear solution, the pH of this solution was adjusted to 6.3 by addition of 0.1 M NaOH solution. To this solution mPEG-SVA (10 mg) and NHS (1 mg) were added. This was followed by addition of EDC (14 mg). The reaction mixture was kept for stirring for 24 h and the resultant solution was dialysed through a dialysis tubing (10,000 MWCO, Sigma Aldrich, USA). The resulting solution was frozen at -80 °C for 8 h and then loaded into the lyophilizer (Daihan Labtech, Namyangju, South Korea). The sample was subjected to freeze drying at -40 °C over a period of 48 h to obtain the modified chitosan in powder form. PEGylation of chitosan was confirmed by FTIR spectroscopic analysis of modified and unmodified polymer [13]. The FTIR spectra of the unmodified and modified chitosan were recorded in the region of 4000 to 400 cm-1 using FTIR 8300 (Shimadzu, Kyoto, Japan) [14]. 3.3. Preparation of nanoparticles: Ionotropic gelation method followed by homogenization and ultrasonication was used for preparing the nanoparticles [15, 16]. PEGylated chitosan was dissolved in acetic acid (1% v/v) to yield a concentration of 2.5% w/v. RC was added to chitosan solution and and the mixture was homogenized at 18,000 rpm for 15 min. During homogenization, a solution of tripolyphosphate (TPP; 2.5% w/v in Milli-Q water) was added drop-wise (0.3 mL/min) to the mixture. Immediately after homogenization, the dispersion was subjected to probe sonication (80 amplitude; pulse 6 sec) for 40 min. The dispersion was maintained on an ice bath to prevent unwarranted rise in temperature. The dispersion was then centrifuged at 20,000 rpm and 4 °C

5

for 45 min to separate the nanoparticles in pellet form. The nanoparticles obtained as pellet were re-dispersed in phosphate buffer of pH 7.4. Different batches of nanoparticles (F1 to F9) were prepared by varying the polymer: TPP and polymer: drug ratios and sonication parameters. The nanoparticles were evaluated for particle size, polydispersity index (PDI), zeta potential and entrapment efficiency. 3.4. Characterization of nanoparticles: Particle size and polydispersity index were determined by Dynamic Light Scattering technique using a ZetaSizer (Nano ZS, Malvern Instruments, UK), equipped with HeNe laser (5mW, 633 nm) as the light source. This technique measures time-dependent fluctuations in the intensity of scattered light as a result of Brownian motion exhibited by the particles. Zeta potential was also determined in the ZetaSizer using a combination of Phase Analysis Light Scattering (PALS) and Laser Doppler Velocimetry (LDV). The nanoparticle formulations were diluted with Milli-Q water and measurements were made in triplicate at 25 °C [17]. For determining the entrapment efficiency, the nanoparticles were lysed in a solution of 1% v/v acetic acid and analysed using HPLC [18]. Briefly, to 100 μL of the nanoparticle suspension, 900 μL of acetic acid solution was added and vortexed. This mixture was then filtered through 0.22 μm syringe filter, diluted and analysed using HPLC. The entrapment efficiency was calculated using the following equation: Percentage entrapment =

Calculated amount of drug in dispersion X 100 Theoretical amount of drug added

------------- Eq. (1)

Shape and surface morphology of the optimized batch of nanoparticles were observed using TEM (Phillips, CM200, Netherlands) by staining the sample with a 2% w/v solution of phosphotungstic solution (pH 6.0). A drop of the formulation was placed on a carbon-coated copper grid. Then the film was negatively stained using the phosphotungstic acid solution and the grid was dried. Thereafter the sample was observed under TEM [19]. 3.5. In vitro drug release testing of nanoparticles: 6

In vitro release of the optimized RC nanoparticles was performed using membrane diffusion method [20]. Vertical type diffusion cells with diffusion area of 1.33 cm2 and receptor compartment capacity of 3.5 mL were used in the study. The donor compartment was kept in contact with the receptor compartment placing a hydrated dialysis tubing (Sigma Aldrich, USA) in between them. This was maintained at room temperature. Nanoparticle formulation (1 mL, containing 0.5 mg of drug) was loaded in the donor compartment. The receptor compartment was filled with 3.5 mL of phosphate buffer pH 7.4 and the entire setup was maintained on a magnetic stirrer at 300 rpm. Samples (0.5 mL) were withdrawn from the receptor compartment at time intervals of 1, 2, 3, 4, 6, 8, 10, 12, 24, 30, 36, 48, 60, 72, 96 and 120 hours. An equal volume of phosphate buffer pH 7.4 was replaced each time into the receptor compartment. The amount of RC released was determined by HPLC.

3.6. Solid state characterisation: Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared (FTIR) analysis were used to determine the compatibility of drug and excipients. X-Ray Diffraction studies were used to detect any polymorphic changes in the drug after incorporation into the nanoformulation [21, 22]. DSC analysis was performed by using DSC-60 Plus (Shimadzu, Kyoto, Japan). The instrument comprised a calorimeter (DSC 60), flow controller (FCL 60), thermal analyzer (TA 60) and operating software TA-60 WS. The samples were placed in a sealed aluminum pan and heated under nitrogen flow (30 mL/min) at a scanning rate of 15 °C/min from 30 to 300 °C. Empty aluminium pan was used as reference. The heat flow as a function of temperature was measured for plain RC, physical mixture of RC + excipients (1:1) and optimized RC nanoparticles and the DSC thermograms were recorded. FT-IR analysis was performed as mentioned in section 3.2. The samples analysed were plain RC, physical mixture

7

of RC + excipients (1:1) and optimized RC nanoparticles. In X Ray Diffraction (XRD) studies, X-ray diffractograms of plain RC and lyophilized RC nanoparticles were obtained using X-ray diffractometer (Rigaku Miniflex 600). The instrument was operated at 15 mA current at 2θ wide angle. The 2θ values were interpreted for any polymorphic conversion in RC after incorporation in sthe nanoformulation. 3.7. In vivo studies: Pharmacokinetics studies Animals: Adult Wistar rats, 6-8 weeks old, weighing 180-220 g were used for the experiments. The animals were housed at 24 – 26 °C, exposed to a daily 12:12 h light: dark cycle and had free access to normal diet and water ad libitum. To reduce the stress associated with the experimental procedures, the rats were handled daily for one week prior to the experimentation. The animal experimental protocol was approved by Institutional Animal Ethics Committee, Kasturba Medical College, Manipal (Approval No.: IAEC/KMC/91/ 2015). The animals were divided into 2 groups and treated with either oral solution or intravenous nanoparticles. Group 1: Oral administration of RC solution (1 mg/kg of RC in phosphate buffered saline (PBS)) Group 2: Intravenous administration of developed nanoparticles (1 mg/kg of RC as nanoparticulate dispersion in PBS) The overnight fasted animals were anesthetized using a mixture of ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively) prior to drug administration [23]. At different time intervals after drug administration (0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 36, 48 and 120 h), ≈ 300 μL of blood was withdrawn by retro orbital puncture into heparinized tubes. The blood samples were immediately centrifuged in a refrigerated centrifuge (Sigma Laborzentrifugen GmbH, Germany) to separate the plasma and the latter was stored at -20 °C. The drug content in the plasma samples was determined by HPLC.

