Preparation and in vitro-in vivo evaluation of surface-modified poly(lactide-co-glycolide) nanoparticles as controlled release carriers for flutamide delivery.

http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, Early Online: 1–9 ! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2014.995731

RESEARCH ARTICLE

Journal of Microencapsulation Downloaded from informahealthcare.com by University of Colorado Libraries on 12/28/14 For personal use only.

Preparation and in vitro–in vivo evaluation of surface-modified poly(lactide-co-glycolide) nanoparticles as controlled release carriers for flutamide delivery Pradum Pundlikrao Ige and Solanki Nirmal Dipsingh Department of Pharmaceutics and Quality Assurance, R C Patel Institute of Pharmaceutical Education and Research, Shirpur, Dhule, Maharashtra, India Abstract

Keywords

This investigation explores the use of methoxy polyethylene glycol (mPEG) functionalised poly(D,L-lactide-co-glycolide) (PLGA) nanocrystals of flutamide (FLT) with enhanced solubility, bioavailability and blood circulation time for targeting prostate cancer. FLT had Log P 3.27, short half life 5–6 h, low water solubility, permeability and bioavailability with extensive firstpass metabolism. FLT-loaded nanocrystals were prepared using nanoprecipitation method with surface coating by mPEG and characterised through differential scanning calorimetry, Fourier transform infrared spectroscopy, X-ray powder diffraction, scanning electronic microscopy, particle size, zeta potential, percent entrapment efficiency (% EE), in vitro dissolution, haemolysis, sterility, bioavailability and stability studies. The percent cumulative drug release and % EE of optimised formulation was found to be 95.21 ± 1.18 and 88.36 ± 1.20, respectively, for 48 h. In addition, FLT-loaded PEGylated PLGA nanocrystals exhibited significantly delayed blood clearance with drug level of about 766.71 ng/mL at 48 h. In conclusion, PEGylated PLGA FLT nanocrystals could be demonstrated as a novel approach to enhance solubility, bioavailability and blood circulation time.

Injectables, nanoparticle, pharmacokinetics, PLGA, zeta potential

Introduction Solubility is one of the crucial factors for drug therapy in any route of administration. Approximately, 70% of newly developed and investigated drugs are poorly water-soluble, which often leads to low bioavailability. Nanoprecipitation has emerged as an important tool in drug delivery to rectify the solubility issues (Abdelwahed et al., 2006; Keck and Muller, 2006; Muller et al., 2006; Sahoo et al., 2008; Mu¨ller and Keck, 2012; Elzoghby et al., 2013). In the past three to four decades, there has been an enormous increase in the incidence of prostate cancer and gradually increase in mortality. The growth of cancer cells is different from normal cell growth. Instead of dying, cancer cells continue to grow and form new abnormal cells. Prostate cancer is the most frequently diagnosed cancer of men in Asia, Europe and the United States. It affects 2.5 million men in the United States alone (Gronberg, 2003; Fizazi and Navone, 2005; Hodgson et al., 2007; Cho et al., 2010; Okamoto et al., 2012). Flutamide (FLT) is an anti-androgenic agent and indicated as monotherapy for the treatment of patients with locally advanced or metastatic prostate cancers. It is a potent inhibitor of

Address for correspondence: Dr. Pradum Pundlikrao Ige, Assistant Professor, Department of Pharmaceutics & Quality Assurance, R.C. Patel Institute of Pharmaceutical Education & Research, Karwandnaka, Shirpur, Dhule 425405, Maharashtra, India. Tel: +91-2563-255189. Mobile no.: +91-9823509648. Fax: +91-2563-252808. E-mail: [email protected]

History Received 10 June 2014 Revised 15 October 2014 Accepted 13 November 2014 Published online 24 December 2014

testosterone-stimulated prostatic DNA synthesis and prevents prostate cancer cells to grow. FLT belongs to Biopharmaceutical Classification System class II drugs and it undergoes extensive first-pass effect through CYP1A2 after oral administration. It has short half-life 5–6 h and log P 3.350 with low solubility and bioavailability (49%); therefore, its clinical application is in conflict (Zuo et al., 2002; Acharya and Sahoo, 2011; Elgindy et al., 2011; Anitha et al., 2012). Different researchers had reported their work deals with the development of sustained drug delivery system (DDS) for FLT using polymeric microparticles and nanoparticles (NPs). These include cross-linked chitosan NPs and methacrylic acid copolymer-based NPs, as oral DDS. In such investigation, sustained DDS for FLT was developed with certain limitations of efficacy and safety (Sternal and Nugara, 2001; Chorny et al., 2002; Svenson, 2004; Cruz et al., 2011; Parveen and Sahoo, 2011; Gref et al., 2012; Jain et al., 2012; Palumbo et al., 2012; Sah et al., 2013; Sharma et al., 2013). Poly(D,L-lactide-co-glycolide) (PLGA) is an excellent synthetic biodegradable copolymer, which has been huge application in formulation of hydrophobic and hydrophilic drugs. It undergoes hydrolysis in the body to produce the biodegradable metabolite monomers, lactic acid and glycolic acid (Moghimi et al., 2001; Kumari et al., 2010; Danhier et al., 2012). There has been a growing interest in the development of a colloidal drug carrier, which is small enough for intravenous administration and possesses an adequate circulation half-life in order release the drug in a continuous and controlled fashion. The parental routes of administration, i.v drug infusion/injection,


