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The treatment of Glioblastoma Xenografts by surfactant conjugated dendritic nanoconjugates
Biomaterials 32 (2011) 6213e6225
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The treatment of Glioblastoma Xenografts by surfactant conjugated dendritic nanoconjugates Virendra Gajbhiye, Narendra K. Jain* Pharmaceutics Research Laboratory, Dept. of Pharmaceutical Sciences, School of Engineering & Technology, Dr. H. S. Gour University, Sagar, MP 470 003, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2011 Accepted 22 April 2011 Available online 26 May 2011
Polysorbate 80 (P80) anchored poly(propyleneimine) (PPI) dendritic nanoconjugate was developed and evaluated for targeting anti-cancer drug, docetaxel (DTX) to the brain tumor. In vitro cytotoxicity studies of free DTX, DTXePPI and DTXeP80-PPI dendrimers were carried out using U87MG human glioblastoma cell line. The in vivo anti-cancer activity in brain tumor bearing rats revealed that DTX loaded P80 conjugated dendrimers reduced the tumor volume extremely significantly (p < 0.0001; more than 50%). The median survival time for brain tumor bearing rats treated with DTXeP80-PPI dendrimers (42 days) was extended very significantly as compared to DTXePPI (23 days; p < 0.001), receptor blocked group (15 days; p < 0.001) and free DTX (18 days; p < 0.001). Gamma scintigraphy and biodistribution studies further confirmed the targeting efficiency and higher biodistribution of ligand conjugated dendrimer into the brain. The results concluded that the developed nanoconjugate has potential to deliver significantly higher amount of drug to brain tumor for improved therapeutic outcome. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Animal model Apolipoprotein Brain Dendrimer Drug delivery
1. Introduction Brain tumors are one of the 10 major causes of death by cancer. Despite of advances in strategies combining surgery, radiotherapy, and chemotherapy, the average 1 year survival of the brain tumor patient has amplified extremely petite over the past three-four decades. This is attributed to the fact that brain tumors, upon diagnosis, are usually already 40e60 cm3 in volume comprising approximately 4e6 1010 cells [1]. Brain tumors often attack or compress on normal brain tissue, and symptoms can result from that pressure. A person might experience different kinds of symptoms depending on wherein the brain a tumor is located [2]. A tumor that initiates in the brain is a primary brain tumor. Cancer cells that start growing elsewhere in the body and then travel to the brain via blood stream develop metastatic brain tumors e.g. cancers of the lung, breast, colon and skin frequently reach to the brain and may cause Glioblastoma multiforme, astrocytoma, medulloblastoma etc. Glioblastoma (gliomas) represents 42% of primary CNS tumors with more than three fourth being malignant. Prevalence of gliomas is approximately 5e10 per 100,000 population [3]. Approximately 20,000 cases of malignant glioma were diagnosed in the U.S. each year with 12,000 deaths estimated in year 2007 [4].
* Corresponding author. E-mail addresses: [email protected] (V. Gajbhiye), [email protected]. in (N.K. Jain). 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.04.057
Delivery of the bioactives to the brain is the utmost challenging task to cope with brain diseases, which attributed to bloodebrain barrier (BBB), blood-CSF barrier and efflux mechanism (P-glycoprotein; P-gp). These barriers control the entry of body as well as foreign compounds to access the brain cells [5]. The BBB prevents the entry of about 98% of small molecules and 100% of large molecules. Therapeutic benefit in glial tumors is often limited due to low permeability of delivery systems across the BBB, drug resistance and poor access into the tumor [6]. The aforementioned biochemical and targeting confronts, hurdle the advancement of effectual brain tumor chemotherapies. The field of drug delivery has fully emerged and came into existence as an ideal approach for drug targeting to brain [6]. It corresponds to a promising non-invasive approach for better drug delivery to the brain through exploitation of the various influx transport systems depicted within the cerebral endothelial, including carrier-mediated transports (CMT), receptor-mediated endocytosis (RME) and adsorptive-mediated endocytosis (AME). Nowadays, nano-sized carriers, especially polymeric (liposomes, nanoparticles and dendrimers) are being used widely and are under investigation with or without coating or coupling with ligands to offer a sustained level of drug and to accomplish cellular target with improved specificity [7e10]. These carriers are widely consented and appreciated due to their controlled drug release profile as well as their special targeting mechanism. Dendrimers are mono-dispersed macromolecules with a regular tree-like branched three-dimensional architecture that have potential for drug delivery, attributed to the properties of the
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macromolecules (1e100 nm), with multivalency, monodisperse, host-guest entrapment, defined molecular weight, large number of peripheral end groups, and interior cavities [11]. Special properties of dendrimers subjected them for immense exploration among the researchers, which has led to their possible exploitation in different pharmaceutical fields like drug delivery [12], gene delivery [13], solubility enhancement [14], diagnosis [15] etc. In addition, dendrimers have also generated interest in drug discovery by virtue of their therapeutic values [16]. Recently, surface functionalized dendrimers are being extensively explored for targeting anti-cancer and anti-HIV drugs [17,18]. The field of nanotechnology mediated anticancer drug targeting has observed emergence of an endowed carrier ’dendrimer’ in the last decade. Dendrimers can be anchored with ligands, that bind exclusively to the receptors over-expressed on BBB and might elicits receptor-mediated endocytosis. Apolipoproteins (e.g. ApoE) are believed to be adsorbed on P80 anchored nanocarriers, which could be responsible for the interaction with LDL receptors on BBB and subsequent endocytosis [19]. This approach was endeavored to utilize for brain delivery of DTX via P80 anchored PPI dendrimers. Thus in the present study, surfactant (polysorbate 80) has been anchored to PPI dendrimers and the nanoconjugate was explored for brain targeting potential of DTX. 2. Materials and methods 2.1. Materials DTX was a benevolent gift from m/s Dabur Pharmaceuticals (New Delhi), India. Carbonyldiimidazole (CDI), Polysorbate 80 and MTT were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ethylenediamine, and acrylonitrile were purchased from CDH (Mumbai), India. Reney Nickel was purchased from Merck (Mumbai), India. All other chemicals were of reagent grade and purchased from CDH, (Mumbai), India. 2.2. Synthesis of 5.0G PPI dendrimers PPI dendrimers (5.0 G) was synthesized by divergent method as reported by us earlier [20,21]. Briefly, double Michael addition reaction was used to produce half generation (eCN terminated) by adding acrylonitrile to the initiator core, ethylenediamine (EDA). It was followed by heterogeneous hydrogenation by means of Reney Nickel catalyst to synthesize full 1.0 G generation (eNH2) dendrimers. The reaction sequence was repeated intermittently to fabricate PPI dendrimers up to fifth generation (5.0 G PPI). The 5.0 G PPI was purified by dialysis against double distilled water in a dialysis tube (MWCO 5 kDa, Sigma, USA) to remove lower generation dendrimers and un-reacted chemicals. FT-IR spectroscopy was carried out using PerkineElmer FT-IR spectroscope (USA). 1H NMR spectroscopy of the dendrimer sample was carried out at 300 MHz in CDCl3 (Bruker DRX, USA). 2.3. Synthesis and characterization of P80 anchored 5.0 G PPI dendrimer This is a debut report on P80 or any type of surfactant conjugation on 5.0 G PPI dendrimers. The conjugation of P80 on 5.0 G PPI dendrimers was carried out with the help of 1,10 carbonyldiaimidazole (CDI) as a catalyst. This is also the debut report on anchoring any type of ligand on 5.0 G PPI dendrimers with the help of CDI. Firstly, free OH group of P80 was converted into a reactive imidazole carbamate by reacting with CDI (Scheme 1). For the above reaction P80 (25 mM) and CDI (25 mM) were dissolved in DMSO and the solution was stirred at 30 C for 1 h to generate a reactive imidazole carbamate [22]. Subsequent addition of a highly nucleophilic group (e.g. NH2) in the solution of reactive imidazole carbamate replaces imidazole and forms a highly stable carbamate. The 5.0 G PPI dendrimers are highly nucleophilic due to 64 terminal NH2 groups, which were added (1 mM) to the solution of reactive imidazole carbamate of P80. The mixture was stirred at room temperature (27 1 C) for 48 h to ensure the completion of reaction. Resulting solution was concentrated under vacuum. P80 anchored dendrimers (P80-PPI) were purified by dialysis against double distilled water in a dialysis tube (MWCO 12 kDa, Sigma, USA) to remove un-reacted P80 and impurities followed by lyophilization (Heto Drywinner, Heto-Holten, Allerod, Denmark). FT-IR and 1H NMR spectroscopies of lyophilized P80-PPI dendrimers were carried out as done for 5.0 G PPI dendrimers. The P80-PPI dendrimers was also characterized for extent of P80 conjugation and for exact molecular weight of the conjugate by Size Exclusion Chromatography (SEC). This experiment was performed using SEC system consisting refractive index detector (Santa Barbara, CA). The analysis was done at room temperature (27 2 C) using two serially aligned TSKGEL columns G3000 PW and G4000 PW. The isocratic mobile phase was PBS (pH 7.4)
at a flow rate of 1 ml/min. Sample concentration was kept 1 mg/ml in PBS, and 100 ml was injected. Molecular weights of 5.0 G PPI and P80-PPI dendrimers were determined using Astra V software (Wyatt Technology Corporation, Santa Barbara, CA). Transmission Electron Microscopy (TEM) was carried out to characterize the size of the conjugated system. The conjugates were added to a carbon grid and stained negatively using osmium tetroxide at 50 kV. The photomicrographs were taken at suitable magnification using a transmission electron microscope (Philips CM12 Electron Microscope, Eindhoven, Netherland). 2.4. Drug loading in formulations The known molar concentrations of P80-PPI dendrimer (10 mM) and DTX (100 mM) were taken in PBS (pH 7.4), and The mixture was magnetically stirred (50 rpm) for 48 h using teflon beads. The solvent was evaporated and dialyzed twice in cellulose dialysis bag (MWCO 1000 Da Sigma, USA) against methanol under sink conditions for 15 min to remove free DTX from the formulations. The free DTX was then estimated spectrophotometrically (lmax 230 nm; UV-1601, Shimadzu, Japan) to determine indirectly [20,21] the amount of drug loaded within the system. Similar procedure was followed for DTX entrapment in plain 5.0 G PPI dendrimer. The dialyzed formulations were lyophilized and used for further characterization. 2.5. Characterization of the drug loaded formulations The drug loaded dendritic system (DTXeP80-PPI) was characterized by differential scanning calorimetry (DSC). Differential scanning calorimetry (DSC) was carried out to evaluate the thermal stability and transformation in crystallinity over a range of temperatures. Free DTX, P80-PPI and DTX loaded P80-PPI dendrimer complex were studied and compared. Different samples were placed in an aluminium pan separately and a lid was crimped. The pan was then positioned in the analyzing cell of a DSC module (DSC Q10 V9.0 Build 275, TA Instruments, USA). The temperature of the DSC module was equilibrated at 35 C and then amplified at a rate of 10 C/min under a N2 gas flush out until the material began to degrade. The temperatures for each peak were obtained from resulting curve, which provided the indication of temperature stability and phase transitions. 2.6. In vitro release Drug release from known amounts of DTX loaded plain PPI, and P80-PPI dendrimers were determined in acid phthalate buffer (pH 4.0) and PBS (pH 7.4) in a modified dissolution method [20,21]. The dialysis bags (MWCO 1000 Da, Sigma, USA) were filled with a known amount of DTX loaded dendrimeric formulations and were placed in 50 ml of medium (PBS:Tween 80 in a ratio of 80:20) at 37 2 C with slow magnetic stirring under sink conditions. Aliquots of 1 ml were withdrawn from the external solution and immediately replenished with an equal volume of fresh medium. The drug concentration was detected spectrophotometrically (lmax 230 nm; UV-1601, Shimadzu, Japan) against the similar blank medium. 2.7. Comparison of hemolytic toxicity The RBC suspension for hemolytic studies was prepared following the reported procedure [20,21]. Briefly, the RBC suspension (5% hematocrit) of the human blood was collected in HiAnticlot blood collection vials (Himedia Labs, Mumbai, India). To 4.5 ml of normal saline 0.5 ml of suitably diluted free DTX, plain 5.0 G PPI, P80-PPI, DTXePPI, and DTXeP80-PPI were added and incubated for 1 h with RBC suspension. This allowed comparison of the hemolysis data of the free drug and different dendritic architectures to assess the effect of conjugation on hemolysis. After centrifugation, supernatants were taken and diluted with an equal volume of normal saline and absorbance was measured at 540 nm. RBC suspension was added to 5 ml of 0.9% NaCl solution (normal saline) and 5 ml distilled water to obtain 0% and 100% hemolysis, respectively. The degree of hemolysis was determined by the following equation: Hemolysisð%Þ ¼
Abs Ab0 100 Ab100 Ab0
where Abs, Ab0 and Ab100 are the absorbance of sample, a solution of 0% hemolysis and a solution of 100% hemolysis, respectively. 2.8. Cell culture In the present study U87MG human glioblastoma cell line, purchased from National Center for Cell Sciences, Pune, India was used. The cell line was cultured in cell culture flask using cell culture medium comprised of Dulbecco’s modified Eagle medium (DMEM; Himedia, Mumbai, India) supplemented with 20% fetal calf serum (FCS), 4 mM/l L-glutamine, 100 mg/ml streptomycin, and 100 U/ml penicillin (DMEM, FCS, glutamine, streptomycin, penicillin purchased from Himedia, Mumbai, India). The cells were cultured under a humidified atmosphere containing 5% CO2 at 37 C (Heracell CO2 Incubator, Hanau, Germany). The cells for the present study were utilized after 15th passage (Supporting Files Figure 1 aeh).
