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Formulation development and in vitro–in vivo assessment of the fourth-generation PPI dendrimer as a cancer-targeting vector Aim: In spite of numerous biopharmaceutical applications of fifth-generation poly(propyleneimine) (PPI) dendrimers, inherent toxicity due to the presence of many peripheral cationic groups is the major issue that limits their applicability. Maximum biocompatibility with minimal toxicity is the key rationale for an ideal drug-delivery system. Keeping this principle in mind, the present investigation aimed to explore the tumor-targeting potential of folate-engineered fourth-generation PPI dendrimers loaded with an anticancer drug, melphalan. Materials & methods: Fourth-generation PPI as well as folate-conjugated fourth-generation PPI dendrimers were synthesized, characterized and loaded with melphalan. Results: Hemolytic toxicity, cytotoxicity, cellular uptake and fluorescence uptake studies reveal that the developed folateconjugated derivative has significantly lower toxicity, as well as demonstrates folate receptor specificity. Discussion & conclusion: The developed nanoconjugates appear to be proficient in carrying as well as site-specific delivery of melphalan, with an improved therapeutic margin and improved safety.
Prashant Kesharwani1, Rakesh K Tekade2 & Narendra K Jain*,1 Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr Hari Singh Gour University, Sagar 470003, Madhya Pradesh, India 2 College of Pharmacy, University of Hawaii at Hilo, 96720 HI, USA *Author for correspondence: jnarendr@ yahoo.co.in 1
Original submitted 11 July 2013; Revised submitted 11 November 2013 Keywords: cancer targeting • fourth-generation PPI dendrimer • melphalan • pharmacodynamics • pharmacokinetics • stability • toxicity
An ideal cancer remedial device can diminish tumors without damaging healthy tissue. Therefore, a distinct capacity to target tumors without affecting healthy cells is essential for the success of any new cancer remedial device. Active targeting is a promising strategy that involves the surface modification of a drug carrier system with a bioactive agent such as an antibody, carrier protein or ligand that shows selective binding and uptake via receptors overexpressed on tumor cells [1] . Folate receptors, which are 38-kDa glycosylphosphotidylinositolanchored proteins, are overexpressed on tumor cells, making them a good option for tumor-targeted drug delivery [2,3] . Various drug-delivery carriers such as polymeric micelles, polymer conjugates, liposomes, nanoparticles and dendrimers are being exploited for selective delivery of
10.2217/NNM.13.210 © 2014 Future Medicine Ltd
various anticancer bioactives at the tumor site [4,5] . Recently, dendrimers are one of the most widely explored carriers for anticancer therapy [6–8] . Dendrimers are essentially 3D, highly branched, monodispersed macromolecules with a 3D nanometric structure, obtained by an iterative sequence of reaction steps producing a precise, unique branching structure [9,10] . Regardless of the wide application of dendrimers, the inherent toxicity due to the surface amine groups limits application of these cationic dendrimers. This toxicity is ascribed to the interaction of their peripheral cationic charge with negatively charged biological membranes [11] . The intensity of toxicity is directly proportional to the number of surface amine groups; the number of amine groups increases as the generation of dendrimer increases. Poly(propyleneimine)
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Research Article Kesharwani, Tekade & Jain (PPI) dendrimers represent one of the most explored classes of dendrimers, with the fifth-generation (5.0G) subtype leading the family. It is well documented that the 5.0G dendrimer is more toxic compared with fourth-generation (4.0G) dendrimers due to an increased number of surface cationic groups. In 2010, Jain and coworkers reported that the 5.0G PPI dendrimer exhibited almost three-times as much red blood cell (RBC) hemolysis compared with the 4.0G PPI dendrimer at a concentration of 1 mg/ml [12] . To keep this toxicity at a minimum, we are exploring lowergeneration (4.0G) PPI dendrimers to deliver an anticancer drug, melphalan (MPN). The biocompatibility, drugloading affinity and tumor-targeting potential may be further enhanced by folate conjugation. The main rationale behind proposing to use lower-generation PPI dendrimers lies in their diminished toxicity compared with higher-generation dendrimers (>5.0G PPI), and thus attaining better efficacy with lower toxicity. As such, various strategies have been proposed in the literature to alleviate the toxicity associated with dendrimers [13] . Among these strategies, surface conjugation of biocompatible moieties, ligands and polyethylene glycol chains, among others, have been widely utilized to reduce dendrimer toxicity. In this line, the surface primary amine group of dendrimers has been successfully conjugated with biocompatible ligands such as folate, dextran, galactose and mannose for tumor targeting [10,14–17] . Among various available ligands [13] , a recent report from our laboratory infers folate-based dendritic conjugates to be the most promising in drug targeting, as well as producing the most biocompatible dendritic formulation [8] . The core objective of this investigation was to assess the 4.0G PPI dendrimer for its drug-targeting propensity by engaging MPN as an anticancer drug. The drug MPN has been selected on account of its insolubility in most frequently used solvents; loading it within the dendrimers may overcome this limitation. In this project, first, 4.0G PPI dendrimers were synthesized and thoroughly characterized, followed by surface modification employing folic acid (FA) as a targeting ligand. 4.0G PPI dendrimer-based formulations were investigated for their drug-loading aptitude, drug release, cell line-based cytotoxicity assay, cell uptake assay, florescence uptake assay, hemolytic toxicity and stability. Bioperformance of the developed formulations was also studied to deduce pharmacokinetic and pharmacodynamic performance in BALB/C mice. It was expected that the developed systems will not only selectively target the drug to the tumor cells but will also reduce the charge-associated toxicity of the dendrimers, which is the major constraint of 5.0G PPI dendrimers.
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Materials & methods FA, dimethylsulfoxide and dichloromethane and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were purchased from Himedia (Mumbai, India). Acrylonitrile and ethylenediamine (EDA) were purchased from Central Drug House (Mumbai, India), and Raney ® nickel was purchased from Fluka (MO, USA). 3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (MO, USA). Analytical-grade reagents were purchased from Merck India Ltd (Mumbai, India). MCF-7, HeLa and KB cells were purchased from the National Center for Cell Sciences (Pune, India). MCF-7 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum and incubated at 37°C in a humidified incubator and 5% CO2 atmosphere. HeLa cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Himedia) supplemented with 10% fetal calf serum, 2 mM glutamine in a 37°C humidified incubator and 5% CO2 atmosphere. KB cells were grown in RPMI-1640 medium supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml) and 10% heat-inactivated fetal bovine serum at 37°C and 5% CO2. The protocol for animal experiments was duly approved by the Institutional Animal Ethics Committee of Dr HS Gour University (Sagar, India; registration no. 379/01/ab/CPCSEA). All experiments were performed on healthy BALB/c mice (average bodyweight: 25.0 ± 2.0 g). Animals were housed and handled in accordance with the institutional guidelines. Throughout the experiment, the animals were housed in laminar flow cages maintained at 22 ± 2°C, 50–60% relative humidity, under a 12-h light–12-h dark cycle. The animals were maintained in these facilities for at least 1 week before the experiment. The rats were permitted free access to tap water and commercialized food (commercial standard chow diet; CJ Feed Co. Ltd, Seoul, Republic of Korea) throughout the experiment. Synthesis of 4.0G PPI dendrimers
The 4.0G PPI dendrimers was prepared by a previously reported divergent method using EDA as an initiator core and acrylonitrile as a branching moiety [6,8,16,18] . Briefly, acrylonitrile was added to an aqueous solution of EDA using a double Michael addition reaction to produce half-generation (-CN terminated) dendrimers. This exothermic reaction results in a rise in temperature to approximately 38°C; it was followed by heating up to 80°C to complete the addition reaction. Consequently, excess of acrylonitrile was removed as
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Development & assessment of fourth-generation PPI dendrimer as a cancer-targeting vector
a water azeotrope through vacuum distillation. The obtained crystalline solid was half-generation PPI dendrimer. The half-generation dendrimers were subjected to heterogeneous hydrogenation using Raney nickel as a catalyst to produce full-generation amine-terminated PPI dendrimers. The iterative sequences of reaction steps were repeated cyclically to produce PPI dendrimers up to the 4.0G dendrimer (4.0G PPI) (Figure 1A) . Synthesis of folate-engineered 4.0G PPI dendrimers
Folate conjugation to 4.0G PPI dendrimers was carried out by a method previously reported by us (Figure 1B) [8] .