8

Plasma concentration vs. time curve was plotted and pharmacokinetic parameters were calculated using PK Solutions 2.0TM Software (Summit Research Services, Montrose, CO, USA). The pharmacokinetic parameters obtained with Group 1 were statistically compared with those obtained with Group 2 by unpaired t-test using GraphPad Prism software (GraphPad, San Diego, USA). 3.8.2. Bioanalytical method for estimation of RC in plasma: RC was estimated in plasma using RP-HPLC (section 3.1). Extraction of the drug from the plasma matrix was achieved by protein precipitation method using chilled acetonitrile. To an aliquot of plasma (250 μL), gliclazide solution (30 μL) was added as internal standard (IS) and vortexed for 2 min. Chilled acetonitrile (precipitating agent) was added and the mixture was again vortexed for 2 min. This was then centrifuged at 20,000 rpm for 10 min at 4 °C. The supernatant (0.5 mL) was collected and the solvent was evaporated using a nitrogen evaporator for 10 min. The residue was reconstituted in 0.5 mL of mobile phase mixture and again centrifuged at 20,000 rpm for 10 min. The supernatant (100 µL) was collected separately and injected into the HPLC. The analysis conditions were similar to those mentioned previously (section 3.1) but the injection volume was 80 µL. Retention time of RC was found to be 6.8 min and that of gliclazide was found to be 12.6 min. No interference was observed from the plasma matrix at the retention time of either RC or gliclazide. The bioanalytical method was validated with respect to linearity (R2>0.997 in the range of 10-1,000 ng/mL), precision (RSD: < 15%) and accuracy (RSD: < 15%). Recovery was found to be consistent and reproducible. 3.8.3. Pharmacodynamic Study: Adult Wistar rats were used for the study. The animals were divided into four groups of six animals each. The animals were fasted overnight and hyperlipidemia was induced in three of the groups using a single intraperitoneal injection of tyloxapol (300 mg/kg in 0.9% saline) [24].

9

Group 1: Untreated (Normal control) Group 2: Intraperitoneal administration of tyloxapol (Disease control) Group 3: Intraperitoneal administration of tyloxapol followed by oral administration of RC solution (1 mg/kg of RC) Group 4: Intraperitoneal administration of tyloxapol followed by i.v. administration of nanoparticle formulation (1 mg/kg of RC) After different treatments as shown above, around 500 μL of blood was withdrawn at different time intervals (2, 4, 6, 8, 10, 12, 24 and 48 h) by retro orbital puncture into blood handling tubes (Vacutainer, Becton Dickinson, NJ, USA). The blood samples were kept aside at room temperature for 30 min. The the serum was separated by centrifuging the blood samples at 2000 g for 10 min and the serum samples were stored at -20 °C until analysis for the levels of cholesterol and triglycerides. The serum cholesterol and serum triglycerides (TGs) were estimated in all the groups using in vitro diagnostic kits (Abcam, MA, USA). Statistical analysis was performed using GraphPad Prism software. Comparisons were made by one-way ANOVA (analysis of variance) followed by Dunnet’s post hoc-test to compare results with control. A ‘p’ value less than 0.05 was considered statistically significant.

4. Results and Discussion: 4.1. PEGylation of chitosan: PEGylation of chitosan can be identified by the use of spectral techniques like FTIR [13]. The IR spectra of mPEG-SVA (Fig. 1A) and chitosan (Fig. 1B) were compared with that of the PEGylated chitosan (Fig. 1C). The IR spectrum of mPEG-SVA depicts the free carboxylic group (C=O) of the PEG portion at 1737.92 cm-1 followed by peaks at 1350.22 cm1

and 1280.78 cm-1 which represent the aromatic N-O and C-N bonds of the valerate portion of

the above molecule. The IR spectrum of chitosan shows a broad peak at 3435.34 cm-1, which

10

represents –OH stretch. In the IR spectrum of the product (PEGylated chitosan), amide bond (-C=ONH2) formation is clearly indicated by the peaks at 1649.19 cm-1 and 1566.25 cm-1; also the C=O group of mPEG SVA at 1737.92 cm-1 has completely disappeared. Moreover, the disappearance of peaks at 1350.22 cm-1 and 1280.78 cm-1 indicates removal of the valerate portion of the mPEG-SVA and attachment of the -NH group of chitosan. The -OH group of chitosan (broad peak at 3435.34 cm-1), methylene group (peak at 2922.25 cm-1) and secondary amine group (3747.81 cm-1) were retained in PEGylated chitosan. Chitosan is a biopolyaminosaccharide cationic polymer that is obtained from the alkaline deacetylation of chitin. Being a natural polymer, it has the advantages of biodegradability, biocompatibility and nontoxicity. Moreover, the presence of reactive amino groups in the backbone of the chitosan structure allows it to chemically conjugate with several molecules, including PEG [25]. PEGylation results in increased aqueous solubility and stability, reduced renal clearance and subsequently prolonged plasma lifetime [9]. In the current study, we have used mPEG-SVA for the synthesis of PEG-chitosan. The PEGylation reaction includes interaction of the succinimidyl valerate portion with the amine group of chitosan to form a stable amide linkage. Addition of EDC and NHS activate the carbonyl group of mPEG-SVA. As the carbonyl carbon has positive charge due to dipole moment, the lone pair of electrons on the amino group of chitosan attaches to the carbon forming a new bond. During this, the NHS group departs from the parent structure being a bulkier group and this gives rise to a stable amide bond [26]. 4.2. Preparation and Optimization of Nanoparticles: Particle size and particle size distribution of nanoparticles are important parameters as they influence i) uptake of the nanoparticles into different cells and tissues, ii) rate of drug release from the nanoparticles, iii) rate of clearance of nanoparticles from vascular compartment and iv) stability of the formulation. Moreover, intravenous administration