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Scheme 1. Synthesis of P80-PPI dendrimers.
2.9. In vitro cytotoxicity assay The in vitro cytotoxicity of free DTX, plain 5.0 G PPI, P80-PPI, DTXePPI, and DTXeP80-PPI were assessed via MTT assay on U87MG cell line. The cells were seeded into 96-well tissue culture plate in a density of 5 103 cells/well containing DMEM
medium and incubated for 48 h to acquire more than 75% confluency. Freshly prepared solutions of samples (100 ml) in DMSO were applied to each well in a concentration range between 0.001 and 10 mM. The culture plates were incubated for 24 and 48 h. After incubation, 20 ml of MTT solution (5 mg/m; in Hank’s Balanced Salt Solution) was added to each well and incubated for 3 h to allow viable cells to
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reduce the yellow MTT to purple formazan crystals. To each well 100 ml of DMSO was added, which dissolves formazan crystals. Absorbance was measured at 570 nm in an ELISA plate reader (Medispec Ins. Ltd, Mumbai, India). Cells without any sample were used as control. The experiment was repeated six times for each sample. Cell viability of each group was compared with control and was expressed as % cell viability using following formula: % cell viability ¼
Atreated 100 Acontrol
where Atreated is the absorbance of treated cells with different samples at 570 nm and Acontrol is the absorbance of control at 570 nm. 2.10. Cell uptake assay using gamma scintigraphy U87MG cell line was used to perform cellular uptake of different formulations. Free DTX, 5.0 G PPI dendrimers and P80-PPI dendrimers were radiolabeled with 99m TcO4 at Department of Nuclear Medicine, JawaharLal Nehru Cancer Hospital & Research Center (JNCHRC), Bhopal (MP) India, by the method reported earlier from our lab [23]. The U87MG cells were seeded in fibronectin coated tissue culture petri dishes at a density of 5 104 cells/plate containing 1 ml DMEM. The petri dishes were divided into five groups, each group containing six petri dishes. The dishes were incubated for 48 h in a humidified atmosphere containing 5% CO2 at 37 C with changing of the medium after every 12th h. First four groups were applied with free 99m TcO4, 99mTcO4eDTX, 99mTcO4ePPI and 99mTcO4eP80ePPI (100 mCi). Fifth group was treated as control. The samples were incubated with the cells for 3 h. After incubation period culture medium was removed and the cells were washed five times with Hank’s Balanced Salt Solution (Himedia, Mumbai, India). Each cell containing petri dishes were provided with 1 ml of culture medium and immediately subjected to measurement of radioactivity with the help of a gamma camera (Siemens E-cam, MEDX Inc, Illinois, USA). The radioactivity was measured in the form of radioactive counts over a duration of 60 s. 2.11. Tumor implantation The tumor implantation was carried out by the reported method [7,24] with slight modification. Male albino rats (Sprague Dawley stain, 8e9 weeks old weighing 250 20 g) were obtained from animal house of Department of Research, JawaharLal Nehru Cancer Hospital and Research Center, Bhopal (MP) India. All the animal studies were conducted in accordance with the protocol approved by the Institutional Animal Ethical Committee of Dr. H. S. Gour University, Sagar (MP) India (registration no.379/01/ab/CPCSEA; 10/87, dated 20-05-2010). The U87MG cells were suspended in serum free DMEM medium in a density of 5 105/10 ml. The cells were implanted stereotactically into right basal ganglia field of animals. Animals were kept under similar laboratory conditions and fed with normal food and water avoiding any type of stress. 2.12. In vivo anti-tumor activity After 8 days of tumor inoculation rats were divided into eight groups each group comprising 12 animals. The animals were administered with different formulations samples intravenously via tail vein according to grouping on every 3rd day of treatment up to one week (i.e. 9, 11, 13th day). The details of samples were as follows: Group 1 ¼ PBS (pH 7.4); Group 2 ¼ 5.0 G PPI dendrimers; Group 3 ¼ P80; Group 4 ¼ P80-PPI; Group 5 ¼ DTX; Group 6 ¼ DTXePPI and Group 7 ¼ DTXeP80-PPI (30 mg/kg equivalent of DTX). Group 1 was treated as control and 8th group was utilized for receptor blockage assay. This assay was based on the principle of initial blockage of LDL receptors by injecting 30 mg/kg cholesterol, followed by treatment with DTXeP80-PPI and subsequent effect on tumor volume. Six animals from each group were sacrificed on 15th day of treatment (n ¼ 6) and tumor volume (cubic milimeters) was measured with the help of Vernier Caliper using formula: V ¼ length width height [7]. Antitumor potential of different formulations are expressed as tumor inhibitory ratio (%T/C; treated versus control), dividing tumor volumes of treated groups by control group and multiplied by 100). Remaining six animals were observed for survival life span using KaplaneMeier survival curve. 2.13. In vivo biodistribution studies The in vivo qualitative biodistribution studies were performed by scanning the glioblastoma bearing rats under a gamma camera. Briefly, the tumor induced animals were divided into four groups (n ¼ 3). First, second, third and forth groups were injected intravenously via tail vein with free 99mTcO4, 99mTcO4eDTX, 99m TcO4ˉePPI and 99mTcO4eP80-PPI, respectively. The animals were subjected to whole body gamma scanning after 1 h. Tissue distribution studies were carried out for quantitative measurement of DTX in different organs. Tumor induced rats were divided into 4 groups, each group comprising 9 animals. First group was fed with PBS (pH 7.4) and treated as control. Second, third and fourth groups were administered with free DTX, DTXePPI and
DTXeP80-PPI, respectively (30 mg/kg) via tail vein. Three animals from each group were sacrificed 1, 4 and 8 h post administration (n ¼ 3). Immediately after sacrificing the animals the tissues viz. brain, liver, kidney, lung, spleen and tumor were carefully removed and weighed. One gram each of different tissues were added with 5 ml of trichloroacetic acid (10% v/v in water) and vortexed for 1 min. The required quantity of ethanol was added and homogenized well in a tissue homogenizer (York Scientific Instrument, New Delhi, India), which also removed the entrapped drug from dendrimers. The homogenates were centrifuged at 5000 rpm (Remi Elektrotech Ltd., Mumbai, India) for 15 min and the supernatant was collected and assayed for DTX content by HPLC method [25] using acetonitrile: methanol: 0.02 M ammonium acetate buffer (pH 5.0) as mobile phase in a ratio of 20:50:30 (v/v/v) at a flow rate of 1 ml/min.
3. Statistical analysis The results are expressed as mean standard deviation and the analysis was performed with SPSS 13 (SPSSÒ, Chicago, USA). Statistical significance is reported as P < 0.05. 4. Results 4.1. Synthesis of 5.0G PPI dendrimers PPI dendrimers (5.0 G) was synthesized following established method and characterized as reported by us earlier [20,21]. The characteristic FT-IR peaks of 5.0 G PPI dendrimers are of CeC bend (1107 cm1); CeN stretch (1309 cm1); CeH bend (1405 cm1); NeH deflection of amine (1663 cm1); and stretching of primary amine (3421 cm1), confirming nitrile terminal groups of dendrimer were converted to amine terminals. The fifth generation PPI dendrimer was further characterized by 1H NMR spectroscopy. Multiplets (m) between 0.9 and 1.2 ppm and 2.2e2.3 correspond to methylene (eCH2) groups of EDA. Multiplets at 2.5e2.8 correspond to eN(CH3). The results matched with the reported synthesis of PPI dendrimers [20,21]. 4.2. Synthesis of P80 anchored 5.0G PPI dendrimer The characterized 5.0 G PPI dendrimers were anchored with P80 with the help of CDI (Scheme 1). FT-IR spectrum exhibited major peak at 1666 cm1 (eCONH stretching), which confirmed the conjugation of P80 with dendrimer periphery (Fig. 1a). Other major peaks in the FT-IR spectrum of nanoconjugate were of eCH2 rocking (661 cm1), eCN stretching (1071 cm1), NH bending of amine (1455 and 1571 cm1), and overlapping broad peak of eOH and eNH stretching (3318 and 3393 cm1). 1H NMR d values at 7.08 and 7.7 ppm strongly evidenced the presence of eCONH bond (Fig. 1b), which further confirmed the conjugation. Primary amine (eNH2) exhibited peak at d value of 5.3 ppm. The d value at 3.6 ppm was attributed to eOH groups present in P80. Characteristic peaks of eCH2 of alkane and eN(CH3) of tertiary amine were present at 0.9e1.2 and 2.5 ppm, respectively. SEC was performed to determine the extent of P80 conjugation and exact molecular weight of the nanoconjugate. The molecular weight was found to be around 23 kDa (Fig. 2b), which suggested that approximately 12 molecules of P80 (molecular weight 1310 Da) conjugated to dendrimer periphery. The increase in molecular weight also evidenced the conjugation. TEM results displayed the nanometric size of P80 conjugated dendritic system, wherein circular nanometric conjugates were observed (Fig. 3 a and b). The results were in agreement with earlier reports [26]. 4.3. Drug loading Equilibrium dialysis method was utilized to entrap DTX in dendrimeric formulations. The percentage drug loading was determined indirectly by estimating the un-entrapped drug,
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spectrophotometrically. The percentage DTX loading was found extremely significant (P < 0.0001) for P80-PPI dendrimers as compared with plain 5.0 G PPI dendrimers (Fig. 4 a and b). The P80PPI dendrimer displayed 67.61 1.44% DTX entrapment, whereas plain 5.0G PPI displayed only 27.47 1.01% DTX loading (data not shown). 4.4. Characterization of the drug loaded formulations The lyophilized drug loaded P80-PPI formulation was characterized for the presence of DTX inside the dendritic system by DSC. The free DTX experienced a sharp endothermic peak (20 mW) at its melting point 230 C (Fig. 5a), while broad endothermic peaks (27 to 46 mW) ranging from 65 to 125 C were observed in case of P80-PPI dendrimer (Fig. 5b). However, the DSC curve of DTXeP80-PPI showed both the endothermic peaks of DTX and P80PPI at around 230 and 125 C (Fig. 5c), which confirmed presence of DTX in the dendritic nanoconstructs. 4.5. In vitro drug release The release studies suggested that both 5.0 G PPI and P80ePPI dendrimers provided burst release of DTX at pH 4.0. The drug release was found to be 98.2 1.32 and 56.4 2.25% for plain 5.0 G PPI and P80ePPI dendrimers, respectively at 4th h (Fig. 6). On the contrary DTX release was significantly (P < 0.0001) retarded from P80-PPI at pH 7.4 (55.7 1.83% at 24th h) as compared to plain 5.0 GPPI dendrimers (97.4 2.05% at 24th h). P80-PPI dendrimeric nanocarrier released 97.7 1.61% of DTX up to 96th h. 4.6. Hemolytic toxicity All major anti-cancer drugs exert adverse effect of hemolysis. DTX also exerts adverse effect like anemia and neutropenia due to hemolytic nature. In addition, the hemolytic toxicity of the cationic dendrimers poses constraint for potential as drug delivery system. This toxicity is attributed to the polycationic nature of the PPI dendrimers [20,21]. DTX displayed almost more than 50% hemolysis at 0.4% w/v concentration (Fig. 7). The 5.0 G PPI dendrimers showed hemolytic toxicity 9.0 0.26, 11.9 0.21, 14.1 0.42 and 17.4 0.83% at 0.1, 0.2, 0.3 and 0.4% w/v concentrations, respectively. However P80 anchoring of dendrimers caused decreased hemolysis of the RBC considerably at all concentrations (1.7 0.35, 2.6 0.52, 3.3 0.42, and 3.9 0.33% at 0.1, 0.2, 0.3 and 0.4% w/v concentrations, respectively). The DTXePPI has shown reduced toxicity as compared to DTX but higher toxicity than plain PPI and DTXeP80-PPI dendrimers. Whereas, DTXeP80-PPI has shown significantly less (p < 0.05) toxicity than PPI, DTX and DTXePPI but higher toxicity than unloaded P80ePPI dendrimers (Fig. 7). The hemolytic toxicity of PPI dendrimers was well in agreement with previous reports [20,21,26] 4.7. In vitro cytotoxicity assay The in vitro cytotoxicity assay was carried out on U87MG cells via MTT assay by incubating different formulations for 24 and 48 h. Unloaded plain 5.0 G PPI and P80-PPI dendrimers have not shown any significant cytotoxic effect after 24 and 48 h incubation even at higher concentration (10 mM). DTX exerted very potent cytotoxic effect and displayed IC50 value of 0.15 mM after incubation for a period of 24 h (Fig. 8a), which was significantly higher (p < 0.001) than DTXePPI (0.9 mM) and DTXeP80-PPI (3.5 mM). DTXePPI has also shown significantly higher (p < 0.001) cytotoxicity and lower IC50 value than DTXeP80-PPI after 24 h.
