loaded PLGA nanoparticles and cytarabine loaded PLGA-mPEG ... Biodistribution studies showed that the PEGylated nanoparticles of cytarabine were.
Journal of Microencapsulation, 2011; 28(8): 729–742 ß 2011 Informa UK Ltd. ISSN 0265-2048 print/ISSN 1464-5246 online DOI: 10.3109/02652048.2011.615949
Long circulating PEGylated PLGA nanoparticles of cytarabine for targeting leukemia Khushwant S. Yadav1, Sheeba Jacob2, Geetanjali Sachdeva2, Krishna Chuttani3, Anil K. Mishra3 and Krutika K. Sawant1 Journal of Microencapsulation Downloaded from informahealthcare.com by HINARI on 08/29/12 For personal use only.
1
TIFAC- Centre of Relevance and Excellence in NDDS, Pharmacy Department, The M. S. University of Baroda, Fatehgunj, Vadodara 390002, Gujarat, India, 2Primate Biology, National Institute for Research in Reproductive Health, Jehangir Merwanji Street, Parel, Mumbai 400012, India, and 3Division of Radiopharmaceuticals and Radiation Biology, Institute of Nuclear Medicine and Allied Sciences, Brig. S.K. Mazumdar Road, Delhi 110 054, India Abstract The present investigation was aimed at developing PEGylated PLGA nanoparticles of cytarabine. PLGA Nanoparticles were prepared by modified nanoprecipitation method, optimized for mean particle size (152 6 nm) and entrapment efficiency (41.1 0.8%) by a 32 factorial design. The PEGylated PLGA nanoparticles of cytarabine had a zeta potential of 7.5 1.3 mV and sustained the release of cytarabine for 48 h by Fickian diffusion. The IC50 values for L1210 cells were 6.5, 5.3, and 2.2 mM for cytarabine, cytarabine loaded PLGA nanoparticles and cytarabine loaded PLGA-mPEG nanoparticles respectively. Confocal microscopy and flow cytometry showed that the nanoparticles were internalized by the L1210 cells and not simply bound to their surface. Biodistribution studies showed that the PEGylated nanoparticles of cytarabine were present in significantly higher concentrations in blood circulation as well as in brain and bones and avoided RES uptake as compared to the free drug. Keywords: long circulating, PEGylated PLGA nanoparticles, cytarabine, cellular uptake, brain, bone, biodistribution
Introduction
As a result, Cyt is usually required to be administered intravenously and is available as multidose vials. Thus, conventional therapy suffers from lack of specificity, that is, it also inhibits normal cell growth which eventually leads to necrosis of normal cells. Hence, improvement in treatment modalities for leukemia requires a drug delivery system which is long circulating in blood so that it can penetrate the desired sites of action i.e. the bone marrow and brain, and can provide sustained release of the drug. The polymeric materials used for the carrier have to be biodegradable and biocompatible. Colloidal drug carriers such as polymeric nanoparticles have recently gained attention for targeting and sustaining the release of the drug. Nanoparticles (NP) are solid or semisolid colloidal particles ranging in size from 10–1000 nm (Couvreur et al., 1995). NP have the ability to
Leukemia, the cancer of the blood, is characterized by the widespread, uncontrolled proliferation of large number of abnormal blood cells, usually of the white cell lineages, which take over the bone marrow and often spill out into the blood stream. In leukemia, non-functioning cells accumulate in the marrow and blood. Moreover, leukemia cells flourish into the hideouts of brain, eventually causing fatal complications. Cytarabine (Cyt) is an antimetabolite used primarily for acute myelogenous leukemia and meningeal leukemia. It is metabolized intracellularly into its active triphosphate form (cytosine arabinoside triphosphate) which damages DNA. However, Cyt is poorly absorbed from gastrointestinal tract with less than 20% bioavailability and has a short half life of 2–4 h (Ho and Frei, 1971).
Address for correspondence: Krutika K. Sawant, Pharmacy Department, Faculty of Technology and Engineering, The Maharaja Sayajirao University of Baroda, Kalabhavan, Vadodara 390001, Gujarat, India. Tel: þ91-2652434187. Fax: þ91-2652418927. E-mail: dr_krutikasawant@rediffmail.com (Received 30 Dec 2010; accepted 18 Jul 2011) http://www.informahealthcare.com/mnc 729
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deliver a wide range of drugs to varying areas of the body for sustained periods of time (Prabha and Labhasetwar, 2004). Nanoparticle preparations, mainly used for intravenous injection, are employed for the sustained release of drugs, and for the passive targeting of anticancer drugs. There are two major challenges faced by intravenously injected particulate system for achieving targeting; one is avoidance of the macrophages of the mononuclear phagocytic system and second is that the carrier should reach the desired site of action (Goppert and Muller, 2003). When administered intravenously, the NP act as foreign particulates and are cleared by resident macrophages of the reticuloendothelial system (RES). Approaches employed to avoid this uptake of the injected particles by the RES include modification of the particle properties such as surface charge and particle size (Moghimi et al., 2001; Yadav et al., 2011). An example of modification of surface charge is PEGylation, which involves the addition of polyethylene glycol over the surface of the NP (Haley and Frenkel, 2008). Poly(lactide-coglycolide) (PLGA) has been one of the most widely used polymers for preparation of biodegradable and biocompatible nanoparticles (Avgoustakis et al., 2003). However, conventional polymeric NPs are rapidly removed from the blood stream after IV administration by the macrophages of the mononuclear phagocyte system and hence require special efforts to overcome this phenomenon. PLGA has been modified by use of methoxy Poly(ethylene glycol) (mPEG) to modify its hydrophilicity and to prepare stealth nanoparticles which could avoid, or at least reduce, the uptake by phagocytes and prolong drug residence time in blood circulation (Gref et al., 1994). Nanoparticles prepared from PEG modified PLGA have been extensively investigated as drug carriers due to their biodegradability, biocompatibility and ability to provide controlled release (Avgoustakis et al., 2003). After intravenous administration, the PLGA-mPEG nanoparticles were reported to remain in the systemic circulation for hours, whereas the PLGA nanoparticles were removed from blood within few minutes (Panagi et al., 2001). The PEG layer provides a steric barrier to the particle and reduces their opsonization, thereby making them long circulating (Moghimi and Davis, 1994). Against this background, the present investigation was aimed at developing long circulating, Cyt loaded PLGAmPEG based biodegradable nanoparticles which would have sustained release. It was hypothesized that the PEGylation of PLGA NP would provide steric barrier for longer blood circulation and therefore facilitate distribution of the drug to the brain and bones.