Research Article
Sigma-Aldrich) against double-distilled water under strict sink conditions for 30 min to remove free drug from the formulation, which was estimated by a HPLC method using mobile-phase methanol:purified water:acetic acid (49.5:49.5:1 v/v) pumped onto the Ultrasphere® ODS (Hichrom Ltd, Theale, UK; 5 µm) column at a flow rate of 2 ml/min, and the analysis was carried out at 260 nm [20] to indirectly determine the amount of drug loaded within the formulations. A similar method was used to load drug within folateconjugated dendrimers [21] . Both of the formulations were lyophilized using 2% lactose as cryoprotectant and used for further characterization. Developed drugloaded formulations thus formed were coded as MPNloaded PPI dendrimers (PPI-MPN) and MPN-loaded FA-conjugated PPI dendrimers (FA-PPI-MPN).
Synthesis of tert-butoxycarbonyl-protected FA
The tert-butoxycarbonyl (t-Boc)-protected FA was synthesized following a reported method [8,19] . In brief, FA (10 mM) was dissolved in a mixture of dimethylsulfoxide:dichloromethane (1:1 v/v), followed by addition of t-Boc (12 mM), and the mixture was continuously stirred in the dark for 48 h. One molar (5 ml) KHSO4 was added to the resultant product, followed by subsequent extraction of the product from the mixture by shaking with three separate portions of ethyl acetate each for 1 h.
Particle size determination
The average particle size and polydispersity index of the formulations (PPI-MPN and FA-PPI-MPN) as 10 w/v solutions were determined in a zetasizer (DTS Version 4.10; Malvern Instruments, Malvern, UK). The particle size distributions are represented by the average size (diameter) and the variance (polydispersity) of the Gaussian distribution function in logarithmic axis mode. In vitro drug-release studies
Conjugation of t-Boc-protected FA to 4.0G PPI dendrimer
The t-Boc-protected FA (4 mM) was activated by adding 1-ethyl-3-(3-dimethylaminopropyl)carbo diimide hydrochloride (5 mM) to it and stirring for 4 h in the dark. The solution of PPI dendrimers was placed in a container, followed by addition of the t-Boc-protected FA solution drop-wise to it, and the mixture was continually stirred in the dark for 48 h (500 rpm; 37 ± 2°C). The resultant conjugate was concentrated under reduced pressure (washed twice with dimethylformamide) and was labeled as folateengineered 4.0G PPI dendrimers (FA-PPI). The resultant conjugation was characterized by Fourier transform infrared spectroscopy (FT-IR), 1H nuclear magnetic resonance (1H-NMR) and MALDI-TOF. Drug loading
The known molar concentration of MPN (100 mM) was dissolved in methanol and mixed with an aqueous solution of 4.0G PPI (10 mM) dendrimer, and the resultant mixture was incubated with slow magnetic stirring (50 rpm) using a Teflon® (Teja Scientific Glass Works, Hyderabad, India) bead for 24 h. The resulting solution was dialyzed twice through a cellulose dialysis bag (molecular weight cut-off: 3.5 kDa;
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The marketed MPN injectable formulation (MMIF; melphalan hydrochloride; Alkeran®; GlaxoSmithKline LLC, Mumbai, India) and MPN-loaded formulations (10 mg) were introduced into a dialysis bag (molecular weight cut-off: 3.5 kDa; Sigma-Aldrich) separately and the end-sealed dialysis bags were submerged in 100 ml of release medium (phosphate-buffered saline [PBS]; pH 7.4; containing 0.1% v/v propylene glycol) and stirred at 200 rpm (37 ± 2°C) for 48 h. At predetermined intervals of time (0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24 and 48 h), 1-ml aliquots were withdrawn and replaced with an equal volume of fresh medium. The concentration of MPN in samples was determined by a HPLC method [20] . Hemolytic toxicity
The degree of reduction of toxicity due to folate conjugation was investigated by a hemolytic toxicity study according to previously reported studies [22] . In short, first, RBCs were collected from human blood and resuspended in normal saline to obtain a 5% RBC suspension, which was used further for hemolytic studies. To the RBC suspension (1 ml), distilled water (5 ml) and normal saline (5 ml) were added separately, which were considered to be 100 and 0% hemolytic, respectively. In a similar fashion, formulations (PPI,
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Figure 1. Synthesis of dendritic formulations. (A) Step-by-step synthesis of 4.0G PPI dendrimers and (B) synthesis of folate-conjugated PPI dendrimers. 1.0G: First generation; 2.0G: Second generation; 3.0G: Third generation; 4.0G: Fourth generation; PPI: Poly(propyleneimine); t-Boc: Tert-butoxycarbonyl.
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Research Article Kesharwani, Tekade & Jain
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Development & assessment of fourth-generation PPI dendrimer as a cancer-targeting vector
PPI-MPN and FA-PPI-MPN) as well as MMIF (0.5 ml) were added to the mixture of normal saline (4.5 ml) and RBC suspension (1 ml) separately. The tubes were allowed to stand for 30 min with gentle intermittent shaking and were centrifuged for 15 min at 3000 rpm in an ultracentrifuge (Microprocessor Research ultracentrifuge PR-23; Remi Elektrotech Ltd, Mumbai, India). The supernatants were pooled and UV spectrophotometric assay was performed at 540 nm. The degree of hemolysis was determined by Equation 1:
MTT cytotoxicity assay
Hemolysis (%) = Abs - Abs0 # 100 Abs100 - Abs0
Cell uptake assay
(Equation 1)
Where Abs, Abs0 and Abs100 are the absorbance of samples, a solution of 0% hemolysis and a solution of 100% hemolysis, respectively. Hematological study
For hematological study, BALB/C mice were divided into four groups, each consisting of three mice. Mice were administered intravenously with free drug, PPI-MPN and FA-PPI-MPN (1 mg/kg bodyweight) separately into all three groups (the fourth group was kept as control) every day up to 7 days. This was followed by the collection of a blood sample, which was analyzed for RBC count, white blood cell (WBC) count and differential count of monocytes, lymphocytes and neutrophils by the pathology laboratory [23] . Stability studies
The influence of accelerated storage conditions on the stability of developed formulations was determined using a previously reported protocol with slight modifications [7,17,24] . Briefly, formulations (10% w/v, 5 ml) were kept in the dark in amber-colored and colorless (light conditions) vials at 0°C, room temperature (25 ± 2°C) and accelerated temperature (60 ± 2°C) in controlled ovens for a period of 5 weeks, and analyzed every week for any visual changes such as precipitation, turbidity, crystallization, color and drug leakage (however, only terminal results were reported). The formulations (PPI-MPN and FA-PPI-MPN) were analyzed for drug content initially and at weekly intervals up to 5 weeks, spectrophotometrically. The percentage drug leakage (release content of the drug) in various formulations was used to analyze the effects of accelerated conditions of storage on the formulations. The amount of residual drug in the dialysis sac was also simultaneously analyzed, which was considered as residual content of the formulation. The study was conducted for all of the formulations of MPN.
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The in vitro cytotoxicity of MMIF as well as the developed formulations was determined by the MTT assay based on the activity of mitochondrial dehydrogenases. Following treatment of cells (HeLa, KB and MCF-7 separately), 20 µl MTT (5 mg/ml) in PBS (pH 7.4) was added to each well, and plates were incubated at 37°C for 1 h. The resulting formazan crystals were dissolved by the addition of dimethylsulfoxide and absorbance was recorded at 560 nm [25] . A cell uptake study was performed on MCF-7 cells by in vitro incubation of cells with various formulations. Briefly, MCF-7 cells were seeded at 2 × 106 cells/ml in six-well plates (Sigma-Aldrich) containing fresh medium and suspended for 24 h in a humidified incubator at 37 ± 2°C with 5% CO2 atmosphere. Cells were washed with PBS after removing the culture medium. Cells were then treated with PBS (×3) followed by treatment with 1 ml of RPMI-1640 medium, and were transferred in 24-well cultured plates. After 6 days of culture, 100 µl of MMIF, PPI-MPN and FA-PPI-MPN containing equivalent amounts of drug were added to the wells. The plates were incubated at 37 ± 2°C for 48 h. The cell suspension was transferred to polycarbonate filters (0.45 µm) after the appropriate time. The wells of the cell culture plates were rinsed with 1 ml PBS (pH 7.4) and the washings were consequently transferred to the filters. The cells were separated from the medium in the form of a pellet by centrifuging the filters at 4000 rpm for 15 min [26] . Pellets were ruptured by adding Triton™ X-100 (Sigma-Aldrich) and the mixture was incubated at 25 ± 2°C for 6 h, and uptake of drug was determined by HPLC [20] . Cell topographic assay
MCF-7 cells (5 × 104) were seeded on six-well plates and incubated at 37 ± 0.5°C under 5% CO2 for a period of 24 h, following which time medium was removed and incubated with 2.5 ml of Nile redloaded formulations (Nile red, PPI-loaded Nile red, folate-conjugated PPI-loaded Nile red) for 2 h. After 2 h, medium was removed, and the cells were gently washed three times with PBS (pH 7.4) and fixed with the help of formaldehyde (2% v/v) in the PBS at room temperature. The cells were then immediately imaged using an Axiovert 40® (Carl Zeiss LLC, NY, USA) inverted microscope at 40× magnification at Nile red 554 nm excitation and 635 nm emission wavelengths with a mercury vapor lamp as a light source for fluorescence imaging.