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After 48 h, DTX has shown insignificant change in cytotoxicity and IC50 value (0.12 mM). However, extremely significant (p < 0.001) decrease in IC50 value of DTXePPI and DTXeP80-PPI was observed (Fig. 8b), which was shifted from 0.9 mM to 0.25 mM and 3.5 mMe0.3 mM for DTXePPI and DTXeP80-PPI, respectively. The difference between IC50 values of DTXePPI and DTXeP80-PPI was found to be insignificant (p ¼ 0.0742) after 48 h incubation (Fig. 8b). 4.8. Cell uptake assay study Cell uptake of radiolabeled 5.0 G PPI and P80-PPI was performed using gamma scintigraphy. The results showed that there was significant uptake of dendritic formulations as compared to free 99m TcO4ˉ and 99mTcO4ˉeDTX. The 5.0 G99PPI and P80-PPI dendrimers showed extremely significant higher (p < 0.0001) counts (6062 29 and 6015 25, respectively) in 60 s at 3rd h than free 99m TcO4ˉ (Fig. 9 a and b), while 99mTcO4eDTX and free 99mTcO4 have shown 2465 26 (Supporting Files Figure 2) and 953 31 counts, respectively in 60 s. The 5.0 G PPI had shown higher uptake than P80ePPI, but the difference was insignificant (p ¼ 0.1006). 4.9. In vivo anti-tumor activity The anti-tumor studies on glioblastoma bearing rats have shown that normal saline, 5.0 G PPI, P80 and P80-PPI do not possess any anti-tumor efficacy. The result suggested that DTXeP80-PPI reduced extremely significant tumor volume than DTXePPI and free DTX (p < 0.0001). The tumor volume at day 15th of tumor inoculation for control, DTX, DTXePPI and DTXeP80-PPI group were found to be 128.8 4.1, 102.6 2.2, 90.8 2.2, and 46.4 2.9 mm3, respectively (Fig. 10). On the contrary the tumor volume of LDL receptor blocked group was found to be 110.5 4.5 mm3. DTXePPI had also shown very significant (p < 0.001) reduction in tumor volume as compared to control and free DTX. The tumor volume ratio (%T/C) was found to be highest for free DTX. The tumor volume ratio for DTX, DTXePPI and DTXeP80ePPI were found to be 79.65 3.9, 70.49 2.7, and 36.02 1.6%, respectively. KaplaneMeier survival curves based on survival time were plotted for different groups. The curves suggested that the median survival time for rats treated with DTX loaded P80 anchored 5.0G PPI dendrimers (42 days) was extended significantly (Fig. 11) as compared to DTXePPI (23 days, p < 0.001), free DTX (18 days p < 0.001), receptor blocked group (15 days, p < 0.001) and control group (14 days, p < 0.001). 4.10. In vivo biodistribution studies The in vivo targeting efficiency was further evaluated by injecting free 99mTcO4, 99mTcO4eDTX, 99mTcO4ePPI and 99m TcO4ˉeP80ePPI into different groups of rats via tail vein. Free 99m TcO4 was selected because it does not cross BBB [27]. The qualitative gamma scanning images after 1 h suggested that free 99m TcO4 failed to cross blood brain barrier (Fig. 12a), while very little amount of radioactivity was seen in brain for 99mTcO4eDTX and 99mTcO4ePPI (Fig. 12b and c). In contrast, 99mTcO4eP80ePPI showed highest amount of radioactivity in brain as compared to free 99mTcO4ˉ, 99mTcO4eDTX and 99mTcO4ePPI (Fig. 12d). The targeting efficiency was further evaluated by biodistribution of DTX from different formulations. The concentration of DTX in different organs and tumor were estimated via HPLC [25] at 1st, 4th and 8th h. The result indicated that more than 7.5, 9 and 10% of injected dose accumulated in brain at 1st, 4th, and 8th h, respectively in case of DTXeP80-PPI (Table 1), while the percentage was
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Fig. 1. (a) FT-IR spectrum of P80-PPI, and (b) 1H NMR spectrum of P80-PPI.
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increased from 1st to 4th and then to 8th h in case of DTXeP80-PPI and DTXePPI, while free DTX had shown increase in concentration from 1st to 4th hr and then again decreased at 8th h. Similar pattern of DTX distribution was seen at the most desirable site, the brain tumor. Here also, DTXeP80-PPI had shown 4.1 and 39 times higher accumulation as compared to DTXePPI and free DTX at 8th h. Free DTX had shown higher accumulation in excretory organs viz. liver and spleen as compared to DTXePPI and DTXeP80-PPI, which was found to increase with time (Table 1). DTXeP80-PPI showed lesser distribution of drug to excretory organs. The DTX concentration was increased in lungs and kidneys from 1st to 4th h in case of all three formulations, but the concentration was decreased at 8th h for all formulations.
Fig. 2. Mass fraction profile of (a) plain 5.0G PPI, and (b) P80-PPI as measured by SEC.
approximately 2, 2.5 and 2.8, respectively for DTXePPI. In contrast, DTX have shown very little tendency to cross blood brain barrier. The percentage accumulated dose for DTX was approximately 0.4, 0.35, and 0.26% at 1st, 4th, and 8th h, respectively. The DTXeP80PPI nanoconjugate had shown approximately 3.5 and 39 times higher penetration of DTX in brain as compared to DTX-PPI and free DTX, respectively at 8th h (Table 1). The distribution of DTX was
Fig. 3. Transmission Electron Microscope images of (a) plain 5.0G PPI, and (b) P80ePPI nanoconjugates.
5. Discussion The blood brain barrier constitutes major hurdle for anti-cancer agents to reach brain tumor cells [5,6]. Apart from this P-gp system plays major role in efflux of P-gp substrate anti-cancer bioactives, which also leads to drug resistance. The DTX is a well known substrate of P-gp efflux system. This peculiarity is attributed to its ineffectiveness for brain tumor chemotherapy, even if possessing potent cytotoxic behavior. The trafficking of DTX in brain has been found to increase by blocking P-gp efflux systems [28]. The objective of the present study was to increase the bioavailability and residence time of DTX for effective brain tumor chemotherapy. In the present study we reported debut conjugation of surfactant and dendritic nano-architecture for achieving the aforementioned goal. Firstly, 5.0 G PPI dendrimers were prepared and characterized according to procedures established by us. The P80 anchoring to 5.0 G PPI dendrimers was carried out using CDI chemistry. The purified conjugate was subjected to characterization by FT-IR and 1 H NMR. FT-IR peak of eCONH stretching at 1666 cm1 primarily suggested presence of eCONH bond between P80 and 5.0 G PPI dendrimers. 1H NMR d values at 7.08 and 7.7 ppm further confirmed the presence of eCONH bond. The formation of eCONH bond must be attributed to CDI chemistry, which formed highly reactive imidazole carbamate with P80 and then substituting the reactive imidazole with highly nucleophilic groups (eNH2 terminals of PPI dendrimers). The conjugation was furthermore evidenced by increase in molecular weight (7.1 kDae23 kDa) estimated by SEC. Increase in molecular weight attributed to conjugation of approximately 12 molecules of P80 on the dendrimer periphery. Electron microscopy further demonstrated nanometric size of conjugate and provided indirect qualitative evidence of increase in size after P80 conjugation. The percentage DTX loading was found extremely significant (P < 0.0001) for P80-PPI dendrimers as compared with plain 5.0 G PPI dendrimers. Non-covalent interactions between DTX and P80-PPI dendrimers, such as hydrophobic interaction and hydrogen bonding, contributed to the physical entrapment of DTX molecules inside dendritic cavities and also in surface P80 layers. Higher entrapment in P80ePPI might have attributed to additional interaction of DTX and P80 at the dendrimer periphery. Sealing of dendrimer periphery with P80 increased the steric hindrance, which might have contributed to higher drug loading. Comparison of FT-IR spectra of free DTX, P80-PPI and DTXeP80-PPI suggested drug loading, which was further supported by DSC curves. The separate DSC curve of DTX in case of DTXeP80-PPI further demonstrated the physical entrapment of DTX in dendrimer interiors and ruled out any drug-dendrimer conjugation possibilities. In vitro DTX release was extremely significantly retarded from DTXeP80-PPI at pH 7.4 than pH 4.0. Primary and tertiary nitrogens at dendrimer periphery and cavities, respectively might have contributed to extended release at pH 7.4 and burst release at pH
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Fig. 4. (a) Schematic diagram of DTX loaded P80ePPI, and (b) molecular model of P80ePPI.
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Fig. 5. Curves of differential scanning calorimetry (a) free DTX, (b) P80-PPI, and (c) DTX loaded P80ePPI.
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4.0. The nitrogens are 2/3rd protonated at pH 7.4, which causes shrinking of the hydrophobic interior of PPI dendrimer. P80 anchoring on dendrimer periphery also further retarded the drug release. Burst release at pH 4.0 may be ascribed to higher protonation of tertiary amines at acidic pH. Higher protonation causes increase in size of dendrimer molecule and expansion of dendrimer branches. This accounts for the inability of dendritic formulation to retain drug molecules at acidic pH. However, as the tumor cells have slightly acidic environment, burst release at acidic pH might be beneficial for anti-cancer drug targeting. Once the drug entrapped dendrimer enters the cancer cells, it may provide rapid release of its content due to acidic pH. The drug release patterns at pH 4.0 and 7.4 were found similar to previous reports [20,21]. Hemolytic toxicity of the developed P80-PPI system was found significantly less compared to 5.0 G PPI and free DTX. DTX is highly cytotoxic drug, which contributed to higher hemolytic activity. The polycationic surface of 5.0 G PPI also resulted in higher hemolytic toxicity. However, shielding of dendrimer periphery reduced the hemolytic toxicity significantly as compared with plain 5.0 G PPI and free drug. The results supported that entrapment of anti-cancer agent may decrease the adverse effect on red blood cells. Higher hemolytic toxicity of DTXeP80-PPI than P80-PPI may be attributed to leaching of DTX from the formulation causing blood dyscrasias. In vitro MTT cytotoxicity assay on U87MG human glioblastoma cells suggested that DTX exerted highest cytotoxic effect as it was indirect contact with brain tumor cells. However the IC50 value was highest for developed DTXeP80-PPI system at 24th h. Sustained release of DTX from P80ePPI system might have contributed to high IC50 value. On the other hand, significantly less IC50 value was observed in case of DTX-PPI, which might have released the drug relatively faster than DTXeP80ePPI due to acidic environment of brain tumor cells. On the contrary, the IC50 was decreased dramatically at 48th h for DTXeP80-PPI system and the difference in IC50 values of DTXeP80-PPI and DTXePPI was not significant. This might have occurred due to complete release of DTX from P80-PPI. The reason for increase in IC50 value was well in agreement with in vitro drug release results, where P80ePPI released 56.4 2.25% of DTX at 4th h, while PPI released 98.2 1.32% of DTX at the same time. The in vitro cell uptake study suggested higher uptake of 5.0 G PPI than P80-PPI, but the difference was insignificant. The higher uptake may be attributed to interaction of positively charged dendrimers with negatively charge cell membrane. The in vivo DTX targeting efficiency was determined via anticancer activity on brain tumor bearing rats. Results demonstrated
Fig. 6. Cumulative percentage DTX released from dendritic formulations (n ¼ 3).