Materials and methods Materials Chemicals and reagents Cyt was obtained as a gift sample from Biocon Ltd., Bangalore, India. Poly (DL lactide-co-glycolide) PLGA 50:50 (inherent viscosity 0.22 dl/g) was obtained as a gift
sample from Boehringer Ingelheim Ltd., Germany. Pluronic F-68 (BASF) was obtained as a gift sample from Alembic Ltd., Vadodara, India. Chloroform, Methanol, Acetone, Potassium dihydrogen phosphate, Disodium hydrogen phosphate, Hydrochloric acid and Sodium hydroxide were obtained from SD Fine Chemicals, Mumbai, India. 6- Coumarin was obtained from Polysciences Inc., USA. 3-(4,5-dimethylthiaol-2-yl)-2,5diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, USA), were obtained as gift samples from NIRRH, Mumbai, India. Fluoromount-G was obtained from Southern Biotech Associates, USA. Polycarbonate membranes (sizes 0.2, 0.45, 2 mm and 25 mm) were obtained from Whatman, USA. Synthetic cellulose membrane having molecular weight cut off (MWCO) of 12 000 daltons was purchased from Himedia Labs, Mumbai, India. Stannous octoate and Monomethoxypoly(ethyleneglycol) (mPEG, molecular weight 5000) were purchased from Sigma, St. Louis, USA. Technetium-99 m (99mTc) as Pertechnetate (TcO4-) was obtained from Regional Center for Radiopharmaceutical division (Northern region), Board of Radiation and Isotope Technology, New Delhi, India. Stannous chloride and Silica gel coated fibre sheets (Gelman Sciences Inc., Ann Arbor, MI, USA) were kindly supplied by INMAS, New Delhi, India. Cell lines and their sub-culturing L1210 mouse leukemia cell lines were obtained as gift samples from National Center for Cell Sciences, Pune, India and was maintained in DMEM media supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin with 10% fetal bovine serum (Sigma, USA; obtained as gift samples from NIRRH, Mumbai, India) at 37 C in a 5% CO2 humidified atmosphere. L1210 cells were grown exponentially as a suspension culture. Animals Balb/c mice of either sex weighing 20–25 g and SpragueDawley rats of either sex weighing 200–250 g were obtained from INMAS, New Delhi. All animal experiments were approved and carried out as per the guidelines of Institutional Animal Ethics Committee, INMAS, New Delhi, India.
Methods Preparation of cytarabine loaded PLGA-mPEG nanoparticles Poly(lactide-co-glycolide)–monomethoxy(polyethyleneglycol) copolymer (PLGA-mPEG) was synthesized by the ring opening polymerization by melting PLGA and mPEG and using stannous octoate as catalyst as described previously by the authors (Yadav et al., 2010). Modified nanoprecipitation method was used for the preparation of nanoparticles as described earlier (Yadav and Sawant, 2010a). Briefly, 5 mg of Cyt was dissolved in an aqueous phase consisting of 0.3 mL of distilled water and 0.6 mL of methanol. Then 25 mg of PLGA-mPEG copolymer was dissolved in appropriate quantity of the non-solvent (chloroform) and this
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Long circulating PEGylated PLGA nanoparticles of cytarabine for targeting leukemia solution was added drop wise to the aqueous phase under stirring. Finally, the resulting emulsion was added drop wise to 10 mL of distilled water containing 0.5% w/v of Pluronic F-68. The non solvent was then allowed to evaporate by stirring over night. Nanoparticles were recovered from the nanodispersion by centrifugation (Sigma Centrifuge, USA) for 30 min at 25 000 rpm, and washed twice with distilled water to remove unentrapped drug. The dispersion was lyophilized (Heto Dry Winner, Denmark) for 24 h to yield freeze dried nanoparticles. Samples were frozen at 70 C and placed immediately in the freeze-drying chamber. Sucrose at 20% w/w of the total solid content was used as cryoprotectant. Cyt loaded PLGA NP were similarly prepared for comparison by substituting the block copolymer with PLGA. To investigate the in vitro cellular uptake of NP, fluorescent NPs were prepared by substituting drug with 6-coumarin (0.01% w/w). Optimization by factorial design Nine batches were prepared as per 32 factorial design to study the effect of two independent variables, ratio of drug and polymer (X1) and volume of the non solvent (X2) on mean particle size (Y1) and % entrapment efficiency (Y2) of the Cyt-PLGA-mPEG nanoparticles. Each factor was tested at three levels designated as 1, 0 and þ1 (Mehta et al., 2007). The values of the factors were transformed to allow easy calculation of co-efficient in the polynomial equation. Interactive multiple regression analysis and F-statistics was utilized in order to evaluate the response (Yadav and Sawant, 2010b). The regression equation for the response was calculated using the Equation (1). Response: Y ¼ b0 þ b1 X1 þ b2 X2 þ b3 X12 2
þ b4 X2 þ b5 X1X2
ð1Þ
Where, Y is the measured response and b is the estimated coefficient for the factor X. The coefficients corresponding to linear effects (X1 and X2), interaction (X1 X2), and the quadratic effects (X12 and X22) were determined from the results of experiments. Contour plots were drawn to explain the relationship between the independent and dependent variables using STATISTICA software at the values of X1 and X2 between 1 and þ1 at predetermined values of particle size and %EE.
Evaluation of nanoparticles Mean particle size (MPS) and zeta potential The freeze dried nanoparticles were dispersed in distilled water for particle size analysis using Malvern Zetasizer 3000 (Malvern Instruments, UK) which measures the size based on photon correlation spectroscopy (PCS). Zeta potential was studied to determine the surface charge on the nanoparticles using Malvern Zetasizer 3000, (Malvern Instruments, UK) by electrophoretic light scattering (ELS). All the measurements were carried out in triplicate.