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Research Article Kesharwani, Tekade & Jain In vivo studies
The tumor implantation was carried out in BALB/c mice with MCF-7 cells (8 × 106 cells/0.2 ml) following a previously reported method with slight modification [27] . Pharmacokinetic studies
The BALB/c mice were divided into three groups with six tumor-induced mice in each. Formulations (PPIMPN, FA-PPI-MPN and MMIF) were solubilized in saline in the required dose (1 mg/kg bodyweight) and injected intravenously into the mice. At prespecified time intervals, a blood sample was collected through the tail vein and centrifuged for 10 min to separate RBCs and serum (3000 rpm). Supernatants were collected and thoroughly mixed with 2 ml ethylacetate, followed by centrifugation for removal of the organic layer, evaporation to dryness and resuspension in the mobile phase for analysis of the drug by a HPLC method [20] . Tissue distribution study
For biodistribution studies, mice were divided into eight groups and each group was administered with the same intravenous dose (1 mg/kg) of different formulations (MMIF, PPI-MPN and FA-PPI-MPN). Two mice from each group were sacrificed at 2, 8 and 24 h and, immediately after sacrificing, the mice organs (i.e., spleen, kidney, liver and tumor) were carefully removed and weighed. Weighed tissue samples were suspended in 2 ml ethylacetate and homogenized for 5 min. The homogenate was transferred to an Eppendorf tube and centrifuged at 3000 rpm for 10 min at 4°C. The organic layer was subsequently transferred into a second tube and evaporated to dryness under vacuum. The compound was resuspended in 1 ml of the mobile phase, vortexed and then stored at deepfreeze conditions (-70 ±2°C) until analyzed for MPN content by a HPLC method [28] . Pharmacodynamic studies
Tumor was induced in BALB/c mice following previously reported methodology [29–31] , and our observations on tumor induction and growth were in agreement with earlier reports from our laboratory [7,32] . Briefly, BALB/c mice were implanted with MCF-7 cells (8 × 106 cells/0.2 ml) subcutaneously, and tumor cells were allowed to grow for another 21 days to develop palpable tumor mass. After detection of palpable tumor, intravenous injection of various formulations (at 1 mg/kg MPN equivalent dose) suspended in PBS (filtered through a 0.22 micron filter; Millipore Corp., MA, USA) were injected through the tail vein of animals at 0 and 7 days.
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Tumor volume was measured with the help of a Vernier Caliper using Equation 2 : Volume (mm3) = length × width2 × 0.5
(Equation 2)
With an assumption that width equals depth and p equals 3 [28]. Statistical analysis
All of the statistical analyses were performed with GraphPad® Instat software (version 3.0; GraphPad Software, CA, USA) using either unpaired t-test or oneway analysis of variance followed by Tukey–Kramer multiple comparison test. A difference of p HeLa > MCF-7. The highest activity of FA-PPI-MPN with respect to KB cells may be due to overexpression of a comparatively greater number of FA molecules compared with the other cell lines. The results of our study are in accordance with previously reported literature [8] . Higher uptake in the case of FA-PPI-MPN is due to its interaction with overexpressed folate receptors on the MCF-7 cells, which are critical to enhanced cellular uptake. Further cell topographic assay also supported the better anticancer activity of the folate-mediated formulations. Higher drug concentrations of the PPI-based formulations as determined by HPLC analysis were found compared with MMIF due to prolonged and sustained properties of PPI dendrimers. Developed folate-engineered formulations showed a 2.87- and 1.53-times increase of the half-life of FA-PPI-MPN compared with MMIF and PPI-MPN, respectively. This extended half-life may be particularly advantageous in targeted cancer therapy. It was observed that folate modification results in an increase in the half-life of loaded drug, and similar outcomes have been noted by Singh et al. [7] and Gupta et al. [34] . The main reason behind the prolonged half-life of drug loaded inside folate-anchored dendrimers is the entanglement of drug in small packets that were created after ligand anchoring. The same aspect was carefully studied under in vitro as well as in vivo settings. The outcome of these investigations
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Figure 8. In vivo analysis of dendritic formulations. (A) Tissue distribution pattern and (B) pharmacodynamic tumor growth analysis. (A,i) 2 h, (A,ii) 8 h and (A,iii) 24 h. The results are expressed as mean ± standard deviation (n = 3). FA-PPI-MPN: Melphalan-loaded folate-conjugated fourth-generation poly(propyleneimine) dendrimer; MMIF: Marketed melphalan injectable formulation; PPI-MPN: Melphalan-loaded fourth-generation poly(propyleneimine) dendrimer.
infers slow release of loaded drug from the modified dendrimer. It is agreed that folate uptake in various organs must lead to rapid clearance of the folate dendrimer, hence one may expect a short half-life with the folate dendrimer. However, in a practical setting, the opposite was observed, and this may be due to the open and easily accessible structure of the naked dendrimer, as well as the presence of extra crevices compared with the folate-decorated dendrimers. The folate-modified dendrimer elicits extra hindrance towards release of loaded drug owing to the surface-labeled folate groups. Biodistribution studies revealed an elevated concentration of MPN in the urine, suggesting the kidney as a major route of drug elimination. The folate-conjugated formulation (FA-PPI-MPN) was found in comparatively much higher amounts at the tumor site compared with PPI-MPN and MMIF. This outcome is in accordance with the hemolytic toxicity and MTT cell line-based assays, which clearly indicate better distribution as well as targeting efficiency of the folate-mediated dendritic formulations.
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The results of the pharmacodynamic studies on tumor-bearing BALB/C mice demonstrated that, compared with control, PPI-MPN and FA-PPI-MPN resulted in a 1.70- and 2.13-times reduction in tumor volume. However, FA-PPI-MPN was 1.25-times more active compared with PPI-MPN, which may be ascribed to ligand-mediated targeting of dendrimers due to surface conjugation of the FA. Overexpressed folate receptors on cancer cells resulting in greater interaction with the conjugates may be another reason behind its anticancer activity. Conclusion At present, the 5.0G PPI dendrimer is the most widely explored dendrimer compared with lower-generation dendrimers due to the presence of numerous surface amino groups, which provide greater opportunity for surface engineering. Another profit is the higher drug loading due to the presence of a larger cavity. However, these surface amino groups are also the cause of additional toxicity, which restrict the application of the 5.0G dendrimer. With respect
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Research Article Kesharwani, Tekade & Jain to the biocompatibility concern, we explored the 4.0G dendrimer in contrast to 5.0G dendrimer formulations, which as per our report can be further enhanced by folate modification. We have described a surface engineering concept based on folate receptors on tumor cells that is adaptable to a wide variety of dendrimer-based platforms. Folate functionalization on the dendrimer periphery significantly enhanced their accumulation in tumor cells, and resulted in improved efficacy of folate-conjugated MPN-loaded 4.0G dendrimers in a tumor-bearing BALB/C mice model. Hemolytic toxicity, cytotoxicity, cellular uptake and fluorescence uptake studies indicate that the folate-conjugated derivative has significantly lower toxicity, as well as demonstrating folate receptor specificity. Further studies regarding optimization of folate-based formulations are currently in progress in our laboratory, in order to validate this system as a potential targeted drug nanotherapeutic. Future perspective In this article, we have developed folate-functionalized 4.0G-based dendritic nanoconjugates. The developed nanoconjugates are able to deliver anticancer drug significantly within an acceptable toxicity limit. The work is believed to shift the attention of drugdelivery scientists throughout the globe towards 4.0G dendrimer-based formulations instead of the widely explored 5.0G dendritic formulations. In addition, it is envisaged that formulation parameters of 4.0G-based
formulations, such as drug loading and anticancer activity, among others, can further be improved by available techniques such as PEGylation, to make the nanosystem more acceptable and fit for drug-delivery applications. With respect to further enhancement of biocompatibility and negligible toxicity, PEGylation of this optimized formulation (FA-PPI-MPN) is currently in progress in our laboratory. It is anticipated that the 4.0G dendrimer-based formulation will generate a safe, effective and stable formulation that will help in successful transformation of dendritic formulations from laboratory to clinical stages. Financial & competing interests disclosure P Kesharwani would like to acknowledge the Indian Council of Medical Research (New Delhi, India) for providing financial assistance to carry out the present experimental studies. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate insti tutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations.