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Fig. 7. Hemolytic toxicity profiles (n ¼ 3).
that DTXeP80ePPI reduced tumor volume more significantly as compared to free drug and DTXePPI. The tumor volume was reduced almost more than 50% in case of DTXeP80-PPI after one week treatment regimen. Higher BBB crossing by P80 anchoring
might have furnished for DTX delivery to tumor. P80 anchored system has also shown most significant tumor volume ratio and significant longer median survival time of brain tumor bearing rats. The receptor blockage assay was carried out to shed light on
Fig. 8. Percentage cell viability of U87MG cells at (a) 24 h, and (b) 48 h (n ¼ 6).
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Fig. 10. Tumor volume of different formulation at 15th day (n ¼ 6).
via fecal route, which might be responsible for higher distribution of free DTX in liver and spleen. The highest concentration of free drug in liver and spleen was found at 8th h as it was being removed from tissues and blood stream. DTX is well known substrate for Pgp efflux system [28] and this might have attributed to less concentration of free DTX in brain at 8th h as compared to 4th h. P80 anchoring has also been reported to block P-gp efflux system [28]. Tumor regression and drug biodistribution results provide further evidence. These observations proffered strong evidence for the brain tumor targeting and treatment strategy. 6. Conclusion In vitro MTT assay, cell uptake assay, anti-tumor efficacy, receptor blockage assay and biodistribution data demonstrated that DTX loaded P80 anchored dendritic nanoconjugate has potential to
Fig. 9. Gamma images of cell uptake assay on U87MG cells (a) radiolabeled PPI, and (b) radiolabeled P80-PPI (Radioactive counts are circled).
principle of LDL receptor-mediated BBB crossing of P80 conjugated system [29]. Receptor blocked group has shown significant higher tumor volume than DTXeP80-PPI group, which strongly supported the LDL receptor-mediated endocytosis. The in vivo targeting efficiency was further assessed by whole body gamma scanning of tumor bearing animals. The images clearly demonstrated higher blood brain crossing of P80 anchored dendritic system. Once injected in the blood stream, P80 anchored nanoparticulate system supposed to act as low density lipoproteins (LDL) [29]. Apoproteins B and/or E from the blood stream get adsorbed on this LDL mimicking carrier [19], subsequently recognized by LDL receptors on brain endothelium and results into receptor-mediated endocytosis (Animation 1). This might be the possible reason behind highest anti-tumor efficacy of P80 anchored PPI dendrimers. Drug biodistribution studies further supported the above statement. The highest concentration of DTX in brain and tumor was estimated for DTXeP80-PPI. DTXeP80-PPI had shown 4.1 and 39 times higher accumulation in brain tumor as compared to DTXePPI and free DTX at 8th h. The major route of DTX excretion is
Fig. 11. KaplaneMeier survival curves for different formulations.
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towards tumor cells. The developed nanoconjugate also decreased the distribution of anti-cancer agent in excretory organs by increasing its deposition at target site and decreased the adverse effect on non-target site (RBCs). Thus it can be concluded that DTX loaded P80 conjugated 5.0 G PPI dendritic nanoconjugate holds strong potential to treat brain tumor in rats, while producing least blood dyscrasias as against non conjugated system and free drug. This is expected to result in optimized drug delivery, in the treatment of brain tumor, a highly desirable contemporary therapeutic challenge. Acknowledgement Virendra Gajbhiye acknowledges University Grant Commission (UGC), New Delhi, India for providing Senior Research Fellowship. Appendix. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biomaterials.2011.04.057. References
Fig. 12. Gamma scintigraphy images for (a) free 99m TcO4ˉePPI, and (d) 99mTcO4ˉeP80-PPI.
99m
TcO4ˉ, (b)
99m
TcO4ˉeDTX, (c)
cross BBB significantly and can deliver significantly higher amount of drug to brain and brain tumor for higher therapeutic outcome. Furthermore, P80 anchoring may lead to blockage of P-gp efflux system to exert highest therapeutic effect of anti-cancer drug Table 1 Biodistribution of DTX in brain tumor bearing rats (n ¼ 3). Organ
Formulation
Brain
DTX DTXePPI DTXeP80-PPI DTX DTXePPI DTXeP80-PPI DTX DTXePPI DTXeP80-PPI DTX DTXePPI DTXeP80-PPI DTX DTXePPI DTXeP80-PPI DTX DTXePPI DTXeP80-PPI
Liver
Spleen
Lung
Kidney
Tumor
Concentration (mg/g tissue) 1h
4h
8h
**34.74 3.7 *152.14 7.1 467.99 14.1 **495.59 15.5 *314.34 11.8 117.97 6.3 407.25 15.2 292.86 7.4 112.34 7.2 312.70 8.6 278.16 7.3 101.27 4.3 340.63 7.6 225.61 6.3 115.73 5.1 ** 17.28 1.1 * 86.58 3.2 213.75 6.5
**29.57 1.9 *187.78 2.4 695.20 5.2 **781.13 6.6 *458.08 5.3 227.62 4.2 618.61 7.1 413.92 5.6 218.84 4.4 456.94 5.2 344.74 5.0 209.88 3.2 491.54 6.7 374.32 4.9 219.85 4.5 ** 19.43 1.0 * 90.75 3.1 376.82 5.3
**20.38 1.2 *224.87 5.9 799.75 8.9 **1125.86 14.9 *618.76 8.7 340.20 5.9 1018.53 20.3 578.23 6.2 329.72 5.7 396.14 5.9 325.21 4.4 200.30 3.6 426.11 5.9 350.49 5.4 205.30 4.8 ** 13.69 1.1 * 97.67 5.2 514.93 6.8
*p<0.001, very significant. **p<0.0001, extremely significant.
[1] Huynh GH, Deen DF, Szoka FC. Barriers to carrier mediated drug and gene delivery to brain tumors. J Control Rel 2006;110:236e59. [2] Armstrong TS, Mendoza T, Gring I, Coco C, Cohen MZ, Erriksen L, et al. Anderson symptom inventory brain tumor module (MDASI-BT). Neuro Oncol 2006;80:27e35. [3] Behin A, Hoang-Xuan K, Carpentier AF, Delattre JY. Primary brain tumours in adults. Lancet 2003;361:323e31. [4] Winer E, Gralow J, Diller L, Karlan B, Loehrer P, Pierce L, et al. Clinical cancer advances 2007: major research advances in cancer treatment, prevention and, screening- a report from the American society of clinical oncology. J Clin Oncol 2009;27:812e26. [5] Eyal S, Hsiao P, Unadkat JD. Drug interactions at blood-brain barrier: fact or fantasy? Pharmacol Therapeut 2009;123:80e104. [6] Beduneau A, Saulnier P, Benoit JP. Active targeting of brain tumors using nanocarriers. Biomaterials 2007;28:4947e67. [7] Ying X, Wen H, Lu W, Du J, Guo J, Tian W, et al. Dual-targeting daunorubicin liposomes improve the therapeutic efficacy of brain glioma in animals. J Control Rel 2010;141:183e92. [8] Carroll R, Bhatia D, Geldenhuys W, Bhatia R, Miladore N, Bishayee A, et al. Brain-targeted delivery of Tempol-loaded nanoparticles for neurological disorders. J Drug Target 2010;18:665e74. [9] Wu G, Barth RF, Yang W, Kawabata S, Zhang L, Green-Church K. Targeted delivery of methotrexate to epidermal growth factor receptorepositive brain tumors by means of cetuximab (IMC-C225) dendrimer bioconjugates. Mol Cancer Ther 2006;5:52e9. [10] Ren Y, Kang CS, Yuan XB, Zhou X, Xu P, Han L, et al. Co-delivery of as-miR-21 and 5-FU by poly(amidoamine) dendrimer attenuates human glioma cell growth in vitro. J Biomater Sci Polym Ed 2010;21:303e14. [11] Gajbhiye V, Kumar PV, Tekade RK, Jain NK. Pharmaceutical and biomedical potential of PEGylated dendrimers. Curr Pharm Design 2007;13:415e29. [12] Patri AK. Targeted drug delivery with dendrimers: comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex. Adv Drug Del Rev 2005;57:2203e14. [13] Dutta T, Agashe HB, Kumar PV, Garg M, Jain NK. Dendrosome-based gene delivery. J Exp Nanosci 2006;1:235e48. [14] Gupta U, Agashe HB, Jain NK. Polypropylene imine dendrimer mediated solubility enhancement: effect of pH and functional groups of hydrophobes. J Pharm Pharmaceu Sci 2007;10:358e67. [15] Kobayashi H, Brechbiel MW. Nano-sized MRI contrast agents with dendrimer cores. Adv Drug Deliv Rev 2005;57:2271e86. [16] Gajbhiye V, Kumar PV, Tekade RK, Jain NK. Dendrimers as therapeutic agents: a systematic review. J Pharm Pharmacol 2009;61:989e1003. [17] Tekade RK, Kumar PV, Jain NK. Dendrimers in oncology: an expanding horizon. Chem Rev 2009;109:49e87. [18] Dutta T, Jain NK. Targeting potential and anti-HIV activity of lamivudine loaded mannosylated poly (propyleneimine) dendrimer. Biochim Biophys Acta 2007;1770:681e6. [19] Blasi P, Giovagnoli S, Schoubben A, Ricci M, Rossi C. Solid lipid nanoparticles for targeted brain drug delivery. Adv Drug Del Rev 2007;59:454e77. [20] Gajbhiye V, Kumar PV, Sharma A, Jain NK. PEGylated PPI dendritic architectures for sustained delivery of anti-inflammatory agent. Curr Nanosci 2008;4:267e77. [21] Gajbhiye V, Kumar PV, Tekade RK, Jain NK. Novel PEGylated nanoparticulate architectures for sustained delivery of H2 receptor antagonist. Euro J Med Chem 2009;44:155e66.
V. Gajbhiye, N.K. Jain / Biomaterials 32 (2011) 6213e6225 [22] Beban M, Miller PS. Preparation of an imidazole-conjugated oligonucleotide. Bioconjug Chem 2000;11:599e603. [23] Agashe HB, Babbar AK, Jain S, Sharma RK, Mishra AK, Asthana A, et al. Investigation on biodistribution of technetium-99m-labeled carbohydratecaoted poly(propylene imine) dendrimers. Nanomedicine: NBM 2007;3: 120e7. [24] Fujita M, Lee BS, Khazenzon NM, Penichet ML, Wowrowsky KA, Patil R, et al. Brain tumor tendem targeting using a combination of monoclonal antibodies attached to biopoly(b-L-malic acid). J Control Rel 2007;122:356e63. [25] Zhao L, Wei Y, Zhong X, Liang Y, Zhang X, Li W, et al. PK and tissue distribution of docetaxel in rabitts after i.v. administration of liposomal and injectable formulations. J Pharm Biomed Anal 2009;49:989e96.
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[26] Gupta U, Dwivedi SKD, Bid HK, Konwar R, Jain NK. Ligand anchored dendrimers based nanoconstructs for effective targeting to cancer cells. Int J Pharm 2010;393:185e96. [27] Rowe JDD, Bello J, Mitnick R, Sood B, Filippi C, Moran J, et al. Accelerated regression of brain metastases in patients receiving whole brain radiation and the topoisomerase II inhibitor, lucanthone. Int J Radia Onco Biol Phys 1999;43:89e93. [28] Kemper EM, Verheij M, Boogerd W, Beijnen JH, van Tellingen O. Improved penetration of docetaxel into brain by co-administration of inhibitors of Pglycoprotein. Euro J Cancer 2004;40:1269e74. [29] Kreuter J, Shamenkov D, Petrov V, Ramge P, Cychutek K, Koch-brandt C, et al. Apolipoprotein-mediated transport of nanoparticle-bound drugs across the blood-brain barrier. J Drug Target 2002;10:317e25.