2

P. P. Ige and S. N. Dipsingh

J Microencapsul, Early Online: 1–9

provide the most rapid effects. There are least limitations on the volume of i.v administration and the therapeutic responses and associated toxicity are more predictable (Laverman et al., 2001; Owens and Peppas, 2006; Fredenberg et al., 2011). NPs of biodegradable polymers are widely investigated for controlled and targeted delivery of various drugs. Surfacemodified polymeric NPs are the new generation of NP to overcome the drawbacks. They are being developed because of biocompatibility, long-standing track record in biomedical applications for sustained drug release devices up to days, weeks or months and ease of parental administration via injection. The NP surface can be modified with methoxy polyethylene glycol (mPEG) to prevent opsonisation, delay their capture by macrophages and thereby allow them to circulate longer in the body and reach the tumour by the enhanced permeation and retention effect (Heffery et al., 1991; Hancock and Zografi, 1997; Jinping et al., 1999; Niopas and Daftsios, 2001; Nesalin et al., 2009; Pamujula et al., 2012; Ige et al., 2013). In this investigation, an attempt has been made to increase the circulation time of PLGA nanocrystals through surface coating with mPEG (Mol. Wt.: 5000). It was characterised by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy, X-ray powder diffraction (XRPD), scanning electronic microscopy (SEM), particle size, zeta potential (ZP), solubility, percent loading efficiency (% LE), percent entrapment efficiency (% EE), in vitro dissolution, haemolytic studies, sterility, in vivo bioavailability and short-term stability studies. Therefore, it was hypothesised to enhance the solubility, bioavailability and blood circulation time via intravenous administration by surface modified polymeric NPs without causing liver toxicity in prostate cancer. Novelty of work is that, for the first time, a thin coating of the mPEG was applied over PLGA-loaded NPs. The importance of the mPEG coating is that it prevents opsonisation and prolongs the circulation time in the blood, therefore, it overcomes the disadvantages of PLGA NPs.

Materials and methods Materials FLT and PLGA were obtained as gift samples from Cipla (I) Ltd. (Bangalore, India) and Wockhardt Ltd. (Aurangabad, India), respectively. Kolliphor P 188, mPEG and EDAC were purchased from Sigma-Aldrich Inc. (St. Louis, MO). All the reagents used were of analytical grade and used without further purification. Methods Development of parental formulation Screening of surfactant for stabilisation of nanocrystals. Various surfactants like PVP K 25, poloxamer 188, Tween 80 and SDS have been explored to stabilise the

surface of FLT nanocrystals. The minimum particle size was obtained with poloxamer 188 and hence, it was chosen as the stabiliser; 5% poloxamer 188 concentration was selected because no further decrease in particle size was observed. Preparation of FLT nanocrystals. FLT nanocrystals were prepared using nanoprecipitation method (Table 1). The formulation was prepared by dissolving PLGA (100 mg) and FLT (10 mg) in 2 mL of acetone. This organic phase was added at the rate of 1 mL/min to 10 mL of 0.5% w/v Poloxamer 188 solution with continuous stirring on a magnetic stirrer at room temperature. Stirring was continued for 3 h and allowed complete evaporation of the organic solvent. Finally, traces of residual solvent were removed under reduced pressure in a rotary flask evaporator at 40 C for 30 min. The nanocrystal suspension was centrifuged (Beckman Coulter, Ultracentrifuge, Brea, CA) with 25 000rpm at 4 C for 30 min. Lyophilisation of prepared nanocrystals. The FLT nanocrystals dispersion with 5% mannitol (cryoprotectant) was kept in a deep freezer at 70 C for 12 h and lyophilised using a lab freeze-dryer (VirTis Benchtop, SP Scientific, Warminster, PA). Each vial was sealed with rubber stopper and freeze-drying was conducted at 90 C for 7 h. Surface modification of FLT nanocrystals using mPEG. Carboxyl groups on the surface of the nanocrystals (10 mg) were activated by resuspending it in 1 mL isotonic 0.1 M MES saline buffer (pH 5.5) and reacted them with EDAC (10 equiv.) and NHS (10 equiv.) for 1 h. Resultant nanocrystals were centrifuged to remove excess EDAC/NHS and the water-soluble iso urea as byproduct. Activated nanocrystals were resuspended in 1 mL PBS buffer and reacted with mPEG for 24 h. A 100-fold excess of mPEG was used to prevent formation of NC-PEG-NC. The coated nanospheres were centrifuged and washed with PBS buffer to remove any unbound PEG. Determination of solubility of FLT If the solubility of a drug is less than 1.0 mg/mL, it affects the bioavailability due to the absorption problem, hence prediction and observation of solubility is essential task. The apparent solubility of FLT was determined in purified water, buffer solution (pH 1.2), buffer solution (pH 6.8), buffer solution (pH 7.4), methanol and PEG 400 at 37 ± 0.5 C. Accurately weighed quantity of drug was added in 10 mL of solvent in glass vials with rubber stoppers. These vials were kept in an orbital shaking incubator (Remi Instruments Ltd., Mumbai, India) maintained at 37 ± 0.5 C for 24 h. After shaking, the vials were kept in an incubator at 37 ± 0.5 C for equilibrium for 12 h. The solution was

Table 1. Compositions of the different formulation of flutamide nanocrystals with their particle size, PDI, zeta potential and percent entrapment efficiency. Batch no. F1 F2 F3 F4 F5 F6 F7 F8 F9 F10

Drug:polymer ratio

Concentration of surfactant %

Addition of organic phase in to aqueous phase (mL/min)

Particle size (nm) ± SD

Polydispersity index (PDI) ± SD

Zeta potential (mV) ± SD

% EE

1:10 1:05 1:10 1:01 1:05 1:05 1:01 1:10 1:01 1:05

0.50 0.50 0.50 0.75 0.75 0.50 1.00 1.00 0.75 0.50

2.0 1.0 1.0 1.0 1.0 3.0 1.0 1.0 2.0 2.0

152.03 ± 1.1 137.7 ± 1.2 104.53 ± 0.92 121.53 ± 0.73 132.9 ± 1.1 135.4 ± 1.31 133.46 ± 0.87 140.4 ± 1.21 124.46 ± 0.68 162.33 ± 1.04