Executive summary • The folate-conjugated fourth-generation poly(propyleneimine) dendrimer displayed significantly less hemolysis than naked dendrimer-based formulations. • The fourth-generation, folate-modified dendritic formulations can be effectively and rapidly taken up by cancer cells due to overexpressed folate receptors. • The fourth generation-based conjugates developed also displayed prolonged half-life and higher concentration of drug in the systemic circulation. • Surface conjugation of folate on the dendrimer periphery exhibited a significant sustained-release pattern in vitro compared with plain dendrimer. • Folate conjugation imparts rigidity to the system and results in greater stability of these systems compared with plain dendrimer. • The folate-conjugated fourth-generation poly(propyleneimine) dendrimer appears to be proficient in the delivery of anticancer drug, with an improved therapeutic margin and safety profile.
References Papers of special note have been highlighted as: • of interest
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and molecular identification as a folate-binding protein. Am. J. Pathol. 142, 557–567 (1993).
1
Kesharwani P, Ghanghoria R, Jain NK. Carbon nanotube exploration in cancer cell lines. Drug Discov. Today 17, 1023–1030 (2012).
3
Lu Y, Low PS. Folate-mediated delivery of macromolecular anticancer therapeutic agents. Adv. Drug Deliv. Rev. 54, 675–693 (2002).
2
Garin-Chesa P, Campbell I, Saigo PE, Lewis JL Jr, Old LJ, Rettig WJ. Trophoblast and ovarian cancer antigen LK26. Sensitivity and specificity in immunopathology
4
Zhang Z, Feng SS. The drug encapsulation efficiency, in vitro drug release, cellular uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl polyethylene
Nanomedicine (Lond.) (2014) 9(15)
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glycol succinate nanoparticles. Biomaterials 27, 4025–4033 (2006). 5
6
Yang T, Choi MK, Cui FD et al. Preparation and evaluation of paclitaxel-loaded PEGylated immunoliposome. J. Control. Release 120, 169–177 (2007). Tekade RK, Dutta T, Gajbhiye V, Jain NK. Exploring dendrimer towards dual drug delivery: pH responsive simultaneous drug-release kinetics. J. Microencapsul. 26, 287–296 (2008).
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17
Bhadra D, Yadav AK, Bhadra S, Jain NK. Glycodendrimeric nanoparticulate carriers of primaquine phosphate for liver targeting. Int. J. Pharm. 295, 221–233 (2005).
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Brabander-van den Berg EMM, Meijer EW. Poly(propylene imine) dendrimers: large scale synthesis by heterogeneously catalyzed hydrogenation. Angew. Chem. Int. Ed. Engl. 32, 1308–1311 (1993).
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Dutta T, Agashe, HB, Garg M, Balakrishnan P, Kabra M, Jain NK. Poly (propyleneimine) dendrimer based nanocontainers for targeting of efavirenz to human monocytes/macrophages in vitro. J. Drug Target. 15, 89–98 (2007).
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Singh P, Gupta U, Asthana A, Jain NK. Folate and folatePEG-PAMAM dendrimers: synthesis, characterization, and targeted anticancer drug delivery potential in tumor bearing mice. Bioconjug. Chem. 19, 2239–2252 (2008).
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Kesharwani P, Tekade RK, Gajbhiye V, Jain K, Jain NK. Cancer targeting potential of some ligand-anchored poly(propylene imine) dendrimers: a comparison. Nanomedicine 7, 295–304 (2011).
Pinguet F, Joulia JM, Martel P, Grosse PY, Astre C, Bressolle F. High-performance liquid chromatographic assay for MPN in human plasma. Application to pharmacokinetic studies. J. Chromatogr. B Biomed. Appl. 686, 43–49 (1996).
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Describes the comparatively higher antitumor targeting potential of folate-mediated fifth-generation poly(propyleneimine) dendrimers.
Describes a simple, rapid and reproducible HPLC method for the analysis of melphalan in plasma.
21
Tomalia DA, Baker H, Dewald JR et al. A new class of polymers: starburst dendritic molecules. Polym. J. 17, 117–132 (1985).
Kumar PV, Asthana A, Dutta T, Jain NK. Intracellular macrophage uptake of rifampicin loaded mannosylated dendrimers. J. Drug Target. 14, 546–556 (2006).
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Singhai AK, Jain S, Jain NK. Evaluation of an aqueous injection of ketoprofen. Pharmazie 52, 149–151 (1997).
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Agrawal P, Gupta U, Jain NK. Glycoconjugated peptide dendrimers-based nanoparticulate system for the delivery of chloroquine phosphate. Biomaterials 28, 3349–3359 (2007).
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Prajapati RN, Tekade RK, Gupta U, Gajbhiye V, Jain NK. Dendimer-mediated solubilization, formulation development and in vitro in vivo assessment of piroxicam. Mol. Pharm. 6, 940–950 (2009).
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Casas A, Battah S, Di Venosa G et al. Sustained and efficient porphyrin generation in vivo using dendrimer conjugates of 5-ALA for photodynamic therapy. J. Control. Release 135, 136–143 (2009).
26
Dutta T, Jain NK. Targeting potential and anti-HIV activity of lamivudine loaded mannosylated poly (propyleneimine) dendrimer. Biochim. Biophys. Acta 1770, 681–686 (2007).
27
Yoo HS, Park TG. Folate-receptor-targeted delivery of doxorubicin nanoaggregates stabilized by doxorubicin– PEG–folate conjugate. J. Control. Release 100, 247–256 (2004).
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Ying X, Wen H, Lu WL et al. Dual-targeting daunorubicin liposomes improve the therapeutic efficacy of brain glioma in animals. J. Control. Release 141, 183–192 (2010).
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Explores poly(amidoamine) dendrimers extensively for diverse medical applications.
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Kesharwani P, Gajbhiye V, Tekade RK, Jain NK. Evaluation of dendrimer safety and efficacy through cell line studies. Curr. Drug Targets 12, 1478–1497 (2011).
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Jain K, Kesharwani P, Gupta U, Jain NK. Dendrimer toxicity: let’s meet the challenge. Int. J. Pharm. 394, 122–142 (2010).
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Demonstrates how surface engineering masks the cationic charge of dendrimers and enhances their biocompatibility.
12
Mishra V, Gupta U, Jain NK. Influence of different generations of poly(propylene imine) dendrimers on human erythrocytes. Pharmazie 65, 891–895 (2010).
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Pioneering work that illustrates the generationdependent toxicity associated with the cationic groups of poly(propyleneimine) dendrimers.
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Tekade RK, Kumar PV, Jain NK. Dendrimers in oncology: an expanding horizon. Chem. Rev. 109, 49–87 (2009).
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Reviews the importance of and strategies involved in dendrimer-mediated cancer therapy.
14
Wiener EC, Konda S, Shadron A, Brechbiel M, Gansow O. Targeting dendrimer-chelates to tumors and tumor cells expressing the high-affinity folate receptor. Invest. Radiol. 32, 748–754 (1997).
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Kothari K, Bapat K, Korde A. Radiochemical and biological studies of 188Re labeled monoclonal antibody – CAMA3C8 specific for breast cancer. Ind. J. Nuc. Med. 19, 6–11 (2004).
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Patri AK, Kukowska-Latallo JF, Baker JR Jr. Targeted drug delivery with dendrimers: comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex. Adv. Drug Deliv. Rev. 57, 2203–2214 (2005).
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Matsuno F, Haruta Y, Kondo M, Tsai H, Barcos M, Seon BK. Induction of lasting complete regression of preformed distinct solid tumors by targeting the tumor vasculature using two new anti-endoglin monoclonal antibodies. Clin. Cancer Res. 5, 371–382 (1999).
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Thakur S, Tekade RK, Kesharwani P, Jain NK. The effect of polyethylene glycol spacer chain length on the tumortargeting potential of folate-modified PPI dendrimers. J. Nanopart. Res. 15, 1625 (2013).
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VanWeelden K, Flanagan L, Binderup L, Tenniswood M, Welsh JE. Apoptotic regression of MCF-7 xenografts in nude mice treated with the vitamin D 3 analog, EB1089. Endocrinology 139, 2102–2110 (1998).
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Gajbhiye V, Jain NK. The treatment of glioblastoma xenografts by surfactant conjugated dendritic nanoconjugates. Biomaterials 32, 6213–6225 (2011).
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Zhao XB, Lee RJ. Tumor-selective targeted delivery of genes and antisense oligodeoxyribonucleotides via the folate receptor. Adv. Drug Deliv. Rev. 56, 1193–1204 (2004).
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Gupta U, Dwivedi SK, Bid HK, Konwar R, Jain NK. Ligand anchored dendrimers based nanoconstructs for effective targeting to cancer cells. Int. J. Pharm. 393, 185–96 (2010).