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Entrapment efficiency The entrapment efficiency was determined by extracting and quantifying the encapsulated drug using a validated UV-spectrophotometric method. Accurately weighed NPs (100 mg) were added to 10 mL of 1:1 mixture of chloroform and methanol and shaken at room temperature. The resulting solution was evaporated to dryness, and the dried residue was reconstituted with 5 mL of phosphate buffer saline (PBS). The reconstituted dispersion was centrifuged at 10 000 rpm for 15 min. In this extraction procedure, the drug was solubilised in phosphate buffer saline and the polymer which was not soluble remained in the pellet. The supernatant was analyzed for drug content using UVspectrophotometer (Shimadzu 1700, Japan) at 271 nm. The % entrapment efficiency (EE) was calculated using the following formula %EE ¼
Amount of drug in the NPs 100 drug added in the formulation
Scanning electron microscopy The freeze dried nanoparticles were fastened onto a brass stub with double-sided adhesive tape. The stub was fixed into a sample holder and placed in the vacuum chamber of a scanning electron microscope (JEOL JSM 1560 LV, Japan) and observed under low vacuum (1023 mm HG).
In-vitro drug release study and drug release kinetics The dialysis bag diffusion technique was used to evaluate the in vitro drug release (Levy and Benita, 1990). The NP dispersion corresponding to 10 mg of cytarabine was placed in a dialysis bag (MWCO 12 000 daltons) which was tied and placed into 200 mL of PBS (pH 7.4) maintained at 37 C with continuous magnetic stirring in a beaker. At predetermined time intervals, aliquots were withdrawn from the acceptor compartment and replaced by the same volume of phosphate buffer saline. The drug content of the samples was determined spectrophotometrically at 271 nm. The tests were carried out three times and cumulative percentage drug release was calculated. The data was statistically analysed using the Sigmastat software (Sigma Stat, USA). Data obtained from in vitro release studies were fitted to various kinetic equations to understand the mechanism of drug release from formulated nanoparticles. The following plots were plotted: Qt vs. t (zero order kinetic model); log(Q0 Qt) vs. t (first order kinetic model,) and log Mt/M1 ¼ nlog t þ k (Peppas equation). Where Qt is the amount of drug released at time t and Q0 is the initial amount of drug present (Korsmeyer et al., 1983). Mt/M1 is the fraction of drug released after time t in respect to amount of drug released at infinite time, k is the rate constant and n is the diffusional exponent which characterizes the transport mechanism (Peppas, 1985).
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Cytotoxicity assay
Flow cytometry
Cytotoxicity was determined by the use of 3-(4,5dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay (Cole, 1986). Briefly, 100 mL of L1210 DU145 cell (5 104 cells/mL) were seeded onto each well of 96-well plate (Corning Incorp. Life Sciences, USA) and incubated for 24 h at 37 C in a humidified CO2 atmosphere. After incubation, 100 mL of DMSO medium containing test sample (pure drug or drug loaded NP at a concentration of 5, 10, 20, 50 and 100 mM) or complete medium for untreated controls were distributed in the 96-well plates and the plates were then incubated at 37 C for 24 h. The culture medium was subsequently removed and medium containing 20 mL MTT reagent (5 mg/mL) was added to each culture well. After 4 h of incubation, the cells were washed carefully with PBS and the crystals were dissolved by the addition of 10% SDS for 20 min with occasional shaking. Finally, absorbance, at 570 test wavelength and 630 reference wavelength, was measured using an automated microplate reader (Labsystem Multiskan, Helsinki, Finland). In each experiment, the test sample was analyzed in six individual wells. Cell survival was estimated as a percentage of the corresponding control. Cellular sensitivity to the drugs has been defined by the term IC50. The IC50 value is defined as the concentration of drugs that inhibited cell growth by 50% for each cell line to different chemotherapeutic agents (Segawa et al., 2005). The concentration of drug or NP formulation required to inhibit cell proliferation by 50% (IC50) was determined by plotting the percentage of cell growth inhibition versus the concentration of pure drug or NP formulation. IC50 means the concentration of drug required to inhibit growth of 50% of the untreated cell population (Mishra and Jain, 2003).
For flow cytometry analysis, cells were incubated with nanoparticles (6-Coumarin was used as fluorescent marker) in DMEM media supplemented with 10% fetal bovine serum (FBS). After 4 h of incubation, the cells were washed with PBS and then harvested for further analysis. The cells (1 104 counts) were analysed by flow cytometry (FACS Calibur, USA) with a forward scattering (FSC) range between 200 and 600 in a linear scale (Yoo and Park, 2004).
Confocal microscopy Confocal microscopy was used to visualize cellular uptake of the nanoparticles by cells as described by Yoo and Park (2004). After initial passage in tissue culture flasks, cells were grown to semi-confluence in DMEM supplemented media in 6-well tissue culture plates on Corning’s circular glass cover-slips at 37 C and in 5% CO2 atmosphere. After filtration, the nanoparticle suspension was incubated with the cells at 37 C for a period of 1, 2, 3, 4 and 24 h. Then media was removed and the plates were washed thrice with sterile PBS. After the final wash, the cells were fixed with 4% (v/v) paraformaldehyde in PBS for 1.0 h at room temperature and were washed four times with PBS. Individual cover-slips were then mounted cell side up on clean glass slides with fluorescence-free glycerol based mounting medium, Fluoromount-G. Differential interference contrast (DIC) and fluorescence images were acquired with a confocal microscope (Zeiss Confocal LSM 410, USA) at an excitation wavelength of 495 nm and an emission wavelength of 520 nm.
Radiolabeling of formulations Cyt, Cyt-PLGA NP and Cyt-PLGA-mPEG NP were radiolabeled with Technetium-99 m (99mTc) as per method described by Theobald (1990). Briefly, the pertechnetate (TcO4-) (2 mCi) was reduced with stannous chloride (in 10% acetic acid) and the pH was adjusted to 6.5 with 0.5 M sodium bicarbonate. The test formulation to be radiolabeled was added to it in a concentration of 1 mg/mL and incubated at room temperature for 10 min.
Labeling efficiency and stability of the 99mTc labeled complexes The labeling efficiency of the 99mTc labeled drug and NP was determined by instant thin layer chromatography using ITLC–SG mini strips as described earlier by the authors (Yadav et al., 2010). The stability of 99mTc labeled complexes was determined in vitro in human serum by ascending ITLC technique (Reddy et al., 2004). The labeled complex (0.1 mL) was incubated with freshly collected human serum (0.4 mL) at 37 C. The stability study was performed by determining the changes in labeling efficiency at regular intervals up to 24 h and analysing the chromatograms in gamma ray spectrometer (GRS23C, Electronics Corporation of India Limited, India).