0.042 ± 0.01 0.045 ± 0.01 0.044 ± 0.05 0.091 ± 0.01 0.045 ± 0.06 0.074 ± 0.01 0.102 ± 0.01 0.22 ± 0.01 0.081 ± 0.05 0.063 ± 0.01

21.06 ± 1.15 30.43 ± 0.77 26.3 ± 1.1 29.73 ± 1.15 31.3 ± 1.05 24.26 ± 1.20 17.6 ± 0.98 20.33 ± 1.25 19.26 ± 1.20 17.6 ± 1.08

66.88 85.5 88.71 52.22 73.78 66.5 88.36 63.8 52.27 73.52

Development of flutamide nanocrystals for i.v administration

DOI: 10.3109/02652048.2014.995731

filtered through 0.45-mm filter (Nylon 66, Millipore, NY11, Billerica, MA), and the filtrate was assayed using UV spectrophotometer at 300 nm, and the solubility was calculated by respective calibration curve.


Measurement of mean particle size, polydispersity index and ZP The mean particle size, polydispersity index (PDI) (Equation (1)) and ZP of all designed batches were characterised by photon correlation spectroscopy using a Zetasizer (Nano ZS 90, Malvern Instruments, Worcestershire, UK). 2 ð1Þ PDI ¼ Zavg

3

188, physical mixture of formulation and lyophilised FLT-loaded PEGylated PLGA nanospheres. XRPD studies X-ray diffraction measurement of pure FLT (A), PLGA-loaded FLT nanocrystals (B) and FLT-loaded PEGylated PLGA nanosphere (C) were performed using X-ray diffractometer (Brucker AXS, D8 Advance, Karlsruhe, Germany) and a Cu-Ka line as a source of radiation. The X-ray tube was run at 40 kV and 30 mA. Samples were mounted in a flat specimen holder, and diffraction patterns were measured using a new Xcelerator detector along with diffracted beam monochromatic. The X-ray diffractogram of each sample was scanned with the diffraction angle increasing from 3 to 60 , 2y angle, at speed 2 /min and at 25 C.

Prior to the measurements, all samples were diluted 100 times with double-distilled water. Based on the Smoluchowski equation, the surface charge of the nanocrystals was determined by measuring the ZP. All measurements were performed in triplicate at 25 C. The mobility is related to the z-potential at the interface using the Smoluchowski equation (Equation (2)) & ð2Þ E ¼ 4"0"r ð1 þ rÞ 6m

Surface topography

where "0 and "r are the relative dielectric constant and the electrical permittivity of a vacuum, respectively, m is the solution viscosity, r is the particle radius and k ¼ (2n0z2e2/"r"0kBT)1/2 is the Debye–Hu¨ckel parameter, n0 is the bulk ionic concentration, z is the valence of the ion, e is the charge of an electron, kB is the Boltzmann constant and T is the absolute temperature. ZP measurements were run at 25 C with electric field strength of 23 V/m.

Haemolysis studies were done to check the interaction of prepared nanospheres with erythrocytes. Primary interaction was studied using Motic microscopic image analysis technique using an optical DMW2-223 digital microscope (Motic Instruments, Toronto, Canada) equipped with a 1/3’’ CCD camera imaging accessory and computer-controlled image analysis software (Motic Images 2000, 1.3 version). In brief, human blood was collected from blood bank (Indira Gandhi Memorial Hospital, Shirpur, India) into tubes containing EDTA solution and was centrifuged at 3000 rpm for 10 min to obtain pelleted erythrocytes, which were washed three times with PBS (pH 7.4). The number of erythrocytes was measured using haemocytometer. PLGA NPs (coated and uncoated) were dispersed in PBS and added into erythrocytes suspension and incubated at 37 C for 2 h. After incubation the suspension was examined under optical microscope to observe haemolysis if any due to PLGA NPs. The images were compared for studying changes in RBC due to NPs. Erythrocytes mixed with saline were taken as control.

Determination of % EE and % LE Percent EE was calculated by determining the amount of unentrapped FLT in the aqueous surfactant solution. The aqueous medium was separated by using Ultra centrifuge; 2 mL of the nanocrystal dispersion of FLT was placed in the allomer tubes, and speed of centrifuge was kept at 15 000rpm for 30 min at 4 C. The concentration of FLT in the aqueous phase was determined using UV visible spectrophotometer (UV 1700, Shimadzu, Kyoto, Japan) at max 300 nm. % EE and % LE were calculated using Equations (3) and (4) amount of drug added amount of drug in supernatant % EE ¼ 100 ð3Þ amount of drug added % LE ¼

amount of drug added amount of drug in supernatant amount of polymer added

100

ð4Þ

Surface morphology of optimised FLT-loaded PEGylated PLGA nanospheres was studied using scanning electron microscope (JSM-6390LV, JEOL, Tokyo, Japan) with 20 kV accelerating voltage. Samples were prepared by placing a droplet of nanocrystal dispersion onto an aluminium specimen stub, dried overnight and sputter-coated with gold prior to imaging. Haemolysis studies

Sterility test Sterility assay was performed according to the European Pharmacopoeia using membrane filtration technique. Ten samples were tested and filtered through a 0.22 m membrane filter. Resultant filtrates were transferred to autoclaved culture media (thioglycolate and casein soya) in conical flask. Incubation was done for one week for thioglycolate and casein soya media at 37 C and room temperature, respectively. In vitro drug release