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Formulation development and in vitro–in vivo assessment of the fourth-generation PPI dendrimer as a cancer-targeting vector Aim: In spite of numerous biopharmaceutical applications of fifth-generation poly(propyleneimine) (PPI) dendrimers, inherent toxicity due to the presence of many peripheral cationic groups is the major issue that limits their applicability. Maximum biocompatibility with minimal toxicity is the key rationale for an ideal drug-delivery system. Keeping this principle in mind, the present investigation aimed to explore the tumor-targeting potential of folate-engineered fourth-generation PPI dendrimers loaded with an anticancer drug, melphalan. Materials & methods: Fourth-generation PPI as well as folate-conjugated fourth-generation PPI dendrimers were synthesized, characterized and loaded with melphalan. Results: Hemolytic toxicity, cytotoxicity, cellular uptake and fluorescence uptake studies reveal that the developed folateconjugated derivative has significantly lower toxicity, as well as demonstrates folate receptor specificity. Discussion & conclusion: The developed nanoconjugates appear to be proficient in carrying as well as site-specific delivery of melphalan, with an improved therapeutic margin and improved safety.
Prashant Kesharwani1, Rakesh K Tekade2 & Narendra K Jain*,1 Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr Hari Singh Gour University, Sagar 470003, Madhya Pradesh, India 2 College of Pharmacy, University of Hawaii at Hilo, 96720 HI, USA *Author for correspondence: jnarendr@ yahoo.co.in 1
Original submitted 11 July 2013; Revised submitted 11 November 2013 Keywords: cancer targeting • fourth-generation PPI dendrimer • melphalan • pharmacodynamics • pharmacokinetics • stability • toxicity
An ideal cancer remedial device can diminish tumors without damaging healthy tissue. Therefore, a distinct capacity to target tumors without affecting healthy cells is essential for the success of any new cancer remedial device. Active targeting is a promising strategy that involves the surface modification of a drug carrier system with a bioactive agent such as an antibody, carrier protein or ligand that shows selective binding and uptake via receptors overexpressed on tumor cells [1] . Folate receptors, which are 38-kDa glycosylphosphotidylinositolanchored proteins, are overexpressed on tumor cells, making them a good option for tumor-targeted drug delivery [2,3] . Various drug-delivery carriers such as polymeric micelles, polymer conjugates, liposomes, nanoparticles and dendrimers are being exploited for selective delivery of
10.2217/NNM.13.210 © 2014 Future Medicine Ltd
various anticancer bioactives at the tumor site [4,5] . Recently, dendrimers are one of the most widely explored carriers for anticancer therapy [6–8] . Dendrimers are essentially 3D, highly branched, monodispersed macromolecules with a 3D nanometric structure, obtained by an iterative sequence of reaction steps producing a precise, unique branching structure [9,10] . Regardless of the wide application of dendrimers, the inherent toxicity due to the surface amine groups limits application of these cationic dendrimers. This toxicity is ascribed to the interaction of their peripheral cationic charge with negatively charged biological membranes [11] . The intensity of toxicity is directly proportional to the number of surface amine groups; the number of amine groups increases as the generation of dendrimer increases. Poly(propyleneimine)
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Research Article Kesharwani, Tekade & Jain (PPI) dendrimers represent one of the most explored classes of dendrimers, with the fifth-generation (5.0G) subtype leading the family. It is well documented that the 5.0G dendrimer is more toxic compared with fourth-generation (4.0G) dendrimers due to an increased number of surface cationic groups. In 2010, Jain and coworkers reported that the 5.0G PPI dendrimer exhibited almost three-times as much red blood cell (RBC) hemolysis compared with the 4.0G PPI dendrimer at a concentration of 1 mg/ml [12] . To keep this toxicity at a minimum, we are exploring lowergeneration (4.0G) PPI dendrimers to deliver an anticancer drug, melphalan (MPN). The biocompatibility, drugloading affinity and tumor-targeting potential may be further enhanced by folate conjugation. The main rationale behind proposing to use lower-generation PPI dendrimers lies in their diminished toxicity compared with higher-generation dendrimers (>5.0G PPI), and thus attaining better efficacy with lower toxicity. As such, various strategies have been proposed in the literature to alleviate the toxicity associated with dendrimers [13] . Among these strategies, surface conjugation of biocompatible moieties, ligands and polyethylene glycol chains, among others, have been widely utilized to reduce dendrimer toxicity. In this line, the surface primary amine group of dendrimers has been successfully conjugated with biocompatible ligands such as folate, dextran, galactose and mannose for tumor targeting [10,14–17] . Among various available ligands [13] , a recent report from our laboratory infers folate-based dendritic conjugates to be the most promising in drug targeting, as well as producing the most biocompatible dendritic formulation [8] . The core objective of this investigation was to assess the 4.0G PPI dendrimer for its drug-targeting propensity by engaging MPN as an anticancer drug. The drug MPN has been selected on account of its insolubility in most frequently used solvents; loading it within the dendrimers may overcome this limitation. In this project, first, 4.0G PPI dendrimers were synthesized and thoroughly characterized, followed by surface modification employing folic acid (FA) as a targeting ligand. 4.0G PPI dendrimer-based formulations were investigated for their drug-loading aptitude, drug release, cell line-based cytotoxicity assay, cell uptake assay, florescence uptake assay, hemolytic toxicity and stability. Bioperformance of the developed formulations was also studied to deduce pharmacokinetic and pharmacodynamic performance in BALB/C mice. It was expected that the developed systems will not only selectively target the drug to the tumor cells but will also reduce the charge-associated toxicity of the dendrimers, which is the major constraint of 5.0G PPI dendrimers.
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Materials & methods FA, dimethylsulfoxide and dichloromethane and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were purchased from Himedia (Mumbai, India). Acrylonitrile and ethylenediamine (EDA) were purchased from Central Drug House (Mumbai, India), and Raney ® nickel was purchased from Fluka (MO, USA). 3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (MO, USA). Analytical-grade reagents were purchased from Merck India Ltd (Mumbai, India). MCF-7, HeLa and KB cells were purchased from the National Center for Cell Sciences (Pune, India). MCF-7 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum and incubated at 37°C in a humidified incubator and 5% CO2 atmosphere. HeLa cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Himedia) supplemented with 10% fetal calf serum, 2 mM glutamine in a 37°C humidified incubator and 5% CO2 atmosphere. KB cells were grown in RPMI-1640 medium supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml) and 10% heat-inactivated fetal bovine serum at 37°C and 5% CO2. The protocol for animal experiments was duly approved by the Institutional Animal Ethics Committee of Dr HS Gour University (Sagar, India; registration no. 379/01/ab/CPCSEA). All experiments were performed on healthy BALB/c mice (average bodyweight: 25.0 ± 2.0 g). Animals were housed and handled in accordance with the institutional guidelines. Throughout the experiment, the animals were housed in laminar flow cages maintained at 22 ± 2°C, 50–60% relative humidity, under a 12-h light–12-h dark cycle. The animals were maintained in these facilities for at least 1 week before the experiment. The rats were permitted free access to tap water and commercialized food (commercial standard chow diet; CJ Feed Co. Ltd, Seoul, Republic of Korea) throughout the experiment. Synthesis of 4.0G PPI dendrimers
The 4.0G PPI dendrimers was prepared by a previously reported divergent method using EDA as an initiator core and acrylonitrile as a branching moiety [6,8,16,18] . Briefly, acrylonitrile was added to an aqueous solution of EDA using a double Michael addition reaction to produce half-generation (-CN terminated) dendrimers. This exothermic reaction results in a rise in temperature to approximately 38°C; it was followed by heating up to 80°C to complete the addition reaction. Consequently, excess of acrylonitrile was removed as
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Development & assessment of fourth-generation PPI dendrimer as a cancer-targeting vector
a water azeotrope through vacuum distillation. The obtained crystalline solid was half-generation PPI dendrimer. The half-generation dendrimers were subjected to heterogeneous hydrogenation using Raney nickel as a catalyst to produce full-generation amine-terminated PPI dendrimers. The iterative sequences of reaction steps were repeated cyclically to produce PPI dendrimers up to the 4.0G dendrimer (4.0G PPI) (Figure 1A) . Synthesis of folate-engineered 4.0G PPI dendrimers
Folate conjugation to 4.0G PPI dendrimers was carried out by a method previously reported by us (Figure 1B) [8] .
Research Article
Sigma-Aldrich) against double-distilled water under strict sink conditions for 30 min to remove free drug from the formulation, which was estimated by a HPLC method using mobile-phase methanol:purified water:acetic acid (49.5:49.5:1 v/v) pumped onto the Ultrasphere® ODS (Hichrom Ltd, Theale, UK; 5 µm) column at a flow rate of 2 ml/min, and the analysis was carried out at 260 nm [20] to indirectly determine the amount of drug loaded within the formulations. A similar method was used to load drug within folateconjugated dendrimers [21] . Both of the formulations were lyophilized using 2% lactose as cryoprotectant and used for further characterization. Developed drugloaded formulations thus formed were coded as MPNloaded PPI dendrimers (PPI-MPN) and MPN-loaded FA-conjugated PPI dendrimers (FA-PPI-MPN).