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The treatment of Glioblastoma Xenografts by surfactant conjugated dendritic nanoconjugates Virendra Gajbhiye, Narendra K. Jain* Pharmaceutics Research Laboratory, Dept. of Pharmaceutical Sciences, School of Engineering & Technology, Dr. H. S. Gour University, Sagar, MP 470 003, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2011 Accepted 22 April 2011 Available online 26 May 2011
Polysorbate 80 (P80) anchored poly(propyleneimine) (PPI) dendritic nanoconjugate was developed and evaluated for targeting anti-cancer drug, docetaxel (DTX) to the brain tumor. In vitro cytotoxicity studies of free DTX, DTXePPI and DTXeP80-PPI dendrimers were carried out using U87MG human glioblastoma cell line. The in vivo anti-cancer activity in brain tumor bearing rats revealed that DTX loaded P80 conjugated dendrimers reduced the tumor volume extremely significantly (p < 0.0001; more than 50%). The median survival time for brain tumor bearing rats treated with DTXeP80-PPI dendrimers (42 days) was extended very significantly as compared to DTXePPI (23 days; p < 0.001), receptor blocked group (15 days; p < 0.001) and free DTX (18 days; p < 0.001). Gamma scintigraphy and biodistribution studies further confirmed the targeting efficiency and higher biodistribution of ligand conjugated dendrimer into the brain. The results concluded that the developed nanoconjugate has potential to deliver significantly higher amount of drug to brain tumor for improved therapeutic outcome. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Animal model Apolipoprotein Brain Dendrimer Drug delivery
1. Introduction Brain tumors are one of the 10 major causes of death by cancer. Despite of advances in strategies combining surgery, radiotherapy, and chemotherapy, the average 1 year survival of the brain tumor patient has amplified extremely petite over the past three-four decades. This is attributed to the fact that brain tumors, upon diagnosis, are usually already 40e60 cm3 in volume comprising approximately 4e6 1010 cells [1]. Brain tumors often attack or compress on normal brain tissue, and symptoms can result from that pressure. A person might experience different kinds of symptoms depending on wherein the brain a tumor is located [2]. A tumor that initiates in the brain is a primary brain tumor. Cancer cells that start growing elsewhere in the body and then travel to the brain via blood stream develop metastatic brain tumors e.g. cancers of the lung, breast, colon and skin frequently reach to the brain and may cause Glioblastoma multiforme, astrocytoma, medulloblastoma etc. Glioblastoma (gliomas) represents 42% of primary CNS tumors with more than three fourth being malignant. Prevalence of gliomas is approximately 5e10 per 100,000 population [3]. Approximately 20,000 cases of malignant glioma were diagnosed in the U.S. each year with 12,000 deaths estimated in year 2007 [4].
* Corresponding author. E-mail addresses: [email protected] (V. Gajbhiye), [email protected]. in (N.K. Jain). 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.04.057
Delivery of the bioactives to the brain is the utmost challenging task to cope with brain diseases, which attributed to bloodebrain barrier (BBB), blood-CSF barrier and efflux mechanism (P-glycoprotein; P-gp). These barriers control the entry of body as well as foreign compounds to access the brain cells [5]. The BBB prevents the entry of about 98% of small molecules and 100% of large molecules. Therapeutic benefit in glial tumors is often limited due to low permeability of delivery systems across the BBB, drug resistance and poor access into the tumor [6]. The aforementioned biochemical and targeting confronts, hurdle the advancement of effectual brain tumor chemotherapies. The field of drug delivery has fully emerged and came into existence as an ideal approach for drug targeting to brain [6]. It corresponds to a promising non-invasive approach for better drug delivery to the brain through exploitation of the various influx transport systems depicted within the cerebral endothelial, including carrier-mediated transports (CMT), receptor-mediated endocytosis (RME) and adsorptive-mediated endocytosis (AME). Nowadays, nano-sized carriers, especially polymeric (liposomes, nanoparticles and dendrimers) are being used widely and are under investigation with or without coating or coupling with ligands to offer a sustained level of drug and to accomplish cellular target with improved specificity [7e10]. These carriers are widely consented and appreciated due to their controlled drug release profile as well as their special targeting mechanism. Dendrimers are mono-dispersed macromolecules with a regular tree-like branched three-dimensional architecture that have potential for drug delivery, attributed to the properties of the
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macromolecules (1e100 nm), with multivalency, monodisperse, host-guest entrapment, defined molecular weight, large number of peripheral end groups, and interior cavities [11]. Special properties of dendrimers subjected them for immense exploration among the researchers, which has led to their possible exploitation in different pharmaceutical fields like drug delivery [12], gene delivery [13], solubility enhancement [14], diagnosis [15] etc. In addition, dendrimers have also generated interest in drug discovery by virtue of their therapeutic values [16]. Recently, surface functionalized dendrimers are being extensively explored for targeting anti-cancer and anti-HIV drugs [17,18]. The field of nanotechnology mediated anticancer drug targeting has observed emergence of an endowed carrier ’dendrimer’ in the last decade. Dendrimers can be anchored with ligands, that bind exclusively to the receptors over-expressed on BBB and might elicits receptor-mediated endocytosis. Apolipoproteins (e.g. ApoE) are believed to be adsorbed on P80 anchored nanocarriers, which could be responsible for the interaction with LDL receptors on BBB and subsequent endocytosis [19]. This approach was endeavored to utilize for brain delivery of DTX via P80 anchored PPI dendrimers. Thus in the present study, surfactant (polysorbate 80) has been anchored to PPI dendrimers and the nanoconjugate was explored for brain targeting potential of DTX. 2. Materials and methods 2.1. Materials DTX was a benevolent gift from m/s Dabur Pharmaceuticals (New Delhi), India. Carbonyldiimidazole (CDI), Polysorbate 80 and MTT were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ethylenediamine, and acrylonitrile were purchased from CDH (Mumbai), India. Reney Nickel was purchased from Merck (Mumbai), India. All other chemicals were of reagent grade and purchased from CDH, (Mumbai), India. 2.2. Synthesis of 5.0G PPI dendrimers PPI dendrimers (5.0 G) was synthesized by divergent method as reported by us earlier [20,21]. Briefly, double Michael addition reaction was used to produce half generation (eCN terminated) by adding acrylonitrile to the initiator core, ethylenediamine (EDA). It was followed by heterogeneous hydrogenation by means of Reney Nickel catalyst to synthesize full 1.0 G generation (eNH2) dendrimers. The reaction sequence was repeated intermittently to fabricate PPI dendrimers up to fifth generation (5.0 G PPI). The 5.0 G PPI was purified by dialysis against double distilled water in a dialysis tube (MWCO 5 kDa, Sigma, USA) to remove lower generation dendrimers and un-reacted chemicals. FT-IR spectroscopy was carried out using PerkineElmer FT-IR spectroscope (USA). 1H NMR spectroscopy of the dendrimer sample was carried out at 300 MHz in CDCl3 (Bruker DRX, USA). 2.3. Synthesis and characterization of P80 anchored 5.0 G PPI dendrimer This is a debut report on P80 or any type of surfactant conjugation on 5.0 G PPI dendrimers. The conjugation of P80 on 5.0 G PPI dendrimers was carried out with the help of 1,10 carbonyldiaimidazole (CDI) as a catalyst. This is also the debut report on anchoring any type of ligand on 5.0 G PPI dendrimers with the help of CDI. Firstly, free OH group of P80 was converted into a reactive imidazole carbamate by reacting with CDI (Scheme 1). For the above reaction P80 (25 mM) and CDI (25 mM) were dissolved in DMSO and the solution was stirred at 30 C for 1 h to generate a reactive imidazole carbamate [22]. Subsequent addition of a highly nucleophilic group (e.g. NH2) in the solution of reactive imidazole carbamate replaces imidazole and forms a highly stable carbamate. The 5.0 G PPI dendrimers are highly nucleophilic due to 64 terminal NH2 groups, which were added (1 mM) to the solution of reactive imidazole carbamate of P80. The mixture was stirred at room temperature (27 1 C) for 48 h to ensure the completion of reaction. Resulting solution was concentrated under vacuum. P80 anchored dendrimers (P80-PPI) were purified by dialysis against double distilled water in a dialysis tube (MWCO 12 kDa, Sigma, USA) to remove un-reacted P80 and impurities followed by lyophilization (Heto Drywinner, Heto-Holten, Allerod, Denmark). FT-IR and 1H NMR spectroscopies of lyophilized P80-PPI dendrimers were carried out as done for 5.0 G PPI dendrimers. The P80-PPI dendrimers was also characterized for extent of P80 conjugation and for exact molecular weight of the conjugate by Size Exclusion Chromatography (SEC). This experiment was performed using SEC system consisting refractive index detector (Santa Barbara, CA). The analysis was done at room temperature (27 2 C) using two serially aligned TSKGEL columns G3000 PW and G4000 PW. The isocratic mobile phase was PBS (pH 7.4)
at a flow rate of 1 ml/min. Sample concentration was kept 1 mg/ml in PBS, and 100 ml was injected. Molecular weights of 5.0 G PPI and P80-PPI dendrimers were determined using Astra V software (Wyatt Technology Corporation, Santa Barbara, CA). Transmission Electron Microscopy (TEM) was carried out to characterize the size of the conjugated system. The conjugates were added to a carbon grid and stained negatively using osmium tetroxide at 50 kV. The photomicrographs were taken at suitable magnification using a transmission electron microscope (Philips CM12 Electron Microscope, Eindhoven, Netherland). 2.4. Drug loading in formulations The known molar concentrations of P80-PPI dendrimer (10 mM) and DTX (100 mM) were taken in PBS (pH 7.4), and The mixture was magnetically stirred (50 rpm) for 48 h using teflon beads. The solvent was evaporated and dialyzed twice in cellulose dialysis bag (MWCO 1000 Da Sigma, USA) against methanol under sink conditions for 15 min to remove free DTX from the formulations. The free DTX was then estimated spectrophotometrically (lmax 230 nm; UV-1601, Shimadzu, Japan) to determine indirectly [20,21] the amount of drug loaded within the system. Similar procedure was followed for DTX entrapment in plain 5.0 G PPI dendrimer. The dialyzed formulations were lyophilized and used for further characterization. 2.5. Characterization of the drug loaded formulations The drug loaded dendritic system (DTXeP80-PPI) was characterized by differential scanning calorimetry (DSC). Differential scanning calorimetry (DSC) was carried out to evaluate the thermal stability and transformation in crystallinity over a range of temperatures. Free DTX, P80-PPI and DTX loaded P80-PPI dendrimer complex were studied and compared. Different samples were placed in an aluminium pan separately and a lid was crimped. The pan was then positioned in the analyzing cell of a DSC module (DSC Q10 V9.0 Build 275, TA Instruments, USA). The temperature of the DSC module was equilibrated at 35 C and then amplified at a rate of 10 C/min under a N2 gas flush out until the material began to degrade. The temperatures for each peak were obtained from resulting curve, which provided the indication of temperature stability and phase transitions. 2.6. In vitro release Drug release from known amounts of DTX loaded plain PPI, and P80-PPI dendrimers were determined in acid phthalate buffer (pH 4.0) and PBS (pH 7.4) in a modified dissolution method [20,21]. The dialysis bags (MWCO 1000 Da, Sigma, USA) were filled with a known amount of DTX loaded dendrimeric formulations and were placed in 50 ml of medium (PBS:Tween 80 in a ratio of 80:20) at 37 2 C with slow magnetic stirring under sink conditions. Aliquots of 1 ml were withdrawn from the external solution and immediately replenished with an equal volume of fresh medium. The drug concentration was detected spectrophotometrically (lmax 230 nm; UV-1601, Shimadzu, Japan) against the similar blank medium. 2.7. Comparison of hemolytic toxicity The RBC suspension for hemolytic studies was prepared following the reported procedure [20,21]. Briefly, the RBC suspension (5% hematocrit) of the human blood was collected in HiAnticlot blood collection vials (Himedia Labs, Mumbai, India). To 4.5 ml of normal saline 0.5 ml of suitably diluted free DTX, plain 5.0 G PPI, P80-PPI, DTXePPI, and DTXeP80-PPI were added and incubated for 1 h with RBC suspension. This allowed comparison of the hemolysis data of the free drug and different dendritic architectures to assess the effect of conjugation on hemolysis. After centrifugation, supernatants were taken and diluted with an equal volume of normal saline and absorbance was measured at 540 nm. RBC suspension was added to 5 ml of 0.9% NaCl solution (normal saline) and 5 ml distilled water to obtain 0% and 100% hemolysis, respectively. The degree of hemolysis was determined by the following equation: Hemolysisð%Þ ¼
Abs Ab0 100 Ab100 Ab0
where Abs, Ab0 and Ab100 are the absorbance of sample, a solution of 0% hemolysis and a solution of 100% hemolysis, respectively. 2.8. Cell culture In the present study U87MG human glioblastoma cell line, purchased from National Center for Cell Sciences, Pune, India was used. The cell line was cultured in cell culture flask using cell culture medium comprised of Dulbecco’s modified Eagle medium (DMEM; Himedia, Mumbai, India) supplemented with 20% fetal calf serum (FCS), 4 mM/l L-glutamine, 100 mg/ml streptomycin, and 100 U/ml penicillin (DMEM, FCS, glutamine, streptomycin, penicillin purchased from Himedia, Mumbai, India). The cells were cultured under a humidified atmosphere containing 5% CO2 at 37 C (Heracell CO2 Incubator, Hanau, Germany). The cells for the present study were utilized after 15th passage (Supporting Files Figure 1 aeh).