Thermal analysis Thermal analysis of pure FLT, PLGA, Poloxamer 88, physical mixture of formulation and optimised formulation of FLT-loaded PEGylated PLGA nanocrystals were performed using DSC (STARe System, Mettler-Toledo, Ku¨snacht, Switzerland). The instrument was calibrated with indium at the heating rate 10 C/min. The samples were analysed in sealed and pin-holed standard 40 mL aluminium crucibles. DSC analysis was carried at a heating rate of 10 C/min from 35 C to 300 C. DSC thermograms were recorded for pure FLT, PLGA, Poloxamer

In vitro release of FLT of nanocrystals was assessed using dialysis bag method. The nanocrystals (equivalent to 20 mg drug) were dispersed in PBS (pH 7.4) and placed in a cellulose ester dialysis membrane (Cut-off 12–14 kDa) and sealed with universal closures. The bags were tied to the paddle of the USP dissolution test apparatus (Electrolab, Bombay, India) and dialysed against 900 mL PBS (pH 7.4) containing 0.2% Tween 80 and 0.02% sodium azide as a preservative. In comparison, the drug solution in a cosolvent mixture of 0.9% w/v NaCl/ethanol/PEG-200 (2:1:3 v/v/v) was dialysed using the same dissolution medium.

4


The entire system was incubated at 37 ± 0.5 C under stirring at 100 rpm. At predetermined time intervals, 5 mL of the medium was removed and replaced with the same volume of dissolution medium. All samples were run in triplicates and filtered through a 0.45-mm membrane filter, and the amount of FLT released was analysed by HPLC.


stability chamber (CHM-10S, Remi Instruments Ltd.) at 25 ± 2 C/60 ± 5% RH for 90 days (Krishna and Mayer, 2000; Panyam and Labhasetwar, 2003). These samples were analysed for MPS, PDI, % EE and in vitro drug release at interval of 30 days.

Result and discussion


Pharmacokinetic studies Male Sprague Dawley rats weighing about 200–250 g were selected for the pharmacokinetic studies and maintained in the Central Animal House at R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Maharashtra, India (Resolution Number RCPIPER/IAEC/2012-2013/09). The animals were housed in polypropylene cages, provided standard pellet diet and water ad libitum and maintained under controlled conditions of temperature and humidity with 12-h light and dark cycle. For the pharmacokinetic evaluation, lyophilised FLT-loaded PLGA NPs and FLT-loaded PEGylated PLGA NPs formulations were compared with the FLT suspension in addition to an untreated group receiving only the co-solvent vehicle. Rats were anesthetised with ether inhalation and injected i.v. via the tail vein with a single dose of FLT solution or FLT-loaded polymeric nanospheres (12 mg FLT/kg) per rat. Blood samples (0.2 mL) were collected from the retro-orbital plexus at predetermined time intervals (30 min, 1, 2, 4, 6, 8, 12, 24 and 48 h) in EDTA-pretreated tubes. Samples were centrifuged immediately at 3000 rpm for 10 min. The plasma samples were diluted to 2 mL with methanol, vortexed for 10 min and centrifuged at 8000 rpm for 20 min; 20 mL of the supernatant was injected into the HPLC column to determine the plasma drug concentration. Data analysis The non-compartmental model was considered as a best suited model for calculation of the different pharmacokinetic parameters. The Cmax and Tmax were directly computed from the plasma concentration vs. time plot. The trapezoidal method was used to calculate the concentration-time curve from time 0 h to 48 h (area under curve [AUC] 0 ! 48). The Kinetica 5 (Thermo Fisher Scientific, Demo version, Mumbai, India) software was employed for study. ANOVA was performed and significant differences between control and treatment groups were assessed using Turkey’s range test. Data are presented as mean ± SD, and differences were considered significant about p50.05. Accelerated stability studies The dried powder of FLT-loaded PEGylated PLGA nanocrystals was filled in an amber-coloured glass vials, sealed and placed in Figure 1. Effect of surfactant concentration on mean particle size and % EE.

We applied the set of different formulation variables, such as organic phase (acetone), concentrations of Poloxamer 188 as stabiliser (0.5%, 0.75% and 1% w/v), ratio of organic phase and aqueous phase (1:10), drug:polymer ratio (1:1, 1:5 and 1:10) and rate of addition of organic phase (1.0 mL/min) for the production of FLT-loaded nanocrystals. The effects of these variables on particle size, ZP and % EE have been studied (Figure 1). Concentration of surfactant had very predominant effect on the particle size of the nanocrystals. The particle size was found to be decrease with increase in the surfactant concentration at a constant amount of polymer. A higher surfactant concentration reduces the surface tension and facilitates partitioning during nanoprecipitation at optimum concentration limit. The decrease in the particle size is accompanied by a tremendous increase in the surface area. Thus, the process of primary coverage of the newer surfaces competes with the agglomeration of the uncovered surface. Therefore, increase in the surfactant concentration in the primary dispersion resulted in rapid coverage of the newly formed particle surface. There was no significant effect seen on the PDI and ZP with the change in surfactant concentration. On the basis of desired MPS, PDI, ZP, % EE and in vitro release pattern, an optimised formulation (Batch F7) was selected by quantitative and sophisticated arrangements. The MPS of all the formulations was between 104.53 ± 0.92 and 162.33 ± 1.04 nm, therefore it has displayed the aforesaid particle size range for intravenous administration. An electric charge on each particle surface forms electrical barrier, which results in ‘‘repulsion phenomenon’’, is the ZP of a particular formulation. The desired ZP was observed in the freeze-dried PLGA and the surface-modified PLGA nanocrystals. There was increase in the negative charge of the surface-modified PLGA nanocrystal that might be due to the PEGylation on the surface of PLGA nanocrystals. Solubility studies The solubility studies were performed in different buffer systems and solvents. The solubility of FLT in buffer systems was found to be 1.53 ± 0.145 mg/mL, in buffer (pH 1.2), 1.22 ± 0.108 mg/mL, in buffer (pH 7.4) 1.44 ± 0.180 mg/mL. In addition, FLT solubility in the different solvents like purified water, methanol and PEG 400 were found to be 1.43 ± 0.125, 67.9 ± 5.080 and

DOI: 10.3109/02652048.2014.995731

63.6 ± 5.100 mg/mL, respectively. The solubility studies revealed that the FLT was poorly soluble in buffer system and water, but highly soluble in organic solvents like methanol and PEG 400.