Synthesis of tert-butoxycarbonyl-protected FA
The tert-butoxycarbonyl (t-Boc)-protected FA was synthesized following a reported method [8,19] . In brief, FA (10 mM) was dissolved in a mixture of dimethylsulfoxide:dichloromethane (1:1 v/v), followed by addition of t-Boc (12 mM), and the mixture was continuously stirred in the dark for 48 h. One molar (5 ml) KHSO4 was added to the resultant product, followed by subsequent extraction of the product from the mixture by shaking with three separate portions of ethyl acetate each for 1 h.
Particle size determination
The average particle size and polydispersity index of the formulations (PPI-MPN and FA-PPI-MPN) as 10 w/v solutions were determined in a zetasizer (DTS Version 4.10; Malvern Instruments, Malvern, UK). The particle size distributions are represented by the average size (diameter) and the variance (polydispersity) of the Gaussian distribution function in logarithmic axis mode. In vitro drug-release studies
Conjugation of t-Boc-protected FA to 4.0G PPI dendrimer
The t-Boc-protected FA (4 mM) was activated by adding 1-ethyl-3-(3-dimethylaminopropyl)carbo diimide hydrochloride (5 mM) to it and stirring for 4 h in the dark. The solution of PPI dendrimers was placed in a container, followed by addition of the t-Boc-protected FA solution drop-wise to it, and the mixture was continually stirred in the dark for 48 h (500 rpm; 37 ± 2°C). The resultant conjugate was concentrated under reduced pressure (washed twice with dimethylformamide) and was labeled as folateengineered 4.0G PPI dendrimers (FA-PPI). The resultant conjugation was characterized by Fourier transform infrared spectroscopy (FT-IR), 1H nuclear magnetic resonance (1H-NMR) and MALDI-TOF. Drug loading
The known molar concentration of MPN (100 mM) was dissolved in methanol and mixed with an aqueous solution of 4.0G PPI (10 mM) dendrimer, and the resultant mixture was incubated with slow magnetic stirring (50 rpm) using a Teflon® (Teja Scientific Glass Works, Hyderabad, India) bead for 24 h. The resulting solution was dialyzed twice through a cellulose dialysis bag (molecular weight cut-off: 3.5 kDa;
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The marketed MPN injectable formulation (MMIF; melphalan hydrochloride; Alkeran®; GlaxoSmithKline LLC, Mumbai, India) and MPN-loaded formulations (10 mg) were introduced into a dialysis bag (molecular weight cut-off: 3.5 kDa; Sigma-Aldrich) separately and the end-sealed dialysis bags were submerged in 100 ml of release medium (phosphate-buffered saline [PBS]; pH 7.4; containing 0.1% v/v propylene glycol) and stirred at 200 rpm (37 ± 2°C) for 48 h. At predetermined intervals of time (0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24 and 48 h), 1-ml aliquots were withdrawn and replaced with an equal volume of fresh medium. The concentration of MPN in samples was determined by a HPLC method [20] . Hemolytic toxicity
The degree of reduction of toxicity due to folate conjugation was investigated by a hemolytic toxicity study according to previously reported studies [22] . In short, first, RBCs were collected from human blood and resuspended in normal saline to obtain a 5% RBC suspension, which was used further for hemolytic studies. To the RBC suspension (1 ml), distilled water (5 ml) and normal saline (5 ml) were added separately, which were considered to be 100 and 0% hemolytic, respectively. In a similar fashion, formulations (PPI,
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Figure 1. Synthesis of dendritic formulations. (A) Step-by-step synthesis of 4.0G PPI dendrimers and (B) synthesis of folate-conjugated PPI dendrimers. 1.0G: First generation; 2.0G: Second generation; 3.0G: Third generation; 4.0G: Fourth generation; PPI: Poly(propyleneimine); t-Boc: Tert-butoxycarbonyl.
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Research Article Kesharwani, Tekade & Jain
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Development & assessment of fourth-generation PPI dendrimer as a cancer-targeting vector
PPI-MPN and FA-PPI-MPN) as well as MMIF (0.5 ml) were added to the mixture of normal saline (4.5 ml) and RBC suspension (1 ml) separately. The tubes were allowed to stand for 30 min with gentle intermittent shaking and were centrifuged for 15 min at 3000 rpm in an ultracentrifuge (Microprocessor Research ultracentrifuge PR-23; Remi Elektrotech Ltd, Mumbai, India). The supernatants were pooled and UV spectrophotometric assay was performed at 540 nm. The degree of hemolysis was determined by Equation 1:
MTT cytotoxicity assay
Hemolysis (%) = Abs - Abs0 # 100 Abs100 - Abs0
Cell uptake assay
(Equation 1)
Where Abs, Abs0 and Abs100 are the absorbance of samples, a solution of 0% hemolysis and a solution of 100% hemolysis, respectively. Hematological study
For hematological study, BALB/C mice were divided into four groups, each consisting of three mice. Mice were administered intravenously with free drug, PPI-MPN and FA-PPI-MPN (1 mg/kg bodyweight) separately into all three groups (the fourth group was kept as control) every day up to 7 days. This was followed by the collection of a blood sample, which was analyzed for RBC count, white blood cell (WBC) count and differential count of monocytes, lymphocytes and neutrophils by the pathology laboratory [23] . Stability studies
The influence of accelerated storage conditions on the stability of developed formulations was determined using a previously reported protocol with slight modifications [7,17,24] . Briefly, formulations (10% w/v, 5 ml) were kept in the dark in amber-colored and colorless (light conditions) vials at 0°C, room temperature (25 ± 2°C) and accelerated temperature (60 ± 2°C) in controlled ovens for a period of 5 weeks, and analyzed every week for any visual changes such as precipitation, turbidity, crystallization, color and drug leakage (however, only terminal results were reported). The formulations (PPI-MPN and FA-PPI-MPN) were analyzed for drug content initially and at weekly intervals up to 5 weeks, spectrophotometrically. The percentage drug leakage (release content of the drug) in various formulations was used to analyze the effects of accelerated conditions of storage on the formulations. The amount of residual drug in the dialysis sac was also simultaneously analyzed, which was considered as residual content of the formulation. The study was conducted for all of the formulations of MPN.
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Research Article
The in vitro cytotoxicity of MMIF as well as the developed formulations was determined by the MTT assay based on the activity of mitochondrial dehydrogenases. Following treatment of cells (HeLa, KB and MCF-7 separately), 20 µl MTT (5 mg/ml) in PBS (pH 7.4) was added to each well, and plates were incubated at 37°C for 1 h. The resulting formazan crystals were dissolved by the addition of dimethylsulfoxide and absorbance was recorded at 560 nm [25] . A cell uptake study was performed on MCF-7 cells by in vitro incubation of cells with various formulations. Briefly, MCF-7 cells were seeded at 2 × 106 cells/ml in six-well plates (Sigma-Aldrich) containing fresh medium and suspended for 24 h in a humidified incubator at 37 ± 2°C with 5% CO2 atmosphere. Cells were washed with PBS after removing the culture medium. Cells were then treated with PBS (×3) followed by treatment with 1 ml of RPMI-1640 medium, and were transferred in 24-well cultured plates. After 6 days of culture, 100 µl of MMIF, PPI-MPN and FA-PPI-MPN containing equivalent amounts of drug were added to the wells. The plates were incubated at 37 ± 2°C for 48 h. The cell suspension was transferred to polycarbonate filters (0.45 µm) after the appropriate time. The wells of the cell culture plates were rinsed with 1 ml PBS (pH 7.4) and the washings were consequently transferred to the filters. The cells were separated from the medium in the form of a pellet by centrifuging the filters at 4000 rpm for 15 min [26] . Pellets were ruptured by adding Triton™ X-100 (Sigma-Aldrich) and the mixture was incubated at 25 ± 2°C for 6 h, and uptake of drug was determined by HPLC [20] . Cell topographic assay
MCF-7 cells (5 × 104) were seeded on six-well plates and incubated at 37 ± 0.5°C under 5% CO2 for a period of 24 h, following which time medium was removed and incubated with 2.5 ml of Nile redloaded formulations (Nile red, PPI-loaded Nile red, folate-conjugated PPI-loaded Nile red) for 2 h. After 2 h, medium was removed, and the cells were gently washed three times with PBS (pH 7.4) and fixed with the help of formaldehyde (2% v/v) in the PBS at room temperature. The cells were then immediately imaged using an Axiovert 40® (Carl Zeiss LLC, NY, USA) inverted microscope at 40× magnification at Nile red 554 nm excitation and 635 nm emission wavelengths with a mercury vapor lamp as a light source for fluorescence imaging.