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Scheme 1. Synthesis of P80-PPI dendrimers.
2.9. In vitro cytotoxicity assay The in vitro cytotoxicity of free DTX, plain 5.0 G PPI, P80-PPI, DTXePPI, and DTXeP80-PPI were assessed via MTT assay on U87MG cell line. The cells were seeded into 96-well tissue culture plate in a density of 5 103 cells/well containing DMEM
medium and incubated for 48 h to acquire more than 75% confluency. Freshly prepared solutions of samples (100 ml) in DMSO were applied to each well in a concentration range between 0.001 and 10 mM. The culture plates were incubated for 24 and 48 h. After incubation, 20 ml of MTT solution (5 mg/m; in Hank’s Balanced Salt Solution) was added to each well and incubated for 3 h to allow viable cells to
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reduce the yellow MTT to purple formazan crystals. To each well 100 ml of DMSO was added, which dissolves formazan crystals. Absorbance was measured at 570 nm in an ELISA plate reader (Medispec Ins. Ltd, Mumbai, India). Cells without any sample were used as control. The experiment was repeated six times for each sample. Cell viability of each group was compared with control and was expressed as % cell viability using following formula: % cell viability ¼
Atreated 100 Acontrol
where Atreated is the absorbance of treated cells with different samples at 570 nm and Acontrol is the absorbance of control at 570 nm. 2.10. Cell uptake assay using gamma scintigraphy U87MG cell line was used to perform cellular uptake of different formulations. Free DTX, 5.0 G PPI dendrimers and P80-PPI dendrimers were radiolabeled with 99m TcO4 at Department of Nuclear Medicine, JawaharLal Nehru Cancer Hospital & Research Center (JNCHRC), Bhopal (MP) India, by the method reported earlier from our lab [23]. The U87MG cells were seeded in fibronectin coated tissue culture petri dishes at a density of 5 104 cells/plate containing 1 ml DMEM. The petri dishes were divided into five groups, each group containing six petri dishes. The dishes were incubated for 48 h in a humidified atmosphere containing 5% CO2 at 37 C with changing of the medium after every 12th h. First four groups were applied with free 99m TcO4, 99mTcO4eDTX, 99mTcO4ePPI and 99mTcO4eP80ePPI (100 mCi). Fifth group was treated as control. The samples were incubated with the cells for 3 h. After incubation period culture medium was removed and the cells were washed five times with Hank’s Balanced Salt Solution (Himedia, Mumbai, India). Each cell containing petri dishes were provided with 1 ml of culture medium and immediately subjected to measurement of radioactivity with the help of a gamma camera (Siemens E-cam, MEDX Inc, Illinois, USA). The radioactivity was measured in the form of radioactive counts over a duration of 60 s. 2.11. Tumor implantation The tumor implantation was carried out by the reported method [7,24] with slight modification. Male albino rats (Sprague Dawley stain, 8e9 weeks old weighing 250 20 g) were obtained from animal house of Department of Research, JawaharLal Nehru Cancer Hospital and Research Center, Bhopal (MP) India. All the animal studies were conducted in accordance with the protocol approved by the Institutional Animal Ethical Committee of Dr. H. S. Gour University, Sagar (MP) India (registration no.379/01/ab/CPCSEA; 10/87, dated 20-05-2010). The U87MG cells were suspended in serum free DMEM medium in a density of 5 105/10 ml. The cells were implanted stereotactically into right basal ganglia field of animals. Animals were kept under similar laboratory conditions and fed with normal food and water avoiding any type of stress. 2.12. In vivo anti-tumor activity After 8 days of tumor inoculation rats were divided into eight groups each group comprising 12 animals. The animals were administered with different formulations samples intravenously via tail vein according to grouping on every 3rd day of treatment up to one week (i.e. 9, 11, 13th day). The details of samples were as follows: Group 1 ¼ PBS (pH 7.4); Group 2 ¼ 5.0 G PPI dendrimers; Group 3 ¼ P80; Group 4 ¼ P80-PPI; Group 5 ¼ DTX; Group 6 ¼ DTXePPI and Group 7 ¼ DTXeP80-PPI (30 mg/kg equivalent of DTX). Group 1 was treated as control and 8th group was utilized for receptor blockage assay. This assay was based on the principle of initial blockage of LDL receptors by injecting 30 mg/kg cholesterol, followed by treatment with DTXeP80-PPI and subsequent effect on tumor volume. Six animals from each group were sacrificed on 15th day of treatment (n ¼ 6) and tumor volume (cubic milimeters) was measured with the help of Vernier Caliper using formula: V ¼ length width height [7]. Antitumor potential of different formulations are expressed as tumor inhibitory ratio (%T/C; treated versus control), dividing tumor volumes of treated groups by control group and multiplied by 100). Remaining six animals were observed for survival life span using KaplaneMeier survival curve. 2.13. In vivo biodistribution studies The in vivo qualitative biodistribution studies were performed by scanning the glioblastoma bearing rats under a gamma camera. Briefly, the tumor induced animals were divided into four groups (n ¼ 3). First, second, third and forth groups were injected intravenously via tail vein with free 99mTcO4, 99mTcO4eDTX, 99m TcO4ˉePPI and 99mTcO4eP80-PPI, respectively. The animals were subjected to whole body gamma scanning after 1 h. Tissue distribution studies were carried out for quantitative measurement of DTX in different organs. Tumor induced rats were divided into 4 groups, each group comprising 9 animals. First group was fed with PBS (pH 7.4) and treated as control. Second, third and fourth groups were administered with free DTX, DTXePPI and
DTXeP80-PPI, respectively (30 mg/kg) via tail vein. Three animals from each group were sacrificed 1, 4 and 8 h post administration (n ¼ 3). Immediately after sacrificing the animals the tissues viz. brain, liver, kidney, lung, spleen and tumor were carefully removed and weighed. One gram each of different tissues were added with 5 ml of trichloroacetic acid (10% v/v in water) and vortexed for 1 min. The required quantity of ethanol was added and homogenized well in a tissue homogenizer (York Scientific Instrument, New Delhi, India), which also removed the entrapped drug from dendrimers. The homogenates were centrifuged at 5000 rpm (Remi Elektrotech Ltd., Mumbai, India) for 15 min and the supernatant was collected and assayed for DTX content by HPLC method [25] using acetonitrile: methanol: 0.02 M ammonium acetate buffer (pH 5.0) as mobile phase in a ratio of 20:50:30 (v/v/v) at a flow rate of 1 ml/min.
3. Statistical analysis The results are expressed as mean standard deviation and the analysis was performed with SPSS 13 (SPSSÒ, Chicago, USA). Statistical significance is reported as P < 0.05. 4. Results 4.1. Synthesis of 5.0G PPI dendrimers PPI dendrimers (5.0 G) was synthesized following established method and characterized as reported by us earlier [20,21]. The characteristic FT-IR peaks of 5.0 G PPI dendrimers are of CeC bend (1107 cm1); CeN stretch (1309 cm1); CeH bend (1405 cm1); NeH deflection of amine (1663 cm1); and stretching of primary amine (3421 cm1), confirming nitrile terminal groups of dendrimer were converted to amine terminals. The fifth generation PPI dendrimer was further characterized by 1H NMR spectroscopy. Multiplets (m) between 0.9 and 1.2 ppm and 2.2e2.3 correspond to methylene (eCH2) groups of EDA. Multiplets at 2.5e2.8 correspond to eN(CH3). The results matched with the reported synthesis of PPI dendrimers [20,21]. 4.2. Synthesis of P80 anchored 5.0G PPI dendrimer The characterized 5.0 G PPI dendrimers were anchored with P80 with the help of CDI (Scheme 1). FT-IR spectrum exhibited major peak at 1666 cm1 (eCONH stretching), which confirmed the conjugation of P80 with dendrimer periphery (Fig. 1a). Other major peaks in the FT-IR spectrum of nanoconjugate were of eCH2 rocking (661 cm1), eCN stretching (1071 cm1), NH bending of amine (1455 and 1571 cm1), and overlapping broad peak of eOH and eNH stretching (3318 and 3393 cm1). 1H NMR d values at 7.08 and 7.7 ppm strongly evidenced the presence of eCONH bond (Fig. 1b), which further confirmed the conjugation. Primary amine (eNH2) exhibited peak at d value of 5.3 ppm. The d value at 3.6 ppm was attributed to eOH groups present in P80. Characteristic peaks of eCH2 of alkane and eN(CH3) of tertiary amine were present at 0.9e1.2 and 2.5 ppm, respectively. SEC was performed to determine the extent of P80 conjugation and exact molecular weight of the nanoconjugate. The molecular weight was found to be around 23 kDa (Fig. 2b), which suggested that approximately 12 molecules of P80 (molecular weight 1310 Da) conjugated to dendrimer periphery. The increase in molecular weight also evidenced the conjugation. TEM results displayed the nanometric size of P80 conjugated dendritic system, wherein circular nanometric conjugates were observed (Fig. 3 a and b). The results were in agreement with earlier reports [26]. 4.3. Drug loading Equilibrium dialysis method was utilized to entrap DTX in dendrimeric formulations. The percentage drug loading was determined indirectly by estimating the un-entrapped drug,
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spectrophotometrically. The percentage DTX loading was found extremely significant (P < 0.0001) for P80-PPI dendrimers as compared with plain 5.0 G PPI dendrimers (Fig. 4 a and b). The P80PPI dendrimer displayed 67.61 1.44% DTX entrapment, whereas plain 5.0G PPI displayed only 27.47 1.01% DTX loading (data not shown). 4.4. Characterization of the drug loaded formulations The lyophilized drug loaded P80-PPI formulation was characterized for the presence of DTX inside the dendritic system by DSC. The free DTX experienced a sharp endothermic peak (20 mW) at its melting point 230 C (Fig. 5a), while broad endothermic peaks (27 to 46 mW) ranging from 65 to 125 C were observed in case of P80-PPI dendrimer (Fig. 5b). However, the DSC curve of DTXeP80-PPI showed both the endothermic peaks of DTX and P80PPI at around 230 and 125 C (Fig. 5c), which confirmed presence of DTX in the dendritic nanoconstructs. 4.5. In vitro drug release The release studies suggested that both 5.0 G PPI and P80ePPI dendrimers provided burst release of DTX at pH 4.0. The drug release was found to be 98.2 1.32 and 56.4 2.25% for plain 5.0 G PPI and P80ePPI dendrimers, respectively at 4th h (Fig. 6). On the contrary DTX release was significantly (P < 0.0001) retarded from P80-PPI at pH 7.4 (55.7 1.83% at 24th h) as compared to plain 5.0 GPPI dendrimers (97.4 2.05% at 24th h). P80-PPI dendrimeric nanocarrier released 97.7 1.61% of DTX up to 96th h. 4.6. Hemolytic toxicity All major anti-cancer drugs exert adverse effect of hemolysis. DTX also exerts adverse effect like anemia and neutropenia due to hemolytic nature. In addition, the hemolytic toxicity of the cationic dendrimers poses constraint for potential as drug delivery system. This toxicity is attributed to the polycationic nature of the PPI dendrimers [20,21]. DTX displayed almost more than 50% hemolysis at 0.4% w/v concentration (Fig. 7). The 5.0 G PPI dendrimers showed hemolytic toxicity 9.0 0.26, 11.9 0.21, 14.1 0.42 and 17.4 0.83% at 0.1, 0.2, 0.3 and 0.4% w/v concentrations, respectively. However P80 anchoring of dendrimers caused decreased hemolysis of the RBC considerably at all concentrations (1.7 0.35, 2.6 0.52, 3.3 0.42, and 3.9 0.33% at 0.1, 0.2, 0.3 and 0.4% w/v concentrations, respectively). The DTXePPI has shown reduced toxicity as compared to DTX but higher toxicity than plain PPI and DTXeP80-PPI dendrimers. Whereas, DTXeP80-PPI has shown significantly less (p < 0.05) toxicity than PPI, DTX and DTXePPI but higher toxicity than unloaded P80ePPI dendrimers (Fig. 7). The hemolytic toxicity of PPI dendrimers was well in agreement with previous reports [20,21,26] 4.7. In vitro cytotoxicity assay The in vitro cytotoxicity assay was carried out on U87MG cells via MTT assay by incubating different formulations for 24 and 48 h. Unloaded plain 5.0 G PPI and P80-PPI dendrimers have not shown any significant cytotoxic effect after 24 and 48 h incubation even at higher concentration (10 mM). DTX exerted very potent cytotoxic effect and displayed IC50 value of 0.15 mM after incubation for a period of 24 h (Fig. 8a), which was significantly higher (p < 0.001) than DTXePPI (0.9 mM) and DTXeP80-PPI (3.5 mM). DTXePPI has also shown significantly higher (p < 0.001) cytotoxicity and lower IC50 value than DTXeP80-PPI after 24 h.