Biodistribution studies Balb/c mice having body weight between 20–25 g were used in groups of four for these studies. Radiolabeled formulations were injected intravenously (100 mL) in the tail vein of the mice. At 1 h, 4 h and 24 h after injection, animals were anaesthetized with chloroform and blood was collected by cardiac puncture in pre-weighed tubes. Mice were then dissected and each organ to be tested (heart, lungs, liver, spleen, kidneys, stomach, intestine, muscle and brain) was removed. The whole organs were weighed and the radioactivity was counted per gram of the tissue/ organ in the gamma ray spectrometer (GRS23C, Electronics Corporation of India Ltd.). The radioactivity remaining in the tail was also measured and subtracted from the total radioactivity dose administered to the mice.
Long circulating PEGylated PLGA nanoparticles of cytarabine for targeting leukemia
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Table 1. Formulation of CYT-PLGA-MPEG NP batches by 32 factorial design: Factors, their levels and transformed values, response: %EE and MPS. Batch no.
Real value
Transformed values 2
Response 2
Drug: Polymer ratio (mg) (X1)
Volume of the Non solvent (mL) (X2)
X1
X2
X1
X2
X1X2
MPS (nm) SD* (Y1)
% EE SD* (Y2)
1:5 1:5 1:5 1:10 1:10 1:10 1:15 1:15 1:15
2 5 8 2 5 8 2 5 8
1 1 1 0 0 0 1 1 1
1 0 1 1 0 1 1 0 1
1 1 1 0 0 0 1 1 1
1 0 1 1 0 1 1 0 1
1 0 1 0 0 0 1 0 1
187 3.1 165 2.3 152 6.5 192 5.2 176 4.8 165 3.2 198 3.1 179 2.3 169 5.7
29.4 0.3 32.5 1.2 41.1 0.4 32.6 1.2 35.7 2.2 38.2 3.1 36.1 0.8 38.4 1.2 41.1 0.8
CPM NP1 CPM NP2 CPM NP3 CPM NP4 CPM NP5 CPM NP6 CPM NP7 CPM NP8 CPM NP9
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Note: *All the tests were carried out in triplicate.
Blood clearance studies Sprague-Dawley rats of either sex weighing between 200–250 g were selected for the blood clearance studies. Nanoparticles containing 200 mCi of 99mTc were injected into the tail vein of the rats. The blood samples were collected after a period of 15 min, 30 min, 1 h, 2 h, 4 h and 24 h from the retro-orbital plexus of rat eye and analyzed for the radioactivity in gamma ray spectrometer. The blood was weighed, and the radioactivity in whole blood was calculated by considering the volume of blood as 7.5% of the total body weight.
The contour plot as shown in Figure 1(a) for MPS was found to be linear indicating that linear relationship existed between X1 and X2 variables. It was concluded from the contour plot that the MPS of 152 nm could be obtained with X1 range from 1 level (1:5) to 0.7 level (1:6) and X2 range from 0.6 (2.5 mL) to 1.0 (8 mL). The % EE of Cyt in PLGA-mPEG NP varied from 29.4 0.3% to 41.1 0.8%. The highest %EE was observed at two levels of X1 be at lowest (1:5) as well as at highest (1:15) level in both cases, X2 was obtained at the highest level (8 mL) in batches CPM NP3 and CPM NP9 respectively. Equation (3) is for the full model. Y2ð%EEÞ ¼ 34:91 þ 2:1X1 þ 3:71X2 þ 0:93X12 þ 0:88X22 2:35X1X2
Statistical analysis Statistical comparisons were made using one way ANOVA by using Microsoft Excel software. The level of significance was considered at p 5 0.05.
Results and discussion Mean particle size and entrapment efficiency Table 1 displays the values of factors, their levels and transformed values and values of both the responses, %EE and MPS as per 32 factorial design. The mean particle size of NP ranged from 152 6 to 198 3 nm. The lowest MPS was observed at lowest level of X1 (1:5) and highest level of X2 (8 mL) in batch CPM NP3. The regression equation for Y1 (MPS) is given by Equation (2). Y1ðMPSÞ ¼ 175:11 7:0X1 15:16X2 2:66X12 þ 3:83X22 þ 1:5X1X2
ð2Þ
The R2 value for the full model was 0.994179, indicating that the model was able to explain 99.42% variability around its mean in the results. Full Model F value of 102.468 was more than the tabulated F value (Ftab ¼ 9.01), indicating that the full model was significant (Hocking, 1976).
ð3Þ
The positive values for X1 and X2 values in Equation (3) showed that % EE greatly depended on the independent variables, namely the drug polymer ratio and volume of non solvent. It was concluded from the linear contour plots (Figure 1(b)) that the % EE of 41% could be achieved with X1 in two different levels (0.2 to 1.0 as well as 0.2 to 1.0) and X2 range at 0.6 to 1.0 level.
Zeta potential of PLGA-mPEG NP The zeta potential values ranged between 7.0 mV to 9.9 mV for Cyt loaded PLGA-mPEG NP. The optimized batch (CPM NP3) had zeta potential value of 7.5 1.3 mV. The low negative zeta potential was attributed to the presence of PEG on the surface. The presence of mPEG on the surface of particles would create a shield between the NP surface and the surrounding medium, thus masking the charged groups on the surface and preventing the particles from aggregation (Konan et al., 2003).
SEM studies The electron micrograph showed discrete particles in the nanometer size range (Figure 2). The spherical shape of the
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Figure 1. Contour plot depicting combined effect of Drug: polymer and volume of non solvent on (a) mean particle size of PEGylated PLGA nanoparticles of Cyt and (b) % EE of PEGylated PLGA nanoparticles of Cyt.
nanoparticles was also confirmed by scanning electron microscopy.
Drug release studies
Figure 2. SEM of CYT-PLGA-MPEG NP. The bar line indicates 50 nm.
In vitro drug release from the pure drug was complete within 2 h, but was sustained up to 2 days from PLGA-mPEG nanoparticles and up to 1 day from PLGA nanoparticles. The release profile is shown in Figure 3. The nanoparticles released the drug slowly without showing any burst release phenomenon, which is generally reported in case of PLGA and PLGA-mPEG particles (Panagi et al., 2001; Soppimath et al., 2001; Bala et al., 2004). The sustained release of the drug was attributed to the PLGA’s property to sustain the release of the drug entrapped in the nanoparticles (Lamprecht et al., 2000; Vandervoort et al., 2004). Comparison of the R2 value of zero order (0.8001) and first order (0.9969) equations
Long circulating PEGylated PLGA nanoparticles of cytarabine for targeting leukemia
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120
Table 2a. In-vitro cytotoxicity of Cytarabine and Cytarabine loaded NP on L1210 cells (% Viability SD) by MTT assay.