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Research Article Kesharwani, Tekade & Jain In vivo studies
The tumor implantation was carried out in BALB/c mice with MCF-7 cells (8 × 106 cells/0.2 ml) following a previously reported method with slight modification [27] . Pharmacokinetic studies
The BALB/c mice were divided into three groups with six tumor-induced mice in each. Formulations (PPIMPN, FA-PPI-MPN and MMIF) were solubilized in saline in the required dose (1 mg/kg bodyweight) and injected intravenously into the mice. At prespecified time intervals, a blood sample was collected through the tail vein and centrifuged for 10 min to separate RBCs and serum (3000 rpm). Supernatants were collected and thoroughly mixed with 2 ml ethylacetate, followed by centrifugation for removal of the organic layer, evaporation to dryness and resuspension in the mobile phase for analysis of the drug by a HPLC method [20] . Tissue distribution study
For biodistribution studies, mice were divided into eight groups and each group was administered with the same intravenous dose (1 mg/kg) of different formulations (MMIF, PPI-MPN and FA-PPI-MPN). Two mice from each group were sacrificed at 2, 8 and 24 h and, immediately after sacrificing, the mice organs (i.e., spleen, kidney, liver and tumor) were carefully removed and weighed. Weighed tissue samples were suspended in 2 ml ethylacetate and homogenized for 5 min. The homogenate was transferred to an Eppendorf tube and centrifuged at 3000 rpm for 10 min at 4°C. The organic layer was subsequently transferred into a second tube and evaporated to dryness under vacuum. The compound was resuspended in 1 ml of the mobile phase, vortexed and then stored at deepfreeze conditions (-70 ±2°C) until analyzed for MPN content by a HPLC method [28] . Pharmacodynamic studies
Tumor was induced in BALB/c mice following previously reported methodology [29–31] , and our observations on tumor induction and growth were in agreement with earlier reports from our laboratory [7,32] . Briefly, BALB/c mice were implanted with MCF-7 cells (8 × 106 cells/0.2 ml) subcutaneously, and tumor cells were allowed to grow for another 21 days to develop palpable tumor mass. After detection of palpable tumor, intravenous injection of various formulations (at 1 mg/kg MPN equivalent dose) suspended in PBS (filtered through a 0.22 micron filter; Millipore Corp., MA, USA) were injected through the tail vein of animals at 0 and 7 days.
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Tumor volume was measured with the help of a Vernier Caliper using Equation 2 : Volume (mm3) = length × width2 × 0.5
(Equation 2)
With an assumption that width equals depth and p equals 3 [28]. Statistical analysis
All of the statistical analyses were performed with GraphPad® Instat software (version 3.0; GraphPad Software, CA, USA) using either unpaired t-test or oneway analysis of variance followed by Tukey–Kramer multiple comparison test. A difference of p HeLa > MCF-7. The highest activity of FA-PPI-MPN with respect to KB cells may be due to overexpression of a comparatively greater number of FA molecules compared with the other cell lines. The results of our study are in accordance with previously reported literature [8] . Higher uptake in the case of FA-PPI-MPN is due to its interaction with overexpressed folate receptors on the MCF-7 cells, which are critical to enhanced cellular uptake. Further cell topographic assay also supported the better anticancer activity of the folate-mediated formulations. Higher drug concentrations of the PPI-based formulations as determined by HPLC analysis were found compared with MMIF due to prolonged and sustained properties of PPI dendrimers. Developed folate-engineered formulations showed a 2.87- and 1.53-times increase of the half-life of FA-PPI-MPN compared with MMIF and PPI-MPN, respectively. This extended half-life may be particularly advantageous in targeted cancer therapy. It was observed that folate modification results in an increase in the half-life of loaded drug, and similar outcomes have been noted by Singh et al. [7] and Gupta et al. [34] . The main reason behind the prolonged half-life of drug loaded inside folate-anchored dendrimers is the entanglement of drug in small packets that were created after ligand anchoring. The same aspect was carefully studied under in vitro as well as in vivo settings. The outcome of these investigations
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Figure 8. In vivo analysis of dendritic formulations. (A) Tissue distribution pattern and (B) pharmacodynamic tumor growth analysis. (A,i) 2 h, (A,ii) 8 h and (A,iii) 24 h. The results are expressed as mean ± standard deviation (n = 3). FA-PPI-MPN: Melphalan-loaded folate-conjugated fourth-generation poly(propyleneimine) dendrimer; MMIF: Marketed melphalan injectable formulation; PPI-MPN: Melphalan-loaded fourth-generation poly(propyleneimine) dendrimer.
infers slow release of loaded drug from the modified dendrimer. It is agreed that folate uptake in various organs must lead to rapid clearance of the folate dendrimer, hence one may expect a short half-life with the folate dendrimer. However, in a practical setting, the opposite was observed, and this may be due to the open and easily accessible structure of the naked dendrimer, as well as the presence of extra crevices compared with the folate-decorated dendrimers. The folate-modified dendrimer elicits extra hindrance towards release of loaded drug owing to the surface-labeled folate groups. Biodistribution studies revealed an elevated concentration of MPN in the urine, suggesting the kidney as a major route of drug elimination. The folate-conjugated formulation (FA-PPI-MPN) was found in comparatively much higher amounts at the tumor site compared with PPI-MPN and MMIF. This outcome is in accordance with the hemolytic toxicity and MTT cell line-based assays, which clearly indicate better distribution as well as targeting efficiency of the folate-mediated dendritic formulations.
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The results of the pharmacodynamic studies on tumor-bearing BALB/C mice demonstrated that, compared with control, PPI-MPN and FA-PPI-MPN resulted in a 1.70- and 2.13-times reduction in tumor volume. However, FA-PPI-MPN was 1.25-times more active compared with PPI-MPN, which may be ascribed to ligand-mediated targeting of dendrimers due to surface conjugation of the FA. Overexpressed folate receptors on cancer cells resulting in greater interaction with the conjugates may be another reason behind its anticancer activity. Conclusion At present, the 5.0G PPI dendrimer is the most widely explored dendrimer compared with lower-generation dendrimers due to the presence of numerous surface amino groups, which provide greater opportunity for surface engineering. Another profit is the higher drug loading due to the presence of a larger cavity. However, these surface amino groups are also the cause of additional toxicity, which restrict the application of the 5.0G dendrimer. With respect
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Research Article Kesharwani, Tekade & Jain to the biocompatibility concern, we explored the 4.0G dendrimer in contrast to 5.0G dendrimer formulations, which as per our report can be further enhanced by folate modification. We have described a surface engineering concept based on folate receptors on tumor cells that is adaptable to a wide variety of dendrimer-based platforms. Folate functionalization on the dendrimer periphery significantly enhanced their accumulation in tumor cells, and resulted in improved efficacy of folate-conjugated MPN-loaded 4.0G dendrimers in a tumor-bearing BALB/C mice model. Hemolytic toxicity, cytotoxicity, cellular uptake and fluorescence uptake studies indicate that the folate-conjugated derivative has significantly lower toxicity, as well as demonstrating folate receptor specificity. Further studies regarding optimization of folate-based formulations are currently in progress in our laboratory, in order to validate this system as a potential targeted drug nanotherapeutic. Future perspective In this article, we have developed folate-functionalized 4.0G-based dendritic nanoconjugates. The developed nanoconjugates are able to deliver anticancer drug significantly within an acceptable toxicity limit. The work is believed to shift the attention of drugdelivery scientists throughout the globe towards 4.0G dendrimer-based formulations instead of the widely explored 5.0G dendritic formulations. In addition, it is envisaged that formulation parameters of 4.0G-based
formulations, such as drug loading and anticancer activity, among others, can further be improved by available techniques such as PEGylation, to make the nanosystem more acceptable and fit for drug-delivery applications. With respect to further enhancement of biocompatibility and negligible toxicity, PEGylation of this optimized formulation (FA-PPI-MPN) is currently in progress in our laboratory. It is anticipated that the 4.0G dendrimer-based formulation will generate a safe, effective and stable formulation that will help in successful transformation of dendritic formulations from laboratory to clinical stages. Financial & competing interests disclosure P Kesharwani would like to acknowledge the Indian Council of Medical Research (New Delhi, India) for providing financial assistance to carry out the present experimental studies. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate insti tutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations.