11

necessitates the nanoparticles to possess low particle size [27, 28]. Here, ionotropic gelation followed by use of homogenization and ultrasonication was used to prepare the nanoparticles. Several batches of nanoparticles were prepared by varying the polymer: TPP and polymer: drug ratios and sonication parameters for the optimization with respect to particle size, polydispersity index (PDI) and entrapment efficiency, as outlined in Table 1. 4.2.1. Optimization of ultrasonication time: Ultrasonication is a widely used method in the preparation of nanoparticles [29-31]. It is effective in breaking up aggregates and decreasing the size and polydispersity index. Earlier studies [32] have shown that ultrasonication of chitosan nanoparticles does not modify the surface properties of chitosan or cause chemical changes. Initial experiments with ultrasonication parameters revealed that sonication time of 40 min with amplitude 80 and pulse 6 sec, produced particles in the desired size range. Hence amplitude of 80 and pulse of 6 sec were used in further batches. It was found that increase in the sonication time from 20 min to 40 min (Batches F1 to F3; Table 1) led to slight increase in the drug encapsulation efficiency from 3.2±0.56% to 5.2±1.02%. However, there was a slight increase in the particle size also from 306±10.87 nm (Batch F1) to 424±27.83 nm (Batch F3). Batch F2 with sonication time of 35 min showed the particle size of 411±23.44 nm. The increase in particle size could be due to the decrease in zeta potential of the particles when the sonication time was increased. Neither the polydispersity index nor the zeta potential was considerably altered. Therefore sonication time of 40 min was continued for further batches. 4.2.2. Optimization of RC:chitosan ratio: Three batches of nanoparticles were prepared by varying the drug content from 2.5 mg to 1.5 mg (Batches F3 to F5; Table 1), with the ratio of chitosan and TPP constant at 12:5. It was observed the decreasing the amount of drug led to decrease in entrapment efficiency (5.2±1.02% and 1.8±0.21% with 2.5 and 1.5 mg of RC, respectively) and increase in particle

12

size (424±27.83 and 539±37.09 with 2.5 and 1.5 mg of RC, respectively). Therefore, 2.5 mg of drug was considered to be optimum for incorporation in the RC nanoparticles. 4.2.3. Optimization of chitosan: TPP ratio: The process for nanoparticle preparation was ionotpropic gelation of chitosan using tripolyphosphate (TPP) as crosslinking agent. Chitosan is a cationic polyelectrolyte and has the tendency to gel upon contact with anions. The inter- and intra-molecular linkages formed between the positively charged amine groups of chitosan with the counter ion TPP are important factors in the process of gelation [15, 33]. The cross-linking of chitosan with TPP occurs ionically at low pH and by the mechanism of deprotonation at high pH. Ionically crosslinked chitosan shows higher swelling ability [34]. It was observed that as the amount of TPP increased from 2.5 mg to 12.5 mg (F6 to F9), the particle size decreased from 605±34.89 nm (PDI: 0.51±0.06) to 188±5.64 nm (PDI: 0.21±0.06). When the TPP quantity was further increased to 25 mg, it led to a slight increase in particle size, but it was not profound. This is in agreement with previous reports [15], where increase in the concentration of chitosan with respect to TPP ratio led to linear increase in size of nanoparticles. However, this is attributed primarily to the concentration of chitosan in solution; when the concentration of chitosan is lower, the viscosity of the resultant solution is also lower, which in turn leads to higher solubilisation of the chitosan in aqueous medium. This also causes increased protonation of the amino groups of the chitosan backbone, allowing greater interaction between chitosan and the anionic material, which in this case is TPP. In the present study, the concentration of the TPP solution was fixed (2.5% w/v), while the quantity of solution was varied to adjust for the quantity of TPP in the respective formulation. To achieve higher TPP quantity per batch, the amount of solution added was higher, which in turn resulted in greater dilution of the chitosan solution. This could be a causative factor for the size effect. Previous studies [15] found that the ideal chitosan:TPP ratio was 5:1 with respect to

13

achieving low particle size. However in our study, the least particle size was given by the chitosan:TPP ratio of ~1:1 (Batch F8, Table 1). The reason for this could be PEGylation, which might have altered the interaction capacity of the chitosan to TPP. It was also observed that increasing the quantity of TPP from 2.5 mg to 12.5 mg led to a near-linear decrease in zeta potential (from 29.8±0.13 mV to 6.47±0.06 mV with 2.5 and 12.5 mg of TPP, respectively). This could be a result of greater electrostatic interactions between positively charged chitosan and negatively charged TPP leading to lesser positive surface charge of the nanoparticles [35]. However, increasing the quantity of TPP beyond 12.5 mg led only to a slight decrease in zeta potential (3.53±0.07 mV), which could indicate saturation of the interactable groups. Another point noted was the increase in entrapment efficiency when the quantity of TPP was increased. This could be a result of better crosslinking between chitosan and TPP, which caused enhanced entrapment of drug in the polymeric matrix (14.1±1.87% with 25 mg of TPP) Out of all the batches executed, batches F8 and F9 were found to consistently produce nanoparticles with required properties viz., size (<200 nm), PDI (<0.3) and entrapment efficiency of around 14%. These two batches were studied for drug release studies. 4.3. In vitro drug release study: In vitro drug release from the nanoparticles was determined using the membrane diffusion method as described by D’Souza and DeLuca [20]. Considering the solubility studies and also the physiologic acceptance of pH 7.4, PBS pH 7.4 was selected as the donor and receptor medium. Batches F8 and F9, with particle size of 139 nm and 188 nm respectively, were selected for this experiment. The results indicated that both the tested batches showed sustained release of drug over a period of 120 h (Fig. 2). Batches F8 and F9 showed cumulative drug release of 14.07 % and 22.02% respectively. The drug release from batch F8 was more

14

prolonged; however, the drug release was found to be very less even after 120 h. Therefore, batch F9 was selected for further evaluation as it exhibited a better extent of drug release (22.02%) besides demonstrating the sustained release. 4.4.