Particle size, PDI and ZP The MPS of the all the formulations were found to be in the range of 104.53 ± 0.92 to 162.33 ± 1.04 nm. The mean particle size of optimized PEGylated formulations was found to be 133.46 ± 0.87 nm. PDI of all the batches (F1–F10) was in the range of 0.042 ± 0.01 to 0.220 ± 0.01, while the PDI of optimised PEGylated formulations was found to be 0.102 ± 0.01. The ZP of optimised FLT loaded a PEGylated PLGA nanocrystal was found to be –34.66 mV. The EE is mainly dependent on the nature of the drug and the polymer phase in which it is encapsulated. EE of all the formulation were obtained from 52.22% to 88.71%. The FLT was entrapped into PLGA NPs due to the hydrophobic nature of PLGA polymer that might leads to high % EE. Percent drug % EE and % LE of the FLT NPs % EE and % LE of the optimised FLT-loaded nanocrystals were found to be 88.36% and 52.29%, respectively. As the FLT is a lipophilic drug and therefore it had greater entrapment in the PLGA matrix. It is revealed that if the polymer concentration was fixed and amount of drug was increased, % EE and % LE were decreased, and after a certain point, the DL was constant. The consequences can be correlated with the volume of the organic phase. If we increase the amount of the drug and the volume of organic solvent is kept constant, the solubilisation capacity of the polymer was decreased. The drug has not been solubilised in the polymer, it gets precipitated and resulted into decrease in the % EE and % LE. DSC studies The DSC thermogram of PLGA, Poloxamer 188, FLT-loaded freeze-dried PLGA nanocrystals and pure FLT are shown in Figure 2. The DSC thermogram showed sharp endothermic peaks for FLT and PLGA at 111.47 C and 53.23 C, respectively. In addition, DSC thermogram of lyophilised PEGylated nanocrystals exhibited an endothermic peak at 139.43 C. The endothermic peak of drug was shifted from 111.47 C to 139.43 C due to the presence of the mannitol, which was used as cryoprotectant during the process of freeze-drying. There were no significant

Figure 2. DSC thermogram of (A) pure PLGA, (B) pure Poloxamer 188, (C) surface modified FLT nanocrystals, (D) pure FLT and (E) physical mixture.


5

differences in the individual endothermic peak and respective endothermic peak observed in the lyophilised PEGylated nanocrystals. Therefore, DSC studies revealed no compatibility issue between excipients and drug. There was no glass transition in thermal effect of pure drug mainly due to its crystalline nature, but the lyophilised FLT-loaded PEGylated nanocrystals had glass transition temperature (Tg) about 203.22 C revealed its amorphous characteristics. In addition, the % crystallinity of pure drug and FLT-loaded surface modified PLGA nanocrystals was found to be 165.34 and 21.79, respectively. There was no significant shift in the position of endothermic peak of DSC thermogram in drug-loaded surface-modified PLGA nanocrystals. The Tg and % crystallinity revealed that FLT exists in amorphous state in freezedried PEGylated nanocrystals. XRPD studies XRPD patterns of the typical crystalline nature of drug defined by observing principal sharp peaks of FLT between 50 and 600 on 2 scale. The FLT-loaded lyophilised PLGA nanocrystals and FLT-loaded lyophilised surface-modified PLGA nanocrystals XRPD peaks of different excipients and drug are shown in Figure 3. The result indicated that the FLT encapsulated in core of surface-modified PLGA nanocrystals and converted the crystalline to amorphous state. Scanning electron microscopy SEM photomicrograph of the pure FLT, FLT-loaded PLGA NPs and FLT-loaded PEGylated NPs are shown in Figure 4. The shape and surface morphology were different among all the formulations. Surface topography of the pure FLT showed irregular shape, but FLT nanocrystals had cone- and needle-like shapes. This might be due to presence of mannitol and poloxamer 188 present over the nanocrystals surface. Poloxamer 188 is polymeric molecule, which might be adsorbed on the particle surface, act as a steric barrier and avoids the close contact of the crystals. Optimised formulation showed non-spherical-shaped particles with porous nature. Haemolysis assay The interaction of cationic polymers with the negatively charged membrane of the erythrocytes can cause cell lysis and release of

6




Figure 3. XRPD of (A) pure FLT, (B) FLTloaded PLGA nanocrystals and (C) FLTloaded surface-modified PLGA nanospheres.

Figure 4. SEM images of (A) pure FLT, (B) FLT-loaded PLGA nanocrystals and (C) FLT-loaded surface-modified PLGA nanospheres.

haemoglobin. The optical microscopic observations of the erythrocytes did not reveal any disruption of cellular membrane or aggregation of the red blood cells. The images of the optical microscope of the formulation are shown in Figure 5. Microscopic

examination revealed changes in the structure of erythrocyte membrane and morphological appearance accompanied with little aggregation after exposure to surface-modified FLT-loaded PLGA nanocrystals.

DOI: 10.3109/02652048.2014.995731


7


Figure 5. Haemolysis study (A) PBS, (B) FLT-loaded PLGA nanocrystals and (C) FLT-loaded surface modified PLGA nanospheres.