100
Concentration (mM)
% Viability (SD)* Cytarabine
CYT-PLGA NP
CYT-PLGA-MPEG NP
100 0.0 69.3 3.7 53.4 1.4 42.1 3.3 36.3 1.2 32.2 2.1
100 0.0 52 1.1 42 1.0 30 1.4 28 2.0 27 1.1
100 0.0 42.7 1.4 34.8 1.1 25.4 2.2 20.9 0.2 15.1 0.7
% Drug released
80
0 5 10 20 50 100
60
40 Cyt
Note: *For standard deviation (SD) n ¼ 6.
CPM NP
20
CPNP
0 0.5
1
2
4
6
12
24
36
Table 2b. Time based Cytotoxicity study of CYT, CYT-PLGA NP and CYTPLGA-MPEG NP on L1210 cells by MTT assay.
48
Tim e hours
Figure 3. In vitro drug release profile of cytarabine pure drug, cytarabine-loaded PLGA-mPEG NP (CPM NP) and cytarabine-loaded PLGA nanoparticles (CPNP).
(a)
Sr. no.
1 2 3
First order plot
2.5
Formulation
IC50 Day 1 mM SD
IC50 Day 3 mM SD
IC50 Day 5 mM SD
CYT CYT-PLGA NP CYT-PLGA-MPEG NP
6.5 0.6 5.3 0.3 2.2 0.2
6.0 0.3 4.7 0.3 1.8 0.1
5.8 0.3 4.4 0.2 0.8 0.1
2 2
R = 0.9969
log % drug rem aining
1.5 1
CP NP
0.5
CPM NP
2
(n value) was 0.411 for PLGA-mPEG NP which was less than 0.43, indicating a Fickian release mechanism from the NPs.
R = 0.9922
0 0
10
20
30
40
50
60
–0.5 –1 Time (h)
(b)
Korsemeyer plot
0.7 0.6
R2 = 0.9846
0.5 0.4 log Mt /M∞
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0
CPM NP
0.3
CPNP
0.2 R2 = 0.9747
0.1 0 –0.1
0
0.5
1
1.5
2
Log time
Figure 4. (a) First-order plot and (b) Korsmeyer-Peppas model for cytarabine-loaded PLGA-mPEG NP (CPM NP) and cytarabine-loaded PLGA Nanoparticles (CPNP).
indicated that the release of Cyt from PLGA-mPEG NP followed first ordered kinetics (Figure 4(a)) indicating concentration dependent release. The Korsmeyer-Peppas model for Cyt loaded PLGA-mPEG NP and Cyt loaded PLGA NP is shown in Figure 4(b). The diffusion exponent
Cytotoxicity studies Cytotoxicity of the pure drug (Cyt), drug loaded PLGA NP (Cyt-PLGA NP) and drug loaded PEGylated PLGA NP (Cyt-PLGA-mPEG NP) was studied on the leukemic cell line L1210. Table 2a shows that the percentage viability of the L1210 cells was decreased as the concentration of the drug was increased and hence the viability is said to be concentration dependent. The % viability of the cells treated with Cyt (32.2 2.1) and Cyt-PLGA NP (27 1.1) were high compared to Cyt-PLGA-mPEG NP (15.1 0.7) at the highest concentration used (100 mM) indicating greater cytotoxicity of the PEGylated PLGA NP. The increase in cytotoxicity may be attributed to an enhanced cellular uptake of Cyt-mPEG PLGA NPs as compared to that of the free drug (Panyam et al., 2004). Table 2b shows that the IC50 values decreased 2.9 times for Cyt-PLGA-mPEG NP compared with free drug. The order of cytotoxicity was Cyt-PLGA-MPEG NP 4 Cyt-PLGA NP 4 Cyt. The two polymers, PLGA and PLGA-mPEG used in the NP formulations were tested at two concentrations, AC (actual concentration used in the NP formulation) and DC (double concentration used in the NP formulation) to determine the effect of concentration on cytotoxicity. It was found that % viability was more than 99% for PLGA and more than 98% for PLGA-mPEG at both the
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Figure 5. Confocal images of L1210 cells (a) Control, (b) PLGA-MPEG NP, (c) Fluorescence image and (d) fluorescence difference image (F–D).
concentrations tested. This confirmed that both the polymers did not show any cytotoxicity on the cells in the concentrations used. Thus the greater cytotoxicity of the Cyt loaded NP can be attributed to the drug itself whose cellular uptake was facilitated by NPs.
completely utilized on first day and did not show any significant effect on the consecutive days. Comparatively, the nanoparticles released the entrapped drug for a longer duration and showed a lower IC50 values upto 5 days. Hence it was concluded that Cyt loaded nanoparticles had long term cytotoxic affect on the L1210 cells.
Long term cytotoxicity study Confocal microscopy A relatively short incubation period used in the MTT-based cytotoxicity assay (24 or 48 h) is not enough to determine long-term cytotoxicity of the nanoparticulate formulations, and a more prolonged incubation time would be required to fully exert the cytotoxicity effect of nanoparticles (Yoo and Park, 2004). Moreover, after the nanoparticles reach the cytoplasm, the drug must be solubilized in a molecularly dissolved state prior to reaching the nucleus to exert its cytotoxic effect. Zhang and Feng (2006) studied cancer cell viability by MTT assay of paclitaxel-loaded PLA-TPGS nanoparticles and found that on the 3rd day, the IC50 values were significantly reduced. Hence, in the present study, time based cytotoxicity on L1210 cell lines were carried out for 5 days and results are shown in Table 2b. It was seen that there was no significant change in the IC50 values of pure drug solution (Cyt) on the third and fifth days. The IC50 value of Cyt-PLGA NP reduced from initial 5.3–4.7 mM and 4.4 mM on the third and fifth day. A significant difference was observed in the IC50 values in case of Cyt-PLGAmPEG NP with respect to pure drug. The IC50 value of Cyt-PLGA-mPEG NP reduced 2.75 times from 2.2 to 0.8 mM in 5 days. This indicates that pure Cyt was
Confocal microscopy of the L1210 cells exposed to PLGAMPEG NPs showed fluorescence activity in the cells within 30 min (Figure 5). The control experiment performed by incubating cells with 6-coumarin solution (Figure 5(a)) showed that their intracellular fluorescence was insignificant compared to that of cells incubated with PLGA-MPEG NPs (Figure 5(b)). Hence, it was concluded that the fluorescence observed inside the cells was only due to the presence of nanoparticles. Figure 5(c) shows a comparison of fluorescence image and Figure 5(d) shows overlap of fluorescence differential image in L1210 cells for uptake of PLGA-mPEG NP. The overlap of fluorescence and differential image gave a clear picture both the cell and the fluorescent NPs and showed that the NPs were clearly within the L1210 cells and not merely adsorbed onto the cell.