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After 48 h, DTX has shown insignificant change in cytotoxicity and IC50 value (0.12 mM). However, extremely significant (p < 0.001) decrease in IC50 value of DTXePPI and DTXeP80-PPI was observed (Fig. 8b), which was shifted from 0.9 mM to 0.25 mM and 3.5 mMe0.3 mM for DTXePPI and DTXeP80-PPI, respectively. The difference between IC50 values of DTXePPI and DTXeP80-PPI was found to be insignificant (p ¼ 0.0742) after 48 h incubation (Fig. 8b). 4.8. Cell uptake assay study Cell uptake of radiolabeled 5.0 G PPI and P80-PPI was performed using gamma scintigraphy. The results showed that there was significant uptake of dendritic formulations as compared to free 99m TcO4ˉ and 99mTcO4ˉeDTX. The 5.0 G99PPI and P80-PPI dendrimers showed extremely significant higher (p < 0.0001) counts (6062 29 and 6015 25, respectively) in 60 s at 3rd h than free 99m TcO4ˉ (Fig. 9 a and b), while 99mTcO4eDTX and free 99mTcO4 have shown 2465 26 (Supporting Files Figure 2) and 953 31 counts, respectively in 60 s. The 5.0 G PPI had shown higher uptake than P80ePPI, but the difference was insignificant (p ¼ 0.1006). 4.9. In vivo anti-tumor activity The anti-tumor studies on glioblastoma bearing rats have shown that normal saline, 5.0 G PPI, P80 and P80-PPI do not possess any anti-tumor efficacy. The result suggested that DTXeP80-PPI reduced extremely significant tumor volume than DTXePPI and free DTX (p < 0.0001). The tumor volume at day 15th of tumor inoculation for control, DTX, DTXePPI and DTXeP80-PPI group were found to be 128.8 4.1, 102.6 2.2, 90.8 2.2, and 46.4 2.9 mm3, respectively (Fig. 10). On the contrary the tumor volume of LDL receptor blocked group was found to be 110.5 4.5 mm3. DTXePPI had also shown very significant (p < 0.001) reduction in tumor volume as compared to control and free DTX. The tumor volume ratio (%T/C) was found to be highest for free DTX. The tumor volume ratio for DTX, DTXePPI and DTXeP80ePPI were found to be 79.65 3.9, 70.49 2.7, and 36.02 1.6%, respectively. KaplaneMeier survival curves based on survival time were plotted for different groups. The curves suggested that the median survival time for rats treated with DTX loaded P80 anchored 5.0G PPI dendrimers (42 days) was extended significantly (Fig. 11) as compared to DTXePPI (23 days, p < 0.001), free DTX (18 days p < 0.001), receptor blocked group (15 days, p < 0.001) and control group (14 days, p < 0.001). 4.10. In vivo biodistribution studies The in vivo targeting efficiency was further evaluated by injecting free 99mTcO4, 99mTcO4eDTX, 99mTcO4ePPI and 99m TcO4ˉeP80ePPI into different groups of rats via tail vein. Free 99m TcO4 was selected because it does not cross BBB [27]. The qualitative gamma scanning images after 1 h suggested that free 99m TcO4 failed to cross blood brain barrier (Fig. 12a), while very little amount of radioactivity was seen in brain for 99mTcO4eDTX and 99mTcO4ePPI (Fig. 12b and c). In contrast, 99mTcO4eP80ePPI showed highest amount of radioactivity in brain as compared to free 99mTcO4ˉ, 99mTcO4eDTX and 99mTcO4ePPI (Fig. 12d). The targeting efficiency was further evaluated by biodistribution of DTX from different formulations. The concentration of DTX in different organs and tumor were estimated via HPLC [25] at 1st, 4th and 8th h. The result indicated that more than 7.5, 9 and 10% of injected dose accumulated in brain at 1st, 4th, and 8th h, respectively in case of DTXeP80-PPI (Table 1), while the percentage was
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Fig. 1. (a) FT-IR spectrum of P80-PPI, and (b) 1H NMR spectrum of P80-PPI.
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increased from 1st to 4th and then to 8th h in case of DTXeP80-PPI and DTXePPI, while free DTX had shown increase in concentration from 1st to 4th hr and then again decreased at 8th h. Similar pattern of DTX distribution was seen at the most desirable site, the brain tumor. Here also, DTXeP80-PPI had shown 4.1 and 39 times higher accumulation as compared to DTXePPI and free DTX at 8th h. Free DTX had shown higher accumulation in excretory organs viz. liver and spleen as compared to DTXePPI and DTXeP80-PPI, which was found to increase with time (Table 1). DTXeP80-PPI showed lesser distribution of drug to excretory organs. The DTX concentration was increased in lungs and kidneys from 1st to 4th h in case of all three formulations, but the concentration was decreased at 8th h for all formulations.
Fig. 2. Mass fraction profile of (a) plain 5.0G PPI, and (b) P80-PPI as measured by SEC.
approximately 2, 2.5 and 2.8, respectively for DTXePPI. In contrast, DTX have shown very little tendency to cross blood brain barrier. The percentage accumulated dose for DTX was approximately 0.4, 0.35, and 0.26% at 1st, 4th, and 8th h, respectively. The DTXeP80PPI nanoconjugate had shown approximately 3.5 and 39 times higher penetration of DTX in brain as compared to DTX-PPI and free DTX, respectively at 8th h (Table 1). The distribution of DTX was
Fig. 3. Transmission Electron Microscope images of (a) plain 5.0G PPI, and (b) P80ePPI nanoconjugates.
5. Discussion The blood brain barrier constitutes major hurdle for anti-cancer agents to reach brain tumor cells [5,6]. Apart from this P-gp system plays major role in efflux of P-gp substrate anti-cancer bioactives, which also leads to drug resistance. The DTX is a well known substrate of P-gp efflux system. This peculiarity is attributed to its ineffectiveness for brain tumor chemotherapy, even if possessing potent cytotoxic behavior. The trafficking of DTX in brain has been found to increase by blocking P-gp efflux systems [28]. The objective of the present study was to increase the bioavailability and residence time of DTX for effective brain tumor chemotherapy. In the present study we reported debut conjugation of surfactant and dendritic nano-architecture for achieving the aforementioned goal. Firstly, 5.0 G PPI dendrimers were prepared and characterized according to procedures established by us. The P80 anchoring to 5.0 G PPI dendrimers was carried out using CDI chemistry. The purified conjugate was subjected to characterization by FT-IR and 1 H NMR. FT-IR peak of eCONH stretching at 1666 cm1 primarily suggested presence of eCONH bond between P80 and 5.0 G PPI dendrimers. 1H NMR d values at 7.08 and 7.7 ppm further confirmed the presence of eCONH bond. The formation of eCONH bond must be attributed to CDI chemistry, which formed highly reactive imidazole carbamate with P80 and then substituting the reactive imidazole with highly nucleophilic groups (eNH2 terminals of PPI dendrimers). The conjugation was furthermore evidenced by increase in molecular weight (7.1 kDae23 kDa) estimated by SEC. Increase in molecular weight attributed to conjugation of approximately 12 molecules of P80 on the dendrimer periphery. Electron microscopy further demonstrated nanometric size of conjugate and provided indirect qualitative evidence of increase in size after P80 conjugation. The percentage DTX loading was found extremely significant (P < 0.0001) for P80-PPI dendrimers as compared with plain 5.0 G PPI dendrimers. Non-covalent interactions between DTX and P80-PPI dendrimers, such as hydrophobic interaction and hydrogen bonding, contributed to the physical entrapment of DTX molecules inside dendritic cavities and also in surface P80 layers. Higher entrapment in P80ePPI might have attributed to additional interaction of DTX and P80 at the dendrimer periphery. Sealing of dendrimer periphery with P80 increased the steric hindrance, which might have contributed to higher drug loading. Comparison of FT-IR spectra of free DTX, P80-PPI and DTXeP80-PPI suggested drug loading, which was further supported by DSC curves. The separate DSC curve of DTX in case of DTXeP80-PPI further demonstrated the physical entrapment of DTX in dendrimer interiors and ruled out any drug-dendrimer conjugation possibilities. In vitro DTX release was extremely significantly retarded from DTXeP80-PPI at pH 7.4 than pH 4.0. Primary and tertiary nitrogens at dendrimer periphery and cavities, respectively might have contributed to extended release at pH 7.4 and burst release at pH
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Fig. 4. (a) Schematic diagram of DTX loaded P80ePPI, and (b) molecular model of P80ePPI.
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Fig. 5. Curves of differential scanning calorimetry (a) free DTX, (b) P80-PPI, and (c) DTX loaded P80ePPI.
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4.0. The nitrogens are 2/3rd protonated at pH 7.4, which causes shrinking of the hydrophobic interior of PPI dendrimer. P80 anchoring on dendrimer periphery also further retarded the drug release. Burst release at pH 4.0 may be ascribed to higher protonation of tertiary amines at acidic pH. Higher protonation causes increase in size of dendrimer molecule and expansion of dendrimer branches. This accounts for the inability of dendritic formulation to retain drug molecules at acidic pH. However, as the tumor cells have slightly acidic environment, burst release at acidic pH might be beneficial for anti-cancer drug targeting. Once the drug entrapped dendrimer enters the cancer cells, it may provide rapid release of its content due to acidic pH. The drug release patterns at pH 4.0 and 7.4 were found similar to previous reports [20,21]. Hemolytic toxicity of the developed P80-PPI system was found significantly less compared to 5.0 G PPI and free DTX. DTX is highly cytotoxic drug, which contributed to higher hemolytic activity. The polycationic surface of 5.0 G PPI also resulted in higher hemolytic toxicity. However, shielding of dendrimer periphery reduced the hemolytic toxicity significantly as compared with plain 5.0 G PPI and free drug. The results supported that entrapment of anti-cancer agent may decrease the adverse effect on red blood cells. Higher hemolytic toxicity of DTXeP80-PPI than P80-PPI may be attributed to leaching of DTX from the formulation causing blood dyscrasias. In vitro MTT cytotoxicity assay on U87MG human glioblastoma cells suggested that DTX exerted highest cytotoxic effect as it was indirect contact with brain tumor cells. However the IC50 value was highest for developed DTXeP80-PPI system at 24th h. Sustained release of DTX from P80ePPI system might have contributed to high IC50 value. On the other hand, significantly less IC50 value was observed in case of DTX-PPI, which might have released the drug relatively faster than DTXeP80ePPI due to acidic environment of brain tumor cells. On the contrary, the IC50 was decreased dramatically at 48th h for DTXeP80-PPI system and the difference in IC50 values of DTXeP80-PPI and DTXePPI was not significant. This might have occurred due to complete release of DTX from P80-PPI. The reason for increase in IC50 value was well in agreement with in vitro drug release results, where P80ePPI released 56.4 2.25% of DTX at 4th h, while PPI released 98.2 1.32% of DTX at the same time. The in vitro cell uptake study suggested higher uptake of 5.0 G PPI than P80-PPI, but the difference was insignificant. The higher uptake may be attributed to interaction of positively charged dendrimers with negatively charge cell membrane. The in vivo DTX targeting efficiency was determined via anticancer activity on brain tumor bearing rats. Results demonstrated
Fig. 6. Cumulative percentage DTX released from dendritic formulations (n ¼ 3).