Transmission Electron Microscopy: The surface morphology of the nanoparticle formulation (F9) was assessed by TEM

(Fig. 3). The nanoparticles were found to be almost spherical in shape, with size < 200 nm, which is consistent with the findings of particle size analysis by ZetaSizer. The TEM analysis also showed that the nanoparticles were not aggregated. The layer observed around surrounding the nanoparticle core could be due to the PEG molecules attached to the chitosan polymer.

4.5.

Solid state characterization of the nanoparticles:

4.5.1. Differential Scanning Calorimetry (DSC): DSC was used to investigate the compatibility status of the drug and excipients and to determine any form conversions of the drug in the nanoparticulate formulation. The DSC thermograms of drug (plain RC), physical mixture of drug+excipients (1:1) and nanoparticulate formulation (Batch F9) are given in Fig. 4A, 4B and 4C, respectively. Plain RC exhibited an endothermic peak at 132.27 °C, corresponding to its melting point (Fig. 4A). RC has shown wide range for its melting point, between 122 and 160 °C [36-38]. In the physical mixture of drug + excipients, the endothermic peak of the drug was very slightly shifted to 135.3 °C (Fig. 4B). In the optimized formulation (F9), the endothermic peak of the drug was broadened, with a melting range over 95.45 °C to 167.45 °C, with the highest intensity at 132.0 °C (Fig. 4C). This wide spread of the melting range may probably be due to almost complete embedment of drug in the polymer matrix of the nanoparticles [39]. These results suggest no chemical

15

interaction between the drug and excipients; however the results reveal the presence of RC in partial amorphous form in its optimized nanoparticles (Batch F9). 4.5.2. FTIR analysis: FTIR analysis is a technique widely used for determination of chemical interactions between drugs and excipients. The samples that were analysed by FTIR were plain RC, physical mixture of drug + excipients (1:1) and nanoparticulate formulation (Batch F9). The IR spectrum of RC (Fig. 5A) exhibited characteristic peaks at 3379.4 cm-1 (carboxylic OH stretch), 2968.55 cm-1 (N-H stretch), 1546.96 cm-1 (C=C stretch), 1437.02 cm-1 and 1383.01 cm-1 (asymmetric and symmetric vibrations of CH3), 1151.54 cm-1 (C-F stretch) and 777.34 cm-1, 642.32 cm-1, 570.95 cm-1 (absorption bands of out of plane C=C of benzene ring). These peaks were retained in the IR spectra of both the physical mixture of RC and polymer (Fig. 5B) and in the IR spectrum of optimized formulation (Fig. 5C). These observations confirm the compatibility between drug and excipients and lack of any chemical interactions [40]. 4.5.3. X Ray Diffraction studies: XRD analysis was performed to identify form conversions of the drug after incorporation in the polymer matrix. The samples analysed by XRD were RC and optimized nanoparticle formulation (Batch F9) and the respective XRD patterns are given in Fig. 6 (A and B). The form of RC used in this study is amorphous, having high dissolution rate and stability [41]. The XRD patterns of RC (Fig. 6A) and PEGylated chitosan (Fig. 6B) did not exhibit any sharp peaks. This confirms the amorphous nature of existence of both the drug and the polymer. The XRD pattern of the Batch 9 formulation (Fig. 6C) also indicates amorphous nature of the drug in formulation. XRD data imply that there is no form conversion of the drug even after incorporation into nanoparticles. However the halo region observed in XRD pattern of nanoparticles indicates more amorphization of drug in its nanoparticulate form, thus supporting the data of DSC results. Solids in amorphous state are composed of molecules in

16

random arrangement unlike crystalline state, requiring lower energy for separating the molecules. This state may be desirable as it enhances the physical stability and rate of dissolution of the drug [18]. 4.6.

In vivo studies:

4.6.1. In vivo drug release study (Pharmacokinetics): The mean plasma concentration vs. time profile observed after oral administration of RC solution and i.v. administration of RC nanoparticles (Batch F9) is depicted in Fig. 7. PK parameters for both routes were determined by using PK Solutions Software by fitting the data as per oral and i.v. bolus modes separately. However with respect to i.v. route, Cmax and Tmax values were calculated using a model-independent method as explained previously for amphotericin nanoparticles, which were administered by i.v. route [42]. The Cmax value for RC nanoparticles (134.60±8.02 ng/mL) was lesser compared to oral RC solution (225.80±10.56 ng/mL) which could be due to the embedment of rosuvastatin in the nanoparticulate structure, which might have impeded the drug release in the immediate instance. It was observed that oral RC solution produced quick absorption of the drug into systemic circulation (Tmax: 2 h); however the plasma drug levels were not sustainable for long. On the other hand, although RC appeared quickly in plasma from the nanoparticles after their i.v. administration (Tmax: 2 h), the drug release was found to be sustained over a long duration of time. In the case of oral RC solution, the drug concentration was not detectable in plasma after 24 h. On the other hand, the plasma drug levels were found in quantifiable concentration for more than 70 h with F9 nanoparticles. Such prolonged release pattern was also observed in the in vitro release study, where F9 nanoparticles had a cumulative drug release of 22.02% even after 120 h. Taken together, this implies that even if the quantity of nanoparticles administered is high, the drug release at each time point will be low, leading to the entire drug being released over a controllable duration of time.

17

The AUC values obtained from RC nanoparticles were ≈3 times higher (p<0.05) as compared to oral RC solution. This could be due to the slower release of RC from nanoparticles and hence is indicative of the superior bioavailability of RC from its nanoparticles. The elimination rate constant (Kel) for RC nanoparticles was significantly (p<0.05) lesser (0.009±0.001 h-1) than that of oral RC solution (0.04±0.003 h-1) indicating that RC was eliminated slowly when administered in nanoparticulate form. This clearly indicates that oral RC solution is rapidly cleared from the blood whereas RC nanoparticles are retained in the blood circulation for longer periods of time, which could be due to PEGylation of the nanoparticles. The t1/2 of RC in humans is reportedly around 20 h, meaning half of the dose is eliminated within this time period. RC is also extensively plasma protein bound and penetrates into extrahepatic tissue, with the potential to cause rhabdomyolysis on chronic administration [43]. Therefore, it would be highly beneficial to administer RC in a dosage form that produces only requisite drug levels in plasma and over a prolonged period. The t1/2 of RC nanoparticles (74.87±3.26 h) showed a significant increase (p<0.05) as compared to RC oral solution (17.30±0.81 h) which may be attributed to the lower plasma clearance of RC from nanoparticles and longer circulation in the blood. The mean residence time (MRT) value of RC nanoparticles was significantly higher (p<0.05) than that of oral RC solution. The higher MRT of RC nanoparticles (25.40±1.02 h) as compared to oral RC solution (100.10±6.21 h) can be ascribed to the lower particle size of the nanoparticles (<200 nm), which helps to avoid recognition by the reticuloendothelial system (RES) and the stealth effect offered by PEGylation which results in longer blood circulation of RC nanoparticles.