Sterility test An optimised formulation was filtered using membrane filtration technique (0.22 mm Nylon 66 Millipore, MY11, Billerica, MA) and aseptically filled in to the flint-coloured vials under laminar air flow in class 100 aseptic area. In sterility tests, none of the tested nanocrystal samples showed any bacterial and/or microbial or fungal growth, neither in the thioglycolate nor in the casein soya medium mainly because of no colour or turbidity was observed after incubation period. Hence, it is confirmed that the surface-modified formulation was free from microorganism or bacteria and fungi. In vitro drug release The percent cumulative drug release (% CDR) of FLT suspension and optimised FLT-loaded PEGylated PLGA nanocrystals were observed in vitro dialysis bag over a time period of 24 h and seven days, respectively (Figure 6). It was observed that, optimised FLTloaded PEGylated PLGA nanocrystals had drug released up to 94.16 ± 2.71% after (168 h) seven days. The in vitro release pattern showed a sustained and continuous release of surfacemodified FLT-nanocrystals. The release profile of FLT nanocrystals was biphasic. This might be due to penetration of aqueous diffusion medium slowly takes place into the polymer layer as they are lipophilic in nature. Then diffusion and relaxation of the polymer would occur and FLT was released. The differential exponent (n) was calculated and the regression coefficient (r2) was determined. Drug release from the optimised FLT-loaded PEG-PLGA nanospheres followed the zero order (r2 ¼ 0.9406) and Peppas models (r2 ¼ 0.9563, n ¼ 0.8405). Values of n lies between 0.43 and 0.85 (spheres) for the optimised formulation indicated non-Fickian transport controlled by diffusion and relaxation of polymer PLGA.

Figure 6. In vitro drug release of FLT suspension and optimized FLTNPs.

In vivo pharmacokinetic studies in rats FLT-loaded PLGA nanocrystals, FLT-loaded PEGylated PLGA nanocrystals and FLT suspension pharmacokinetic profiles (12 mg/kg) are illustrated in Figure 7. The drug circulation time was extended to 48 h for FLT-loaded PEGylated PLGA nanocrystals, and it was substantially longer than that of FLT suspension. PLGA nanocrystals were quickly removed from the circulating system after i.v. administration with a plasma drug concentration about 540.98 and 740.99 ng/mL at 4 h and 16 h, respectively. On the contrary, FLT-loaded PEGylated PLGA nanocrystals exhibited significantly delayed blood clearance with a plasma drug concentration about 766.71 ng/mL at 48 h after i.v. administration. AUC of FLT-loaded PLGA nanocrystals and FLTloaded PEGylated PLGA nanocrystals was increased about 2.88folds and 6.57-folds, respectively. It is revealed that the prolonged

Figure 7. The mean plasma concentration–time curve after a single i.v dose (12 mg/kg) administration of the optimized formulation of FLT suspension, FLT-loaded PLGA NPs and FLT-loaded PEGylated PLGA NPs in rats (n ¼ 6).

Tmax and improved half life with desired blood circulation time via intravenous administration in rats. The surface modified FLTloaded PLGA nanospheres could extend the half-life of FLT from 1.3 h to 14.4 h. There was an inverse relationship between the nanocarrier clearance and its prolonged circulation time. Therefore, it appears that the longer half-life and pronounced increase in the blood residence time of FLT-loaded Pegylated PLGA nanocrystals.

8



Table 2. Accelerated stability parameters of optimized FLT-loaded PEGylated PLGA NPs (n ¼ 3). Test period Stability parameter

Zero month

One month

Two months

Three months

CDR (%) MPS (nm) PDI EE (%)

94.16 ± 2.71 133.46 ± 0.87 0.102 ± 0.01 88.36 ± 1.20

95.21 ± 2.14 130.09 ± 1.07 0.105 ± 0.035 87.56 ± 0.92

95.63 ± 1.09 135.21 ± 1.05 0.102 ± 0.020 89.32 ± 1380

95.32 ± 1.15 134.42 ± 1.13 0.103 ± 0.027 84.45 ± 1.18


Accelerated stability studies Based on measurement of particle size, PDI, % EE and in vitro drug release accelerated stability studies of optimised formulation was carried out at an interval of 30 days (Table 2). Particle size optimised formulation was found to be in the range of 130.09 ± 1.07 nm to 135.21 ± 1.05 nm. The EE (%) of the optimised batch was found to be between 84.45 ± 1.18 and 88.36 ± 1.20. There were no significant changes in particle size, % EE, PDI and % CDR after 90 days of storage. Therefore, optimised formulation was found to be stable at 25 ± 2 C/60± 5% RH in short-term stability studies. The hydrophilic shell of mPEG could also retain their plasma drug concentration for a longer time as compared with PLGA nanocrystals. Based on the unique findings of the pharmacokinetic studies, mPEG hydrophilic shell that suppress opsonisation through generating a steric barrier preventing hydrophobic interactions of plasma opsonins with the nanocrystal surface and thus inhibiting the uptake by RES. Furthermore, PEGylated PLGA nanocrystals was found to be substantially prolong the drug systemic circulation compared with PLGA nanocrystals. FLT was successfully encapsulated into PLGA NPs by nanoprecipitation method, and surface modification was done using mPEG to avoid the opsonisation through i.v. administration. The sterilisation of the optimised formulation was done using membrane filtration method (0.22 mm) and filled by aseptic technique. The in vitro release pattern showed a sustained and continuous drug release from the optimised formulation of surface modified NPs for (168 h) seven days. The pharmacokinetic studies revealed that optimised formulation had prolonged Tmax and enhanced half-life in comparison with simple FLT suspension. It can be concluded that mPEG-functionalised PLGA nanocrystals have advantages over uncoated PLGA nanocrystals. Hence, mPEG functionalised PLGA NPs are promising for use in prostate cancer therapy.