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Fig. 7. Hemolytic toxicity profiles (n ¼ 3).
that DTXeP80ePPI reduced tumor volume more significantly as compared to free drug and DTXePPI. The tumor volume was reduced almost more than 50% in case of DTXeP80-PPI after one week treatment regimen. Higher BBB crossing by P80 anchoring
might have furnished for DTX delivery to tumor. P80 anchored system has also shown most significant tumor volume ratio and significant longer median survival time of brain tumor bearing rats. The receptor blockage assay was carried out to shed light on
Fig. 8. Percentage cell viability of U87MG cells at (a) 24 h, and (b) 48 h (n ¼ 6).
V. Gajbhiye, N.K. Jain / Biomaterials 32 (2011) 6213e6225
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Fig. 10. Tumor volume of different formulation at 15th day (n ¼ 6).
via fecal route, which might be responsible for higher distribution of free DTX in liver and spleen. The highest concentration of free drug in liver and spleen was found at 8th h as it was being removed from tissues and blood stream. DTX is well known substrate for Pgp efflux system [28] and this might have attributed to less concentration of free DTX in brain at 8th h as compared to 4th h. P80 anchoring has also been reported to block P-gp efflux system [28]. Tumor regression and drug biodistribution results provide further evidence. These observations proffered strong evidence for the brain tumor targeting and treatment strategy. 6. Conclusion In vitro MTT assay, cell uptake assay, anti-tumor efficacy, receptor blockage assay and biodistribution data demonstrated that DTX loaded P80 anchored dendritic nanoconjugate has potential to
Fig. 9. Gamma images of cell uptake assay on U87MG cells (a) radiolabeled PPI, and (b) radiolabeled P80-PPI (Radioactive counts are circled).
principle of LDL receptor-mediated BBB crossing of P80 conjugated system [29]. Receptor blocked group has shown significant higher tumor volume than DTXeP80-PPI group, which strongly supported the LDL receptor-mediated endocytosis. The in vivo targeting efficiency was further assessed by whole body gamma scanning of tumor bearing animals. The images clearly demonstrated higher blood brain crossing of P80 anchored dendritic system. Once injected in the blood stream, P80 anchored nanoparticulate system supposed to act as low density lipoproteins (LDL) [29]. Apoproteins B and/or E from the blood stream get adsorbed on this LDL mimicking carrier [19], subsequently recognized by LDL receptors on brain endothelium and results into receptor-mediated endocytosis (Animation 1). This might be the possible reason behind highest anti-tumor efficacy of P80 anchored PPI dendrimers. Drug biodistribution studies further supported the above statement. The highest concentration of DTX in brain and tumor was estimated for DTXeP80-PPI. DTXeP80-PPI had shown 4.1 and 39 times higher accumulation in brain tumor as compared to DTXePPI and free DTX at 8th h. The major route of DTX excretion is
Fig. 11. KaplaneMeier survival curves for different formulations.
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towards tumor cells. The developed nanoconjugate also decreased the distribution of anti-cancer agent in excretory organs by increasing its deposition at target site and decreased the adverse effect on non-target site (RBCs). Thus it can be concluded that DTX loaded P80 conjugated 5.0 G PPI dendritic nanoconjugate holds strong potential to treat brain tumor in rats, while producing least blood dyscrasias as against non conjugated system and free drug. This is expected to result in optimized drug delivery, in the treatment of brain tumor, a highly desirable contemporary therapeutic challenge. Acknowledgement Virendra Gajbhiye acknowledges University Grant Commission (UGC), New Delhi, India for providing Senior Research Fellowship. Appendix. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biomaterials.2011.04.057. References
Fig. 12. Gamma scintigraphy images for (a) free 99m TcO4ˉePPI, and (d) 99mTcO4ˉeP80-PPI.
99m
TcO4ˉ, (b)
99m
TcO4ˉeDTX, (c)
cross BBB significantly and can deliver significantly higher amount of drug to brain and brain tumor for higher therapeutic outcome. Furthermore, P80 anchoring may lead to blockage of P-gp efflux system to exert highest therapeutic effect of anti-cancer drug Table 1 Biodistribution of DTX in brain tumor bearing rats (n ¼ 3). Organ
Formulation
Brain
DTX DTXePPI DTXeP80-PPI DTX DTXePPI DTXeP80-PPI DTX DTXePPI DTXeP80-PPI DTX DTXePPI DTXeP80-PPI DTX DTXePPI DTXeP80-PPI DTX DTXePPI DTXeP80-PPI
Liver
Spleen
Lung
Kidney
Tumor
Concentration (mg/g tissue) 1h
4h
8h
**34.74 3.7 *152.14 7.1 467.99 14.1 **495.59 15.5 *314.34 11.8 117.97 6.3 407.25 15.2 292.86 7.4 112.34 7.2 312.70 8.6 278.16 7.3 101.27 4.3 340.63 7.6 225.61 6.3 115.73 5.1 ** 17.28 1.1 * 86.58 3.2 213.75 6.5
**29.57 1.9 *187.78 2.4 695.20 5.2 **781.13 6.6 *458.08 5.3 227.62 4.2 618.61 7.1 413.92 5.6 218.84 4.4 456.94 5.2 344.74 5.0 209.88 3.2 491.54 6.7 374.32 4.9 219.85 4.5 ** 19.43 1.0 * 90.75 3.1 376.82 5.3
**20.38 1.2 *224.87 5.9 799.75 8.9 **1125.86 14.9 *618.76 8.7 340.20 5.9 1018.53 20.3 578.23 6.2 329.72 5.7 396.14 5.9 325.21 4.4 200.30 3.6 426.11 5.9 350.49 5.4 205.30 4.8 ** 13.69 1.1 * 97.67 5.2 514.93 6.8
*p<0.001, very significant. **p<0.0001, extremely significant.
[1] Huynh GH, Deen DF, Szoka FC. Barriers to carrier mediated drug and gene delivery to brain tumors. J Control Rel 2006;110:236e59. [2] Armstrong TS, Mendoza T, Gring I, Coco C, Cohen MZ, Erriksen L, et al. Anderson symptom inventory brain tumor module (MDASI-BT). Neuro Oncol 2006;80:27e35. [3] Behin A, Hoang-Xuan K, Carpentier AF, Delattre JY. Primary brain tumours in adults. Lancet 2003;361:323e31. [4] Winer E, Gralow J, Diller L, Karlan B, Loehrer P, Pierce L, et al. Clinical cancer advances 2007: major research advances in cancer treatment, prevention and, screening- a report from the American society of clinical oncology. J Clin Oncol 2009;27:812e26. [5] Eyal S, Hsiao P, Unadkat JD. Drug interactions at blood-brain barrier: fact or fantasy? Pharmacol Therapeut 2009;123:80e104. [6] Beduneau A, Saulnier P, Benoit JP. Active targeting of brain tumors using nanocarriers. Biomaterials 2007;28:4947e67. [7] Ying X, Wen H, Lu W, Du J, Guo J, Tian W, et al. Dual-targeting daunorubicin liposomes improve the therapeutic efficacy of brain glioma in animals. J Control Rel 2010;141:183e92. [8] Carroll R, Bhatia D, Geldenhuys W, Bhatia R, Miladore N, Bishayee A, et al. Brain-targeted delivery of Tempol-loaded nanoparticles for neurological disorders. J Drug Target 2010;18:665e74. [9] Wu G, Barth RF, Yang W, Kawabata S, Zhang L, Green-Church K. Targeted delivery of methotrexate to epidermal growth factor receptorepositive brain tumors by means of cetuximab (IMC-C225) dendrimer bioconjugates. Mol Cancer Ther 2006;5:52e9. [10] Ren Y, Kang CS, Yuan XB, Zhou X, Xu P, Han L, et al. Co-delivery of as-miR-21 and 5-FU by poly(amidoamine) dendrimer attenuates human glioma cell growth in vitro. J Biomater Sci Polym Ed 2010;21:303e14. [11] Gajbhiye V, Kumar PV, Tekade RK, Jain NK. Pharmaceutical and biomedical potential of PEGylated dendrimers. Curr Pharm Design 2007;13:415e29. [12] Patri AK. Targeted drug delivery with dendrimers: comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex. Adv Drug Del Rev 2005;57:2203e14. [13] Dutta T, Agashe HB, Kumar PV, Garg M, Jain NK. Dendrosome-based gene delivery. J Exp Nanosci 2006;1:235e48. [14] Gupta U, Agashe HB, Jain NK. Polypropylene imine dendrimer mediated solubility enhancement: effect of pH and functional groups of hydrophobes. J Pharm Pharmaceu Sci 2007;10:358e67. [15] Kobayashi H, Brechbiel MW. Nano-sized MRI contrast agents with dendrimer cores. Adv Drug Deliv Rev 2005;57:2271e86. [16] Gajbhiye V, Kumar PV, Tekade RK, Jain NK. Dendrimers as therapeutic agents: a systematic review. J Pharm Pharmacol 2009;61:989e1003. [17] Tekade RK, Kumar PV, Jain NK. Dendrimers in oncology: an expanding horizon. Chem Rev 2009;109:49e87. [18] Dutta T, Jain NK. Targeting potential and anti-HIV activity of lamivudine loaded mannosylated poly (propyleneimine) dendrimer. Biochim Biophys Acta 2007;1770:681e6. [19] Blasi P, Giovagnoli S, Schoubben A, Ricci M, Rossi C. Solid lipid nanoparticles for targeted brain drug delivery. Adv Drug Del Rev 2007;59:454e77. [20] Gajbhiye V, Kumar PV, Sharma A, Jain NK. PEGylated PPI dendritic architectures for sustained delivery of anti-inflammatory agent. Curr Nanosci 2008;4:267e77. [21] Gajbhiye V, Kumar PV, Tekade RK, Jain NK. Novel PEGylated nanoparticulate architectures for sustained delivery of H2 receptor antagonist. Euro J Med Chem 2009;44:155e66.
V. Gajbhiye, N.K. Jain / Biomaterials 32 (2011) 6213e6225 [22] Beban M, Miller PS. Preparation of an imidazole-conjugated oligonucleotide. Bioconjug Chem 2000;11:599e603. [23] Agashe HB, Babbar AK, Jain S, Sharma RK, Mishra AK, Asthana A, et al. Investigation on biodistribution of technetium-99m-labeled carbohydratecaoted poly(propylene imine) dendrimers. Nanomedicine: NBM 2007;3: 120e7. [24] Fujita M, Lee BS, Khazenzon NM, Penichet ML, Wowrowsky KA, Patil R, et al. Brain tumor tendem targeting using a combination of monoclonal antibodies attached to biopoly(b-L-malic acid). J Control Rel 2007;122:356e63. [25] Zhao L, Wei Y, Zhong X, Liang Y, Zhang X, Li W, et al. PK and tissue distribution of docetaxel in rabitts after i.v. administration of liposomal and injectable formulations. J Pharm Biomed Anal 2009;49:989e96.
6225
[26] Gupta U, Dwivedi SKD, Bid HK, Konwar R, Jain NK. Ligand anchored dendrimers based nanoconstructs for effective targeting to cancer cells. Int J Pharm 2010;393:185e96. [27] Rowe JDD, Bello J, Mitnick R, Sood B, Filippi C, Moran J, et al. Accelerated regression of brain metastases in patients receiving whole brain radiation and the topoisomerase II inhibitor, lucanthone. Int J Radia Onco Biol Phys 1999;43:89e93. [28] Kemper EM, Verheij M, Boogerd W, Beijnen JH, van Tellingen O. Improved penetration of docetaxel into brain by co-administration of inhibitors of Pglycoprotein. Euro J Cancer 2004;40:1269e74. [29] Kreuter J, Shamenkov D, Petrov V, Ramge P, Cychutek K, Koch-brandt C, et al. Apolipoprotein-mediated transport of nanoparticle-bound drugs across the blood-brain barrier. J Drug Target 2002;10:317e25.