18

Hence, the results demonstrated that RC nanoparticles achieved better pharmacokinetic profile in comparison with oral RC solution which is evident from the significantly (p<0.05) higher AUC, half-life and MRT values for optimized nanoparticles (Batch F9). 4.6.2. Pharmacodynamics study: The pharmacodynamics activity of the optimized RC nanoparticles in comparison to oral administration of RC solution was determined using ‘tyloxapol-induced hyperlipidemia model’ in male Wistar rats [44]. Tyloxapol (Triton WR 1339) is a polymeric non-ionic detergent which upon intraperitoneal administration in animals, blocks the activity of lipoprotein lipase and reduces the clearance of triglycerides from plasma. This agent has been used successfully in several studies to induce hypercholesterolemia in animals [24, 45]. The increase in serum cholesterol produced by single administration of tyloxapol starts from the 6th hour and continues up to 24 h after administration. Therefore, the study was conducted up to 48 h, with sampling at 0 (to measure baseline cholesterol and triglycerides), 2, 4, 6, 8, 12, 24 and 48 h. 4.6.2.1. Comparison of serum cholesterol levels after treatments: Considerable elevation was observed in serum cholesterol (268.36±21.90 mg/dL), in Group 2 of animals, that were administered with tyloxapol alone (Disease control) (Table 2). At 48 h, the serum cholesterol levels in this group were found to decrease to baseline levels. In the animals that were administered with standard treatment (Group 3; RC solution orally along with tyloxapol), the surge in serum cholesterol was much lower with considerable decrease at 12 h (142.36±17.65 mg/dL). However, in the animals of Group 4 (administered with Batch F9 nanoparticles along with tyloxapol), the decrease in cholesterol level (98.9±3.70 mg/dL) was much more substantial when compared with that of Group 3. Moreover, at 24 h also the serum cholesterol level in Group 4 (87.03±4.62 mg/dL) was less than that observed in Group 3 (115.73±6.97 mg/dL).

19

4.6.2.2. Comparison of serum triglyceride levels after treatments: In Group 2 of animals that were administered only tyloxapol (Disease control), there was a significant elevation (p<0.05) in serum triglyceride levels (560.75±56.31 mg/dl) at 12 h (Table 3). In Group 2 (disease control), the increase in serum triglyceride level started at the 4th h, reached the peak at the 12th h and decreased by 48 h. It was noteworthy to observe that in the animals of Group 3 (administered with RC solution orally) the decrease in serum triglycerides at the 12th h was not substantial. On the contrary, in Group 4 of animals (administered with batch F9 of nanoparticles) there was a much greater reduction in the serum triglycerides compared to group 2. In Group 4, the serum triglyceride level came down to baseline levels by 24 h in this group (148.66±12.06 mg/dl). These results clearly indicate the greater benefit of RC nanoparticles compared to RC solution, as demonstrated by the better in vivo performance of the former. Nanoparticles have been documented for their ability to enhance in vivo efficiency of several bio-active compounds. This may be attributed to the unique characteristics of nanoparticles such as size, shape, surface charge, hydrophilicity etc. which determine their specific functions [45]. The superior pharmacodynamic activity of the RC nanoparticles may be attributed to the smaller size (<200 nm) of the particles. In addition, PEGylation of nanoparticles helps to avoid its opsonisation from the blood thus ensuring that these RC nanoparticles circulate in the blood for a long period of time and maintaining sustained release. In the previous reports, chitosan nanoparticles loaded with drugs did not reduce the recognition of the nanoparticles by the macrophages and hence long circulation property was not achieved with nanoparticles of plain chitosan; whereas, nanoparticles of PEGylated chitosan circulated continuously in blood for longer period of time as compared to plain chitosan nanoparticles [9, 10]. Hence we prepared and evaluated the nanoparticles of PEGylated chitosan loaded with rosuvastatin in this study. Furthermore chitosan itself has been reported to reduce serum cholesterol levels and hence

20

finds its use as a suitable excipient in RC nanoparticles of the present study [6]. Hence the better pharmacodynamic activity of the RC nanoparticles could be due to the synergistic action of chitosan as well as RC, coupled with nanoscale architecture.

5.

Conclusion: Rosuvastatin calcium-loaded nanoparticles were successfully formulated using

PEGylated chitosan by following ionotropic gelation method. In vitro drug release demonstrated that the nanoparticles exhibited a highly sustained release. Higher AUC, elimination half-life and MRT values were observed with in vivo pharmacokinetics of RC nanoparticles which indicate that the nanoparticles are retained in the circulation for longer period of time. Also, in vivo efficacy studies confirmed that RC nanoparticles substantially lowered the serum cholesterol and triglyceride levels as compared to oral RC solution. However long-term in vivo performance evaluation studies are required to confirm these results.

Acknowledgements Authors express sincere thanks to Indian Council of Medical Research (ICMR), Government of India, New Delhi for financial assistance (Grant No.: 35/6/2013-BMS). The authors are thankful to Manipal University, Manipal, India for providing the necessary facilities.

21

References: 1.

R-G. Potur, L.S. Moisuc, N. Saraficeanu, T. Boita, L. Macovei, A-D. Telisça. Stable pharmaceutical composition comprising amorphous rosuvastatin calcium. EP 2805714 A1. November 26, 2014

2.

A.J. Lusis, Atherosclerosis, Nature. 407 (2000) 233–241

3.

S.P. Adams, S.S. Sekhon, J.M. Wright, Lipid-lowering efficacy of rosuvastatin, Cochrane Database Syst. Rev. (2014) CD010254

4.

L. Calza, Long-term use of rosuvastatin: a critical risk benefit appraisal and comparison with other antihyperlipidemics, Drug. Healthc. Patient Saf. 1 (2009) 25–33

5.

T. Kean, M. Thanou, Biodegradation, biodistribution and toxicity of chitosan, Adv. Drug Deliv. Rev. 62 (2010) 3–11

6.