Flow cytometry Flow cytometry study was carried out on L1210 cell lines in order to confirm the cellular uptake of nanoparticles.
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Long circulating PEGylated PLGA nanoparticles of cytarabine for targeting leukemia
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Figure 6. Flow cytometric scan of L1210 cell lines using (a) Control, (b) PLGA NPs and (c) PLGA-MPEG NPs at 37oC. M1 marks the population of cells having fluorescence under control, M2 marks the population of cells with fluorescence intensity after uptake.
A significant change in the fluorescence intensity of the PLGA NP (Figure 6(b)) and PLGA-mPEG NP (Figure 6(c)) was seen compared to untreated control (Figure 6(a)). The fluorescence intensity profile shifted to the right side for the nanoparticles, indicating that the nanoparticles internalized within the cells (Park et al., 2010). It can be concluded that incubation of the L1210 cells with NPs caused substantial accumulation of cells in the M2 phase. Quantitative analysis of the events distribution revealed that only 16.81% of the cells were in the M2 phase for untreated control cells (Figure 6(a)), whereas the M2 population was increased to 47.81 and 51.60% for the PLGA-NP, and PLGA-mPEG NP treated cells, respectively (Figure 6(b) and (c)). These data suggest that the nanoparticulate formulations were effective in increasing the cellular uptake of the NPs and comparing the two, PEGylated NP had higher uptake than the PLGA NPs. The PEGylation of the NP was responsible for the enhanced uptake of these NP by the cells. The surface modified NP was made hydrophilic by the presence of PEG layer.
Radiolabeling studies The radiolabeled complexes of cytarabine (99mTc-Cyt), cytarabine loaded PLGA Nanoparticles (99mTc-Cyt-PLGA
Table 3a. Effect of amount of stannous chloride on the labeling efficiency of Cytarabine. Stannous chloride (mg)
Cytarabine
25 50 75 100 125 150
% Labeled
% Colloids
% Free
87.23 97.42 98.43 99.19 94.14 92.58
5.02 1.32 0.18 0.12 4.41 6.12
7.75 1.26 1.39 0.69 1.45 1.30
Table 3b. Effect of amount of stannous chloride on the labeling efficiency of cytarabine loaded PLGA NPs. Stannous chloride (mg) 25 50 75 100 125 150
Cyt-PLGA NP
Cyt-PLGA-MPEG NP
% Labeled
% Colloids
% Free
% Labeled
% Colloids
% Free
78.23 87.32 95.55 97.28 99.15 97.12
4.98 3.78 2.89 1.42 0.55 2.45
16.79 8.90 1.56 1.30 0.30 0.43
82.32 89.25 92.54 97.10 98.10 96.10
7.38 5.43 4.78 1.50 0.49 2.15
10.00 5.30 2.70 1.40 0.29 0.59
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K. S. Yadav et al.
NP) and cytarabine loaded PLGA-mPEG NP (99mTc-CytPLGA-mPEG NP) were prepared with 99mTc by direct labeling procedure using stannous chloride method. All formulations had labeling efficiency of more than 98% at pH 6.5. Table 3a and b shows the effect of amount of stan-
Table 4. Stability data of 99mTc-CYT,
99m
Tc-CYT-PLGA NP in serum.
Time
% Radiolabeled 99m
15 min 30 min 60 min 2.0 h 4.0 h 8.0 h 24.0 h
99m
Tc-CYT
99m
Tc-CYTPLGA NP
99.19 99.12 99.08 99.05 99.01 98.13 98.18
Tc-CYT-PLGAMPEG NP
99.15 99.08 98.67 98.55 97.45 97.28 97.12
98.10 97.45 97.03 96.84 96.43 96.12 96.02
Table 5. Biodistribution studies of 99mTc-Cytarabine in Balb/c mice. 25
Organ/tissue
Percentage injected dose/gram of organ/tissue 1h
Blood Heart Lungs Liver Spleen Kidney Stomach Intestine Muscle Brain Bone
4h
Blood 1h 4h
20
24 h
Mean
SD
Mean
SD
Mean
SD
1.30 0.60 4.68 13.37 15.53 1.81 0.91 0.28 0.52 0.04 0.08
0.08 0.03 0.50 4.13 3.60 0.06 0.10 0.10 0.07 0.01 0.03
1.20 0.80 3.23 23.21 13.23 0.21 0.81 0.16 0.62 0.05 0.09
0.10 0.007 1.60 5.03 3.70 0.20 0.10 0.20 0.40 0.01 0.02
0.20 1.10 2.30 4.43 3.53 0.54 0.72 0.05 0.72 0.07 0.12
0.08 0.06 0.30 1.08 1.03 0.12 0.10 0.02 0.12 0.01 0.02
Percentage injected dose
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nous chloride on the labeling efficiency of the formulations. It was seen that 100 mg/mL of stannous chloride was optimal for Cyt as it provided labeling efficiency of 99.19%. The highest labeling efficiency of 99.15% and 98.10% for 99mTc-Cyt-PLGA NP and 99mTc-Cyt-PLGAmPEG NP, respectively, was obtained by using 125 mg of stannous chloride. Radiocolloids are formed during the process of labeling and distribute extensively to the organs of RES when injected in animal; therefore they may interfere in the results of the biodistribution studies (Banerjee et al., 2005). Radiocolloid formation during the labeling was less than 1.0% in all the formulations (Table 3a and b) and hence was not expected to interference during the biodistribution studies. Stability of 99mTc-Cyt, 99mTc-Cyt-PLGA NP and 99mTcCyt-PLGA-mPEG NP were studied in serum for 24 h. The results obtained are shown in Table 4. It was seen that the labeled complexes were stable up to 24 h as the labeling efficiency in 24 h was 98.18%, 97.12% and 96.02%
24h
15
10
5
0 99m Tc-Cyt
99m Tc-Cyt-PLGA NP
99m Tc-Cyt-PLGA-MPEG NP
Figure 7. Blood concentrations of 99mTc-Cyt-PLGA NP and 99mTc-CytPLGA-mPEG NP in Balb/c mice at 1, 4 and 24 h interval post injection. Each value is the mean (SD) of four mice.