Acknowledgements The authors are grateful to Cipla (I) Ltd, Bangalore, India, and Wockhardt Ltd., Aurangabad, India, for the gift sample of Flutamide and PLGA. The authors would like to thank Dr. S. J. Surana, Principal (R C Patel Institute of Pharmaceutical Education & Research, Shripur, Maharashtra, India) for providing the facilities necessary to carry out our research work.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

References Abdelwahed W, Degobert G, Stainmesse S, Fessi H. Freeze-drying of nanoparticles: Formulation, process and storage considerations. Adv Drug Del Rev, 2006;58:1688–713.

Acharya S, Sahoo S. PLGA nanoparticles containing various anticancer agents and tumour delivery by EPR effect. Adv Drug Del Rev, 2011; 63:170–83. Anitha A, Uthaman S, Nair S, Jayakumar R, Lakshmanan V. Enhanced delivery system of flutamide loaded chitosan-dextran sulphate nanoparticles for prostate cancer. J Biomed Nanotechnol, 2012;9:1–13. Cho HS, Dong Z, Pauletti GM, Zhang J, Xu H, Gu H, Wang L, Ewing RC, Huth C, Wang F, Shi D. Fluorescent, superparamagnetic nanospheres for drug storage, targeting, and imaging: A multifunctional nanocarrier system for cancer diagnosis and treatment. ACS Nano, 2010;4: 5398–404. Chorny M, Fishbein I, Danenberg H, Golomb G. Lipophilic drug loaded nanospheres prepared by nanoprecipitation: Effect of formulation variables on size, drug recovery and release kinetic. J Control Release, 2002;83:389–400. Cruz L, Tacken P, Fokkink R, Figdor C. The influence of PEG chain length and targeting moiety on antibody-mediated delivery of nanoparticle vaccines to human dendritic cells. Biomaterials, 2011; 32:6791–803. Danhier F, Ansorena E, Silva J, Coco R. PLGA-based nanoparticles: An overview of biomedical applications. J Cont Release, 2012;161: 505–22. Elgindy N, Elkhodairy K, Molokhia A, Elzoghby A. Biopolymeric microparticles combined with lyophilized monophase dispersions for controlled flutamide releas. Int J Pharm, 2011;411:113–19. Elzoghby A, Helmy M, Samy W. Spray-dried casein-based micelles as a vehicle for solubilization and controlled delivery of flutamide: Formulation, characterization, and in vivo pharmacokinetics. Eur J Pharm Biopharm, 2013;84(3):487–96. Fizazi K, Navone NM. Preclinical models of prostate cancer. Bull Cancer 2005;92:129–41. Fredenberg S, Wahlgren M, Reslow M, Axelsson A. The mechanisms of drug release in poly (lactide-co-glycolic acid) based drug delivery systems – A review. Int J Pharm, 2011;415:34–52. Gref R, Domb A, Quellec P, Blunk T, Mu¨ller R, Verbavatz J. The controlled intravenous delivery of drugs using PEG-coated sterically stabilized nanospheres. Adv Drug Deliv Rev, 2012;64:316–26. Gronberg H. Prostate cancer epidemiology. Lancet, 2003;361:859–64. Hancock BC, Zografi G. Characteristics and significance of the amorphous state in pharmaceutical systems. J Pharm Sci, 1997;86: 1–12. Heffery J, Davis SS, O’Hagan DT. The preparation and characterization of poly (lactide-co-glycolide) microparticles. I. Oil-in-water emulsion solvent evaporation. Int J Pharm, 1991;77:169–75. Hodgson MC, Astapova I, Hollenberg AN, Balk SP. Activity of androgen receptor antagonist bicalutamide in prostate cancer cells is independent of NCoR and SMRT Corepressors. Cancer Res, 2007;67: 8388–95. Ige PP, Baria RK, Gattani SG. Fabrication of fenofibrate nanocrystals by probe sonication method for enhancement of dissolution rate and oral bioavailability. Colloids Surf B Biointerfaces, 2013;108:366–73. Jain V, Swarnakar N, Mishra P, Verma A, Kaul A, Jain N. Paclitaxel loaded PEGylated gleceryl monooleate based nanoparticulate carriers in chemotherapy. Biomaterials, 2012;33:7206–20. Jinping W, Xiangyuan Y, Delin L, Duan L. Bioavailability and pharmacokinetics of flutamide tablets studied by determination of its active metabolites in serum in Chinese subjects. J Chinese Pharm Sci, 1999;8(3):181–4. Keck CM, Muller RH. Drug nanocrystals of poorly soluble drugs produced by high pressure homogenization. Eur J Pharm Biopharm, 2006;62:3–16. Krishna R, Mayer LD. Multidrug resistance in cancer. Mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs. Eur J Pharm Sci, 2000;11:265–83. Kumari A, Yadav S, Yadav S. Biodegradable polymeric nanoparticles based drug delivery systems. Colloids Surf B Biointerfaces, 2010;75: 1–18. Laverman P, Carstens MG, Moghimi SM. Recognition and clearance of methoxy poly(ethylene glycol) 2000-grafted liposomes by macrophages with enhanced capacity: Implications in experimental and clinical oncology. Biochem Biophys Acta, 2001;1523:227–9. Moghimi SM, Hunter AC, Murray JC. Long circulating and targetspecific nanoparticles: Theory to practice. Pharmacol Rev, 2001;53: 283–318.