C.M. Gallaher, J. Munion, R. Hesslink, J. Wise, D.D. Gallaher, Cholesterol reduction by glucomannan and chitosan is mediated by changes in cholesterol absorption and bile acid and fat excretion in rats, J. Nutr. 130 (2000) 2753–9

7.

H. Zhang, Y. Tao, J. Guo, Y. Hu, Z. Su, Hypolipidemic effects of chitosan nanoparticles in hyperlipidemia rats induced by high fat diet, Int. Immunopharmacol. 11 (2011) 457– 61

8.

J. Suh, K.-L. Choy, S.K. Lai, J.S. Suk, B.C. Tang, S. Prabhu, J. Hanes, PEGylation of nanoparticles improves their cytoplasmic transport., Int. J. Nanomedicine. 2 (2007) 735– 41

9.

J. Chen, L. Huang, H. Lai, C. Lu, M. Fang, Q. Zhang, X. Lou, Methotrexate-Loaded PEGylated chitosan nanoparticles: Synthesis, characterization, and in vitro and in vivo antitumoral activity, Mol. Pharmaceutics. 7 (2014) 2213–23.

22

10.

X. G. Zhang, D. Y. Zhang, D. Y. Teng, Z. M. Wu, X. Wang, Z. Wang, D. M. Yu, C. Z. Li, PEG-grafted chitosan nanoparticles as an injectable carrier for sustained protein release, J. Mater. Sci: Mater. Med. 19 (2008) 3525–33.

11.

M.J. Roberts, M.D. Bentley, J.M. Harris, Chemistry for peptide and protein PEGylation, Adv. Drug Deliv. Rev. 54 (2002) 459–76

12.

C. Prego, D. Torres, E. Fernandez-Megia, R. Novoa-Carballal, E. Quiñoá, M.J. Alonso, Chitosan-PEG nanocapsules as new carriers for oral peptide delivery. Effect of chitosan pegylation degree, J. Control. Release. 111 (2006) 299–308

13.

I.M. Deygen, E.V. Kudryashova, New versatile approach for analysis of PEG content in conjugates and complexes with biomacromolecules based on FTIR spectroscopy, Colloids Surfaces B Biointerfaces. 141 (2016) 36–43

14.

K.S. Avadhani, J. Manikkath, M. Tiwari, M. Chandrasekhar, A. Godavarthi, S.M. Vidya, R.C. Hariharapura, G. Kalthur, N. Udupa, S. Mutalik, Skin delivery of epigallocatechin3-gallate (EGCG) and hyaluronic acid loaded nano-transfersomes for antioxidant and anti-aging effects in UV radiation induced skin damage., Drug Deliv. 24 (2017) 61–74

15.

P. Calvo, C. Remuñan-López, J.L. Vila-Jato, M.J. Alonso, Chitosan and chitosan/ethylene oxide-propylene oxide block copolymer nanoparticles as novel carriers for proteins and vaccines., Pharm. Res. 14 (1997) 1431–6

16.

A. Rampino, M. Borgogna, P. Blasi, B. Bellich, A. Cesàro, Chitosan nanoparticles: preparation, size evolution and stability, Int. J. Pharm. 455 (2013) 219–28

17.

J. Manikkath, A.R. Hegde, G. Kalthur, H.S. Parekh, S. Mutalik, Influence of peptide dendrimers and sonophoresis on the transdermal delivery of ketoprofen, Int. J. Pharm. 521 (2017) 110–9

23

18.

S. Natesan, C. Ponnusamy, A. Sugumaran, S. Chelladurai, S. Shanmugam Palaniappan, R. Palanichamy, Artemisinin loaded chitosan magnetic nanoparticles for the efficient targeting to the breast cancer, Int. J. Biol. Macromol. 104 (2017). 1853-59

19.

A.B. Shreya, R.S. Managuli, J. Menon, L. Kondapalli, A.R. Hegde, K. Avadhani, P.K. Shetty, M. Amirthalingam, G. Kalthur, S. Mutalik, Nano-transfersomal formulations for transdermal delivery of asenapine maleate: in vitro and in vivo performance evaluations., J. Liposome Res. 26 (2016) 221–32

20.

S.S. D’Souza, P.P. DeLuca, Methods to assess in vitro drug release from injectable polymeric particulate systems., Pharm. Res. 23 (2006) 460–74

21.

P.K. Shetty, J. Manikkath, K. Tupally, G. Kokil, A.R. Hegde, S.Y. Raut, H.S. Parekh, S. Mutalik, Skin Delivery of EGCG and Silibinin: Potential of Peptide Dendrimers for Enhanced Skin Permeation and Deposition., AAPS PharmSciTech. 6 (2017). 2346-57

22.

A.R. Hegde, P. V Rewatkar, J. Manikkath, K. Tupally, H.S. Parekh, S. Mutalik, Peptide dendrimer-conjugates of ketoprofen: Synthesis and ex vivo and in vivo evaluations of passive diffusion, sonophoresis and iontophoresis for skin delivery, Eur. J. Pharm. Sci. 102 (2017) 237–49

23.

P.A. Flecknell, Anaesthesia of animals for biomedical research. Br. J. Anaesth. 71 (1993) 885–9

24.

L.C. Bertges, C.A. Mourão Jr, J.B. Souza, V. Antônio, C. Cardoso, Hyperlipidemia induced by Triton WR1339 (Tyloxapol) in Wistar rats, Rev Bras Cien Med Saúde. 1 (2011) 32–4

25.

S.-H. Chen, C.-T. Tsao, C.-H. Chang, Y.-T. Lai, M.-F. Wu, C.-N. Chuang, H.-C. Chou, C.-K. Wang, K.-H. Hsieh, Assessment of reinforced poly(ethylene glycol) chitosan hydrogels as dressings in a mouse skin wound defect model, Mater. Sci. Eng. C. 33 (2013) 2584–94

24

26.

L.A. Canalle, D.W.P.M. Löwik, J.C.M. van Hest, Polypeptide-polymer bioconjugates., Chem. Soc. Rev. 39 (2010) 329–53

27.

W.H. De Jong, P.J.A. Borm, Drug delivery and nanoparticles:applications and hazards., Int. J. Nanomedicine. 3 (2008) 133–49

28.

G. Sonavane, K. Tomoda, K. Makino, Biodistribution of colloidal gold nanoparticles after intravenous administration: Effect of particle size, Colloids Surfaces B Biointerfaces. 66 (2008) 274–80

29.