99m
Notes: Tc indicates Technetium-99m. Each value is the mean (SD) of % injected dose in 4 mice.
Table 6. Biodistribution Studies of 99mTc-Cyt-PLGA NP and 99mTc-Cyt-PLGA-MPEG NP in Balb/c mice. Organ/tissue
Percentage injected dose/gram of organ/tissue 99m
99m
Tc-Cyt-PLGA NP
1h
Blood Heart Lungs Liver Spleen Kidney Stomach Intestine Muscle Brain Bone Notes:
99m
Tc-Cyt-PLGA-MPEG NP
4h
24 h
1h
4h
24 h
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
2.30 0.62 3.21 4.21 3.82 0.61 0.82 0.18 0.50 0.05 0.21
0.08 0.03 0.02 0.63 0.60 0.08 0.12 0.07 0.06 0.01 0.03
1.20 0.70 2.20 3.10 3.10 0.42 0.73 0.16 0.68 0.05 0.15
0.10 0.007 0.05 0.63 0.70 0.20 0.15 0.06 0.41 0.01 0.02
0.40 0.98 1.67 2.86 3.00 0.91 0.56 0.15 0.08 0.07 0.08
0.08 0.06 0.40 1.02 0.02 0.04 0.09 0.02 0.02 0.01 0.02
20.75 0.53 1.21 1.24 1.02 0.21 0.52 0.13 0.48 0.12 0.18
1.18 0.08 0.02 0.33 0.30 0.06 0.10 0.30 0.07 0.05 0.03
20.02 0.43 0.80 2.02 2.08 0.30 0.40 0.70 0.09 1.24 0.49
1.07 0.07 0.05 0.13 0.04 0.20 0.10 0.20 0.02 0.07 0.02
18.21 0.35 0.60 0.71 1.23 1.00 0.22 0.58 0.02 1.62 0.52
1.08 0.05 0.20 0.04 0.02 0.02 0.10 0.2 0.02 0.05 0.02
Tc indicates Technetium-99m. Each value is the mean (SD) of % injected dose in 4 mice.
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Long circulating PEGylated PLGA nanoparticles of cytarabine for targeting leukemia
Figure 8. (a) Liver, (b) spleen and (c) lung concentrations of 99mTc-Cyt-PLGA NP and 99mTc-Cyt-PLGA-mPEG NP in Balb/c mice at 1, 4 and 24 h interval post injection. Each value is the mean (SD) of four mice.
for 99mTc-Cyt, 99mTc-Cyt-PLGA NP and 99mTc-Cyt-PLGAmPEG NP, respectively. This indicated that the radiolabeled formulations could be used in animals for carrying out biodistribution studies.
Biodistribution studies The biodistribution of intravenously injected 99mTc labeled formulations was performed in Balb/c mice. The radioactivity was determined as the percentage injected dose/ gram of organ or tissue after 1, 4 and 24 h post injection. Table 5 shows biodistribution pattern of 99mTc-Cyt indicating greater radioactivity of free cytarabine in lungs, liver
and spleen, which are organs of the RES, than in the blood. The biodistribution pattern of conventional cytarabine loaded PLGA nanoparticles, 99mTc-Cyt-PLGA NP and pegylated PLGA, 99mTc-Cyt-PLGA-mPEG NP is shown in Table 6. After a period of 1 h post injection, the concentration of pure drug, 99mTc-Cyt-PLGA NP and 99mTc-Cyt-PLGAmPEG NP was 1.30%, 2.3% and 20.75% of the ID respectively in the blood (Figure 7). This indicated that the PEGylated PLGA NP increased the uptake by 15.9 folds whereas, the conventional PLGA NP increased the uptake to only 1.7 folds with respect to the pure drug. Even after 24 h, it was observed that there was no significant increase in concentration in case of the pure drug and PLGA NP and
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K. S. Yadav et al. 1.8 1.6
Percentage of injected dose
1.4
Brain 1h 4h 24h
1.2 1 0.8 0.6 0.4 0.2 0 99mTc-Cyt-PLGA NP
99mTc-Cyt-PLGA-MPEG NP
Figure 9. Brain concentrations of 99mTc-Cyt-PLGA NP and 99mTc-CytPLGA-mPEG NP in Balb/c mice at 1, 4 and 24 h interval post injection. Each value is the mean ( SD) of four mice.
0.6
1h
Bone
4h
0.5 Percentage of injected dose
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99mTc-Cyt
24h
0.4
0.3
0.2
0.1
0 99mTc-Cyt
99mTc-Cyt-PLGA NP
99mTc-Cyt-PLGA-MPEG NP
Figure 10. Bone concentrations of 99mTc-Cyt-PLGA NP and 99mTc-CytPLGA-mPEG NP in Balb/c mice at 1, 4 and 24 h interval post injection. Each value is the mean (SD) of four mice.