Executive summary • The folate-conjugated fourth-generation poly(propyleneimine) dendrimer displayed significantly less hemolysis than naked dendrimer-based formulations. • The fourth-generation, folate-modified dendritic formulations can be effectively and rapidly taken up by cancer cells due to overexpressed folate receptors. • The fourth generation-based conjugates developed also displayed prolonged half-life and higher concentration of drug in the systemic circulation. • Surface conjugation of folate on the dendrimer periphery exhibited a significant sustained-release pattern in vitro compared with plain dendrimer. • Folate conjugation imparts rigidity to the system and results in greater stability of these systems compared with plain dendrimer. • The folate-conjugated fourth-generation poly(propyleneimine) dendrimer appears to be proficient in the delivery of anticancer drug, with an improved therapeutic margin and safety profile.
References Papers of special note have been highlighted as: • of interest
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and molecular identification as a folate-binding protein. Am. J. Pathol. 142, 557–567 (1993).
1
Kesharwani P, Ghanghoria R, Jain NK. Carbon nanotube exploration in cancer cell lines. Drug Discov. Today 17, 1023–1030 (2012).
3
Lu Y, Low PS. Folate-mediated delivery of macromolecular anticancer therapeutic agents. Adv. Drug Deliv. Rev. 54, 675–693 (2002).
2
Garin-Chesa P, Campbell I, Saigo PE, Lewis JL Jr, Old LJ, Rettig WJ. Trophoblast and ovarian cancer antigen LK26. Sensitivity and specificity in immunopathology
4
Zhang Z, Feng SS. The drug encapsulation efficiency, in vitro drug release, cellular uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl polyethylene
Nanomedicine (Lond.) (2014) 9(15)
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Development & assessment of fourth-generation PPI dendrimer as a cancer-targeting vector
glycol succinate nanoparticles. Biomaterials 27, 4025–4033 (2006). 5
6
Yang T, Choi MK, Cui FD et al. Preparation and evaluation of paclitaxel-loaded PEGylated immunoliposome. J. Control. Release 120, 169–177 (2007). Tekade RK, Dutta T, Gajbhiye V, Jain NK. Exploring dendrimer towards dual drug delivery: pH responsive simultaneous drug-release kinetics. J. Microencapsul. 26, 287–296 (2008).
Research Article
17
Bhadra D, Yadav AK, Bhadra S, Jain NK. Glycodendrimeric nanoparticulate carriers of primaquine phosphate for liver targeting. Int. J. Pharm. 295, 221–233 (2005).
18
Brabander-van den Berg EMM, Meijer EW. Poly(propylene imine) dendrimers: large scale synthesis by heterogeneously catalyzed hydrogenation. Angew. Chem. Int. Ed. Engl. 32, 1308–1311 (1993).
19
Dutta T, Agashe, HB, Garg M, Balakrishnan P, Kabra M, Jain NK. Poly (propyleneimine) dendrimer based nanocontainers for targeting of efavirenz to human monocytes/macrophages in vitro. J. Drug Target. 15, 89–98 (2007).
7
Singh P, Gupta U, Asthana A, Jain NK. Folate and folatePEG-PAMAM dendrimers: synthesis, characterization, and targeted anticancer drug delivery potential in tumor bearing mice. Bioconjug. Chem. 19, 2239–2252 (2008).
20
8
Kesharwani P, Tekade RK, Gajbhiye V, Jain K, Jain NK. Cancer targeting potential of some ligand-anchored poly(propylene imine) dendrimers: a comparison. Nanomedicine 7, 295–304 (2011).
Pinguet F, Joulia JM, Martel P, Grosse PY, Astre C, Bressolle F. High-performance liquid chromatographic assay for MPN in human plasma. Application to pharmacokinetic studies. J. Chromatogr. B Biomed. Appl. 686, 43–49 (1996).
•
•
Describes the comparatively higher antitumor targeting potential of folate-mediated fifth-generation poly(propyleneimine) dendrimers.
Describes a simple, rapid and reproducible HPLC method for the analysis of melphalan in plasma.
21
Tomalia DA, Baker H, Dewald JR et al. A new class of polymers: starburst dendritic molecules. Polym. J. 17, 117–132 (1985).
Kumar PV, Asthana A, Dutta T, Jain NK. Intracellular macrophage uptake of rifampicin loaded mannosylated dendrimers. J. Drug Target. 14, 546–556 (2006).
22
Singhai AK, Jain S, Jain NK. Evaluation of an aqueous injection of ketoprofen. Pharmazie 52, 149–151 (1997).
23
Agrawal P, Gupta U, Jain NK. Glycoconjugated peptide dendrimers-based nanoparticulate system for the delivery of chloroquine phosphate. Biomaterials 28, 3349–3359 (2007).
24
Prajapati RN, Tekade RK, Gupta U, Gajbhiye V, Jain NK. Dendimer-mediated solubilization, formulation development and in vitro in vivo assessment of piroxicam. Mol. Pharm. 6, 940–950 (2009).
25
Casas A, Battah S, Di Venosa G et al. Sustained and efficient porphyrin generation in vivo using dendrimer conjugates of 5-ALA for photodynamic therapy. J. Control. Release 135, 136–143 (2009).
26
Dutta T, Jain NK. Targeting potential and anti-HIV activity of lamivudine loaded mannosylated poly (propyleneimine) dendrimer. Biochim. Biophys. Acta 1770, 681–686 (2007).
27
Yoo HS, Park TG. Folate-receptor-targeted delivery of doxorubicin nanoaggregates stabilized by doxorubicin– PEG–folate conjugate. J. Control. Release 100, 247–256 (2004).
28
Ying X, Wen H, Lu WL et al. Dual-targeting daunorubicin liposomes improve the therapeutic efficacy of brain glioma in animals. J. Control. Release 141, 183–192 (2010).
9
•
Explores poly(amidoamine) dendrimers extensively for diverse medical applications.
10
Kesharwani P, Gajbhiye V, Tekade RK, Jain NK. Evaluation of dendrimer safety and efficacy through cell line studies. Curr. Drug Targets 12, 1478–1497 (2011).
11
Jain K, Kesharwani P, Gupta U, Jain NK. Dendrimer toxicity: let’s meet the challenge. Int. J. Pharm. 394, 122–142 (2010).
•
Demonstrates how surface engineering masks the cationic charge of dendrimers and enhances their biocompatibility.
12
Mishra V, Gupta U, Jain NK. Influence of different generations of poly(propylene imine) dendrimers on human erythrocytes. Pharmazie 65, 891–895 (2010).
•
Pioneering work that illustrates the generationdependent toxicity associated with the cationic groups of poly(propyleneimine) dendrimers.
13
Tekade RK, Kumar PV, Jain NK. Dendrimers in oncology: an expanding horizon. Chem. Rev. 109, 49–87 (2009).
•
Reviews the importance of and strategies involved in dendrimer-mediated cancer therapy.
14
Wiener EC, Konda S, Shadron A, Brechbiel M, Gansow O. Targeting dendrimer-chelates to tumors and tumor cells expressing the high-affinity folate receptor. Invest. Radiol. 32, 748–754 (1997).
29
Kothari K, Bapat K, Korde A. Radiochemical and biological studies of 188Re labeled monoclonal antibody – CAMA3C8 specific for breast cancer. Ind. J. Nuc. Med. 19, 6–11 (2004).
15
Patri AK, Kukowska-Latallo JF, Baker JR Jr. Targeted drug delivery with dendrimers: comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex. Adv. Drug Deliv. Rev. 57, 2203–2214 (2005).
30
Matsuno F, Haruta Y, Kondo M, Tsai H, Barcos M, Seon BK. Induction of lasting complete regression of preformed distinct solid tumors by targeting the tumor vasculature using two new anti-endoglin monoclonal antibodies. Clin. Cancer Res. 5, 371–382 (1999).
16
Thakur S, Tekade RK, Kesharwani P, Jain NK. The effect of polyethylene glycol spacer chain length on the tumortargeting potential of folate-modified PPI dendrimers. J. Nanopart. Res. 15, 1625 (2013).
31
VanWeelden K, Flanagan L, Binderup L, Tenniswood M, Welsh JE. Apoptotic regression of MCF-7 xenografts in nude mice treated with the vitamin D 3 analog, EB1089. Endocrinology 139, 2102–2110 (1998).
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2307
Research Article Kesharwani, Tekade & Jain
2308
32
Gajbhiye V, Jain NK. The treatment of glioblastoma xenografts by surfactant conjugated dendritic nanoconjugates. Biomaterials 32, 6213–6225 (2011).
33
Zhao XB, Lee RJ. Tumor-selective targeted delivery of genes and antisense oligodeoxyribonucleotides via the folate receptor. Adv. Drug Deliv. Rev. 56, 1193–1204 (2004).
Nanomedicine (Lond.) (2014) 9(15)
34
Gupta U, Dwivedi SK, Bid HK, Konwar R, Jain NK. Ligand anchored dendrimers based nanoconstructs for effective targeting to cancer cells. Int. J. Pharm. 393, 185–96 (2010).
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