Q. Wang, H. Xia, C. Zhang, Preparation of polymer/inorganic nanoparticles composites through ultrasonic irradiation, J. Appl. Polym. Sci. 80 (2001) 1478–88

30.

G. Fang, B. Tang, Z. Liu, J. Gou, Y. Zhang, H. Xu, X. Tang, Novel hydrophobin-coated docetaxel nanoparticles for intravenous delivery: In vitro characteristics and in vivo performance, Eur. J. Pharm. Sci. 60 (2014) 1–9

31.

P. Verma, M. Ahuja, Optimization, characterization and evaluation of chitosan-tailored cubic nanoparticles of clotrimazole., Int. J. Biol. Macromol. 73 (2015) 138–45

32.

E.S.K. Tang, M. Huang, L.Y. Lim, Ultrasonication of chitosan and chitosan nanoparticles., Int. J. Pharm. 265 (2003) 103–14

33.

Y. Xu, Y. Du, Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles., Int. J. Pharm. 250 (2003) 215–26

34.

D.R. Bhumkar, V.B. Pokharkar, Studies on effect of pH on cross-linking of chitosan with sodium tripolyphosphate: A technical note, AAPS PharmSciTech. 7 (2006) E138–E143

35.

R. Asasutjarit, T. Theerachayanan, P. Kewsuwan, S. Veeranodha, A. Fuongfuchat, G.C. Ritthidej, Development and Evaluation of Diclofenac Sodium Loaded-N-Trimethyl Chitosan Nanoparticles for Ophthalmic Use, AAPS PharmSciTech. 16 (2015) 1013–24

25

36.

K.A. Dhoranwala, P. Shah, S. Shah, Formulation Optimization of Rosuvastatin CalciumLoaded Solid Lipid Nanoparticles by 32 Full-Factorial Design, NanoWorld J. 1 (2015). 112-21

37.

www.chemicalbook.com/ChemicalProductProperty_EN_CB9482678.htm.

(accessed

20.06.2017) 38.

www.lktlabs.com/product/rosuvastatin-calcium. (accessed 20.06.2017)

39.

A.D. Chonkar, J.V. Rao, R.S. Managuli, S. Mutalik, S. Dengale, P. Jain, N. Udupa, Development of fast dissolving oral films containing lercanidipine HCl nanoparticles in semicrystalline polymeric matrix for enhanced dissolution and ex vivo permeation., Eur. J. Pharm. Biopharm. 103 (2016) 179–91

40.

A.P. Chiriac, A. Diaconu, L.E. Nita, N. Tudorachi, L. Mititelu-Tartau, A. Creteanu, O. Dragostin, D. Rusu, G. Popa, The influence of excipients on physical and pharmaceutical properties of oral lyophilisates containing a pregabalin-acetaminophen combination, Expert Opin. Drug Deliv. 14 (2017) 589–99

41.

F. Blatter, P. A. van der Schaaf, M. Szelagiewicz. Crystalline forms of rosuvastatin calcium salt. US 7932387 B2. April 26, 2011

42.

M.A. Radwan, B.T. AlQuadeib, L. Šiller, M.C. Wright, B. Horrocks, Oral administration of amphotericin B nanoparticles: antifungal activity, bioavailability and toxicity in rats, Drug Deliv. 24 (2017) 40–50

43.

A.A. Adeneye, O.O. Adeyemi, E.O. Agbaje, Anti-obesity and antihyperlipidaemic effect of Hunteria umbellata seed extract in experimental hyperlipidaemia, J. Ethnopharmacol. 130 (2010) 307–14

44.

N. Kumar, J. Mudgal, V.K. Parihar, P.G. Nayak, N.G. Kutty, C.M. Rao, Sesamol treatment reduces plasma cholesterol and triacylglycerol levels in mouse models of acute and chronic hyperlipidemia., Lipids. 48 (2013) 633–8

26

45.

B. Haley, E. Frenkel, Nanoparticles for drug delivery in cancer treatment, Urol. Oncol. 26 (2008) 57–64

27

List of figures: Fig. 1. FT-IR spectra of A) mPEG-SVA B) chitosan and C) PEGylated chitosan Fig. 2. In vitro drug release from the RC nanoparticles (in PBS) Fig 3. TEM image of batch F9 of nanoparticles Fig. 4. DSC thermograms of A) RC B) RC + excipients (1:1) C) Optimized nanoparticulate formulation (F9) Fig. 5. FT-IR spectra of A) RC B) RC + excipients (1:1) C) Optimized nanoparticulate formulation (F9) Fig. 6. XRD patterns of A) RC B) PEGylated chitosan C) Optimized nanoparticulate formulation (F9) Fig. 7. Plasma concentration–time profiles for the different treatments

28

2359.02

75

667.39

3441.12

459.07

3865.48

82.5

3740.10

90 %T

842.92 954.80

1464.02

1350.22

60

1280.78

2885.60

1737.92

67.5

52.5

45

1111.03

A 37.5

30 4000 M

3750

3500

3250

3000

2750

2500

2250

2000

1750

2250

2000

1750

2250

2000

1750

1500

1250

1000

750

500 1/cm

90

3861.62

%T

461.00

2922.25

3728.53

85

80

1539.25

2360.95

70

1643.41

3446.91

671.25

75

1020.38

B

65

60 4000 C1

3750

3500

3250

3000

2750

2500

1500

1250

1000

750

500 1/cm

90

3865.48

82.5

3747.81

%T

2922.25

665.46

75

2362.88

67.5

4000 C1M

3750

3500

3250

3000

2750

2500

1566.25

C

3435.34

45

1080.17

52.5

1500

1250

1026.16

1649.19

1411.94

60

1000

750

500 1/cm

Fig. 1A-C Journal: International Journal of Biological Macromolecules Authors: Mukund Hirpara et al.

29

30

F9 F8

Cumulative drug released (%)

25

20

15

10

5

0 0

20

40

60

Time (h)

80

100

Fig. 2 Journal: International Journal of Biological Macromolecules Authors: Mukund Hirpara et al.

30

120

140

Fig. 3 Journal: International Journal of Biological Macromolecules Authors: Mukund Hirpara et al.

31

A

B

C

Fig. 4A-C Journal: International Journal of Biological Macromolecules Authors: Mukund Hirpara et al.