their concentrations remained comparatively much lower than the PEGylated PLGA NP. In contrast, there was a progressive increase in the blood concentrations from the PEGylated PLGA NP upto 24 h, indicating their long circulating behavior. The concentration of 99mTc-Cyt-PLGAmPEG NP was 91 folds (18.21% ID) that of the pure drug after 24 h post injection. The biodistribution pattern of the two NP formulations was similar in heart, kidney, stomach, intestine and muscle. There was no significant difference (p 4 0.05) between the two when compared to free drug. The RES organs are very active during the early hours of intravenous administration and a high radioactivity of the labeled free drug (99mTc-Cyt) was found in liver (11 fold), spleen (15 fold) and lung (3 fold) just after 1 h of administration as compared to the PEG coated nanoparticulate drug (99mTc-Cyt-PLGA-MPEG NP) (Figure 8). After an hour 13.37% of ID of 99mTc-Cyt was found in liver when only 1.24% of ID of 99mTc-Cyt-PLGA-MPEG NP was uptaken in the liver. After a circulation time of 4 hours about 23.21% of ID of 99mTc-Cyt was taken up by the liver whereas
only about 2% of ID was found by using the PEGylated, labeled drug (99mTc-Cyt-PLGA-MPEG NP). Thus, the technetium-mediated radioactivity was 11-fold higher in livers of animals treated with 99mTc-Cyt. An enhanced radioactivity was additionally found in spleen (6 fold) and lung (3fold). Cyt is rapidly and extensive metabolized mainly in the liver (Ho and Frei, 1971). Uptake of 99mTc-Cyt was rapid in the liver, reached to a maximum of 23.21% of ID in 4 h and then reduced to 5.03% of ID. As the drug is not metabolized in the lungs, comparatively it was uptaken in the lungs to a lesser extent (3.23% of ID) in 4 h. Comparing the radioactivity of PEGylated nanoparticles in liver and spleen, it was seen that the 99mTc-Cyt-PLGAMPEG NP showed a higher splenic uptake (1.23% of ID) than in liver (0.73% of ID) in 24 h. This was attributed to the property of surface modified nanoparticles as they tend to have more of splenic clearance than through liver. It has been shown that nanoparticles coated with PEG have a higher splenic clearance due to higher initial circulatory levels (Stolnik, 1994). The results indicated that the PEGylated NPs avoided uptake by the RES organs. There was no significant difference (p 5 0.05) in the uptake of 99mTc-Cyt and 99mTc-Cyt-PLGA NP in the brain (Figure 9). This indicates that the conventional PLGA NPs were not able to cross the blood brain barrier and were therefore not able to increase the concentration of cytarabine in the brain. In contrast, the PEGylated NPs of Cyt showed drastic increase (p 4 0.05) in the %ID of the NPs in the mice brain as compared to free drug. In the first hour it which was about 3 times more than that of the free drug. The concentration of 99mTc-Cyt-PLGA-mPEG NP increased about 25 folds in 4 h and about 23 folds in 24 h post injection as compared to the pure drug. This indicated the ability of the PEGylated NP to cross the BBB and provide sustained release of the entrapped drug. Interestingly the PLGA NP also accumulated in the bones, although it was about times 6.5 times less than PEGylated NP in 24 h (Figure 10). PEGylated NPs showed an increase in bone accumulation over a period of 24 h. There was an increase in the concentration of 99mTc-CytPLGA-mPEG NP by 2.2 times (0.18% ID), 5.4 times (0.49% of ID) and 4.3 times (0.52% of ID) in 1, 4 and 24 h, respectively, compared to free drug 99mTc-Cyt in bone of the mice. Thus, the overall results of the biodistribution study showed that there was a great difference in the biodistribution pattern of the PLGA NP and PEGylated NP. Although this marked increase in blood circulation time and reduced liver uptake by the PEGylated PLGA nanoparticles have been reported earlier (Gref et al., 1994; Stolnik et al., 1994; Brigger et al., 2002), we compared the biodistribution pattern of Cyt loaded PLGA NP, PEGlyated NP and pure drug in brain and bones. After intravenous administration, the PLGA-mPEG nanoparticles remained in the systemic circulation for 24 h and could be detected in brain and bones, which are hideouts of the leukemia cells. It was seen that the distribution pattern followed by the PLGA NP was similar to that of the pure drug except for the fact that PLGA NP was available at a lower concentration in liver than the pure drug. The reason behind this
Long circulating PEGylated PLGA nanoparticles of cytarabine for targeting leukemia
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Percentage injected dose in blood
Blood clearance in rat 40
99mTc-Cyt
35
99mTc-Cyt-PLGA NP
30
99mTc-Cyt-PLGAmPEG NP
25 20
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by confocal and flow cytometry studies. The biodistribution and blood clearance studies showed that PLGA-mPEG NP of Cyt had reduced uptake by the RES due to the steric barrier created by presence of PEG on the surface of the NP were present in the circulation for a longer time. Moreover the NP targeted the hideouts places of leukemic cells, bones and brain, in which the free drugs concentration was negligible. Hence the developed nanoparticulate formulations of cytarabine can be potentially useful clinically for improved treatment of leukemia.
15
Acknowledgements
10 5 0 15 min 30 min
1h
2h
4h
24h
Time Figure 11. Blood clearance of 99m Tc-Cyt, 99mTc-Cyt-PLGA NP and 99mTcCyt-PLGA-mPEG NP in rats.
could be that the PLGA-NP is expected to have more of spleen than hepatic clearance (Illum and Davis, 1984). The study concluded that neither the pure drug nor the conventional PLGA NP were able to provide prolonged blood circulation and higher drug concentration in brain and bones as compared to the PEGlyated NP. PEGylation of NPs allows them to avoid uptake by the RES organs and therefore they remain in the blood circulation for an extended period of time.
Blood clearance studies The blood clearance profile of 99mTc-Cyt, 99mTc-Cyt-PLGA NP and 99mTc-Cyt-PLGA-mPEG NP in rats is shown in Figure 11. When 99mTc-Cyt was injected in rat, only 2.35% ID was seen in blood in 15 min, which reduced to 1.23% ID in 12 h and to 0.07% ID in 24 h. A similar pattern was seen for PLGA NP (2.15% ID in 1 h and 0.21% ID in 24 h). However, the concentration of 99mTc-Cyt-PLGA-mPEG NP in blood was 32.25% ID in 15 min which was maintained upto 12.27% ID till 24 h. Thus, our results are in accordance with those of Gref et al. (1994) who reported that PLGA-mPEG NP remained in the circulation at higher concentrations as compared to PLGA NP.
Conclusion In the present investigation PEGylated PLGA NPs loaded with cytarabine were prepared by a modified nanoprecipitation method which showed a sustained release of the drug in vitro for 2 days. The cytotoxicity studies of the NP on L1210 cells showed that the PEGylated NP of Cyt showed a sustained cytotoxic effect till 5 days on the L1210 cells. The uptake of the NP by the cells was shown
The authors gratefully acknowledge Dr. C.P. Puri, Director, NIRRH, Mumbai for providing the cell line study facilities; Dr. M. S. Patole NCCS, Pune for providing the cell lines; Biocon Ltd., Bangalore for gift sample of Cytarabine; Boehringer Ingelheim Limited, Germany for gift sample of PLGA and Alembic Ltd, Vadodara for gift sample of Pluronic F-68.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
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