DOI: 10.3109/02652048.2014.995731

Muller RH, Runge S, Ravelli V, Mehnert W, Thunemann AF, Souto SB. Oral bioavailability of cyclosporine: Solid lipid nanoparticles versus drug nanocrystals. Int J Pharm, 2006;317:82–9. Mu¨ller RH, Keck CM. Twenty years of drug nanocrystals: Where are we, and where do we go? Eur J Pharm Biopharm, 2012;80:1–3. Nesalin J, Gowthamarajan K, Somashekhara C. Formulation and evaluation of nanoparticles containing flutamide. Int J Chem Tech Res, 2009;1:1331–4. Niopas I, Daftsios A. Determination of 2-hydroxyflutamide in human plasma by high-performance liquid chromatography and its application to pharmacokinetic studies. J Chrom B, 2001;759:179–83. Okamoto R, Delansoren R, Wakimoto N, Doan NB, Akagi T, Shen M, Ho QC, Said JW, Koeffer HP. Inecalcitol, an analog of 1a, 25(OH)2 D3, induced growth arrest of androgen dependent prostate cancer cells. Int J Cancer, 2012;130:2464–73. Owens D, Peppas N. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticle. Int J Pharm, 2006;307:93–102. Palumbo P, Andrea P, Daniela T, Pasquale P. Fluorinated and pegylated polyaspartamide derivatives to increase solubility and efficacy of flutamide. J Drug Target, 2012;20:433–41. Pamujula S, Hazari S, Bolden G, Graves R, Chinta A, Dash S. Cellular delivery of PEGylated PLGA nanoparticles. J Pharm Pharmacol, 2012; 64(1):61–7.


9

Panyam J, Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Del Rev, 2003;55:339–47. Parveen S, Sahoo SK. Long circulating chitosan/PEG blended PLGA nanoparticle for tumor drug delivery. Eur J Pharmacol, 2011;670: 372–83. Sah H, Thoma LA, Desu HR, Sah E, Wood GL. Concept and practices used to develop functional PLGA based nanoparticulate systems. Int J Nanomed, 2013;8:747–65. Sahoo NG, Abbas A, Li CM. Micro/nanoparticle design and fabrication for pharmaceutical drug preparation and delivery applications. Curr Drug Ther, 2008;3:78–97. Sharma G, Modgil A, Layek B, Arora K, Sun C, Law B, Singh J. Cell penetrating peptide tethered bi-ligand liposomes for delivery to brain in vivo: Biodistribution and transfection. J Control Release, 2013;167: 1–10. Sternal R, Nugara N. 2001. Flutamide. In: Brittain HG, ed. Analytical profiles of drug substances and excipents, vol. 27. San Diego: Academic Press, pp. 118–57. Svenson S. 2004. Overview: Carrier-based drug delivery. In: Svenson S, ed. Carrier-based drug delivery. ACS Symposium Series 879. Washington, DC: American Chemical Society, pp. 2–23. Zuo Z, Tam Y, Diakur J, Wiebe L. Hydroxypropyl-B-cyclodextrinflutamide inclusion complex. II. Oral and intravenous pharmacokinetics of flutamide in the rat. J Pharm Pharm Sci, 2002;5(3):292–8.

Construction and evaluation of controlled-release delivery system of Abamectin using porous silica nanoparticles as carriers.

Preparation and characterization of amphiphilic calixarene nanoparticles as delivery carriers for paclitaxel.

Preparation of solid lipid nanoparticles as drug carriers for levothyroxine sodium with in vitro drug delivery kinetic characterization.

Cross-linked chitosan microspheres: preparation and evaluation as a matrix for the controlled release of pharmaceuticals.

Silk fibroin nanoparticles prepared by electrospray as controlled release carriers of cisplatin.

Preparation and optimization of chlorophene-loaded nanospheres as controlled release antimicrobial delivery systems.

Bovine serum albumin nanoparticles as controlled release carrier for local drug delivery to the inner ear.

Preparation, characterization and in vivo evaluation of nanoemulsions for the controlled delivery of the antiobesity agent N-oleoylethanolamine.

Nano-engineered erythrocyte ghosts as inhalational carriers for delivery of fasudil: preparation and characterization.

Nanostructured lipid carriers based nanogel for meloxicam delivery: mechanistic, in-vivo and stability evaluation.

Nanoparticles for controlled delivery and sustained release of chlorhexidine in the oral environment.

Preparation and evaluation of a controlled drug release of repaglinide through matrix pellets: in vitro and in vivo studies.

Hyaluronic acid based micelle for articular delivery of triamcinolone, preparation, in vitro and in vivo evaluation.

Preparation of albumin based nanoparticles for delivery of fisetin and evaluation of its cytotoxic activity.

Polymeric nanocarriers for magnetic targeted drug delivery: preparation, characterization, and in vitro and in vivo evaluation.

Preparation and in vivo evaluation of multifunctional ⁹⁰Y-labeled magnetic nanoparticles designed for cancer therapy.

Liquid Crystalline Nanoparticles as an Ophthalmic Delivery System for Tetrandrine: Development, Characterization, and In Vitro and In Vivo Evaluation.

in vivo evaluation.

Functional nanovalves on protein-coated nanoparticles for in vitro and in vivo controlled drug delivery.

Preparation, characterization, and safety evaluation of poly(lactide-co-glycolide) nanoparticles for protein delivery into macrophages.

131I-Traced PLGA-Lipid Nanoparticles as Drug Delivery Carriers for the Targeted Chemotherapeutic Treatment of Melanoma.

Preparation of gelatin films incorporated with tea polyphenol nanoparticles for enhancing controlled-release antioxidant properties.

Novel nanoliposomal delivery system for polydatin: preparation, characterization, and in vivo evaluation.

Lipid nanoparticles for oral delivery of raloxifene: optimization, stability, in vivo evaluation and uptake mechanism.