32

45

4000 F1

3750

3500

3250

3000

95

87.5

B

2750 1750

2500

2250 1332.81

2000 1500

52.5

C

1750

1500

1200

1250 966.34

1074.35

1230.58

1350

1153.43

1500 1050 900

1250

750

1000

1000

33

600

750

750 503.44

2000 1650

617.24 565.16

717.54

2500 1800

1460.11 1446.61 1431.18 1384.89

2100 1950

889.21

1512.19

1683.86

1381.08

1546.96

1153.47

964.44

1334.78

1437.02

1508.38

1600.97

A

1026.16

3000 2400

1647.21

97.5 1836.23

0

1087.89

3500 2700

1546.91

1741.72

3000

2331.94

10

1219.05

82.5 2372.52

2358.94

1228.70

20

1411.94

75 2864.39

4000 RC 3300

1654.98

2926.11

3844.13

3600

2968.45 2931.80

3739.97

3900 D

3282.84

3558.67

2966.62

1068.60

516.94

570.95

775.41

844.85

3381.33

30

1570.11

3284.88

92.5 3618.46

40

1159.26

3421.83

3624.37

3753.60

900.79

640.39

717.54

2357.09

%T 90

80

70

60

50

-10 450 1/cm

102.5

100 %T

90

500 1/cm

%T 90

67.5

60

500 1/cm

Fig. 5A-C Journal: International Journal of Biological Macromolecules Authors: Mukund Hirpara et al.

A

B

C

Fig. 6A-C Journal: International Journal of Biological Macromolecules Authors: Mukund Hirpara et al.

34

Plasma conc. of rosuvastatin (ng/mL)

250 Drug solution (oral) i.v. Batch F9 NPs

200

150

100

50

0 0

10

20

30

40 Time (h)

50

Fig. 7. Journal: International Journal of Biological Macromolecules Authors: Mukund Hirpara et al.

35

60

70

80

Table 1: Optimization trials of RC nanoparticles

Batch No.

RC (mg)

MCHT (mg)

TPP (mg)

Sonication time (min)

ZP (mV)

EE (%)

F1

2.5

12

5

20

306±10.87 0.49±0.07

18.3±0.13

3.2±0.56

F2

2.5

12

5

35

411±23.44 0.35±0.13

16.1±0.56

4.6±0.65

F3

2.5

12

5

40

424±27.83 0.40±0.11

17.0±0.26

5.2±1.02

F4

2

12

5

40

455±24.56 0.59±0.09

16.1±0.14

3.6±0.52

F5

1.5

12

5

40

539±37.09 0.60±0.05

19.8±0.06

1.8±0.21

F6

2.5

12

2.5

40

605±34.89 0.51±0.06

29.8±0.13

3.3±0.45

F7

2.5

12

7.5

40

311±6.72

0.40±0.08

11.7±0.15

6.7±1.73

F8

2.5

12

12.5

40

139±4.35

0.24±0.04

6.47±0.06

14.0±2.66

F9

2.5

12

25

40

188±5.64

0.21±0.06

6.23±0.07

14.1±1.87

Average size (nm)

PDI

Values represented as Mean ± SD (n=3); RC: Rosuvastatin calcium; MCHT: Modified chitosan; TPP: Tripolyphosphate; PDI: Polydispersity index; ZP: Zeta potential; EE: Entrapment efficiency

36

Table 2. Pharmacokinetics parameters obtained after administering rats with RC solution or nanoparticles Parameters

RC solution Batch F9 - NP (Oral) (i.v.)

Cmax (ng/mL)

225.80±10.56

134.60±8.02

Tmax (h)

2.00±0.00

2.00±0.00

AUC0-t (ng.h/mL)

1688.40±82.89

4655.30±221.66*

AUC0-I (ng.h/mL)

2802.50±106.55 8825.50±322.28*

Kel (L/h)

0.04±0.003

0.009±0.001*

t1/2 (h)

17.30±0.81

74.87±3.26*

MRT (h)

25.40±1.02

100.10±6.21*

PK=Pharmacokinetics; NP=Nanoparticles; i.v.=Intravenous; Cmax=maxiumum concentration of drug in plasma; Tmax=Time for maximum drug concentration in plasma; AUC=Area under curve; Kel=Elimination rate constant; t1/2=Elimination half-life; MRT=Mean residential time * Significantly different (p<0.05) from respective PK parameter

37

Table 3. Serum cholesterol levels after different treatments Serum Cholesterol Levels (mg/dL) Time Normal Disease (h) RC solution (Oral) Control Control

Batch F9 – NP (i.v.)

0

93.35±3.45

94.87±3.602

94.56±3.76

87.5±2.31

2

94.77±3.508

92.90±2.91

85.37±4.74

84.2±0.92

4

90.12±1.906

98.47±4.35

75.46±4.05

81.01±2.31

6

85.65±1.20

145.78±6.90a

93.84±6.04b

85.01±4.16

8

93.45±1.74

156.35±5.36a

110.23±2.38b

87.96±6.48b

12

90.34±3.26

268.36±21.90a

142.36±17.65b

98.9±3.703b

24

87.34±4.34

185.02±12.03a

115.73±6.97b

87.03±4.62b

48

91.20±0.672

89.37±1.83

89.06±2.39

88.42±1.388

Values represented as Mean ± SD (n=6) Significantly different (p<0.05) from Normal Control b* Significantly different (p<0.05) from Disease Control a*

38

Table 4. Serum triglyceride levels after different treatments Time (h)

Serum triglyceride levels (mg/dL) Normal Control

Disease Control

RC solution (Oral)

Batch F9 – NP (i.v.)

0

152.13±8.94 132.48±3.67

145.78±3.07

119.37±5.98

2

146.78±2.35 146.57±19.04

119.44±5.95

100.79±5.04

4

132.47±1.25 156.89±1.74

135.80±9.73 a

154.60±5.69

115.59±2.83 b

6

126.49±3.27 302.45±7.08

8

132.58±3.46 410.89±19.53a

387±18.42b

133.86±10.39b

12

137.75±4.98 560.75±56.31a

478.49±10.20b

411.65±11.02b

24

148.78±4.37 380.57±0.57a

291.39±33.41b

148.66±12.06b

48

136.93±1.36 178.56±19.03

104.57±22.05

100.09±13.86

Values represented as Mean ± SD (n=6) a Significantly different (p<0.05) from Normal Control b Significantly different (p<0.05) from Disease Control

39

103.94±0.005b