Bioconjugate Chem. 2008, 19, 2239–2252
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Folate and Folate-PEG-PAMAM Dendrimers: Synthesis, Characterization, and Targeted Anticancer Drug Delivery Potential in Tumor Bearing Mice Prateek Singh, Umesh Gupta, Abhay Asthana, and Narendra K. Jain* Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour University Sagar (M.P.) 470003 India. Received March 25, 2008; Revised Manuscript Received August 8, 2008
Ligand-mediated targeting of drugs especially in anticancer drug delivery is an effective approach. Dendrimers, due to unique surface topologies, can be a choice in this context. In the present study, PAMAM (polyamidoamine) dendrimers up to fourth generation were synthesized and characterized through infrared (IR), nuclear magnetic resonance (NMR), electrospray ionization (ESI) mass spectrometric, and transmission electron microscopic (TEM) techniques. Primary amines present on the dendritic surface were conjugated through folic acid and folic acid-PEG (poly(ethylene glycol))-NHS (N-hydroxysuccinimide) conjugates. Tumor in mice was induced through the use of KB cell culture. Prepared dendritic conjugates were evaluated for the anticancer drug delivery potential using 5-FU (5-fluorouracil) in tumor-bearing mice. Approximately 31% of 5-FU was loaded in folate-PEG-dendritic conjugates. Results indicated that folate-PEG-dendrimer conjugate was significantly safe and effective in tumor targeting compared to a non-PEGylated formulation. Tailoring of dendrimers via PEG-folic acid reduced hemolytic toxicity, which led to a sustained drug release pattern as well as highest accumulation in the tumor area.
INTRODUCTION Nanoparticulate drug delivery systems containing anticancer agents have received much attention recently due to their unique accumulation behavior at the tumor site. Targeted anticancer drug delivery includes drug delivery by avoiding reticuloendothelial system (RES), utilizing the enhanced permeability and retention (EPR) effect and tumor specific targeting (1-3). Polymers have been used as drug carriers in the last 2-3 decades due to their ability to control drug release, to sustain circulation times, and to increase drug solubility. These drug carriers demonstrated higher affinity for preferential accumulation in tumor tissue as a result of EPR effect (4, 5). Polymeric drug carriers encapsulate, complex, or conjugate drug molecules to deliver them to the desired site. In this context, applicability of dendrimers to deliver anticancer drugs and other bioactives is well-reported in the literature (6-13). These are a relatively new type of polymer known for their three-dimensional, monodispersed, highly branched, macromolecular nanoscopic (1-100 nm) architecture with a number of reactive end groups (14). Apart from drug delivery, dendrimers have recently been used successfully in the field of biomedicine and targeting (DNA and gene delivery, cancer diagonosis), catalysis, as lightharvesting devices, optical sensors, and so forth. Dendrimers have been reported to host both hydrophilic and hydrophobic drugs with high drug payload (15-22). Earlier dendrimers have successfully been delivered via transdermal, intravenous, and opthalamic routes, proving their transfection capabilities (23-25). The solubilization property of dendrimer makes them a promising carrier for insoluble drug delivery (26). However, toxicity problems with the surface cationic charge of dendrimers like polyamidoamine (PAMAM) and polypropyleneimine (PPI) pose a hurdle in their biomedical potential (25-28). Recently, surface-engineered/conjugated dendrimers, with drug molecules or targeting ligands or both, are receiving much attention as an alternative to the toxicity problems (25-29). In some reports, * Corresponding author.
[email protected] (Narendra K. Jain);
[email protected] (Umesh Gupta), Tel./Fax.: +91-7582264712.
dendrimer-poly(ethylene glycol) (PEG) grafts were designed as a drug carrier that possesses an increased interior and biocompatible surface for the encapsulation of drugs like 5-fluorouracil, chloroquine phosphate, and so forth (6, 30, 31). In another study, PEGylated dendrimers encapsulating anticancer drugs adriamycin and methotrexate were prepared by extraction with chloroform from the mixtures of the PEGylated dendrimers and varying amounts of drug. Among the PEGylated dendrimers synthesized, the highest ability was achieved by the fourthgeneration dendrimer having the PEG graft with average molecular weight of 2000 Da that could retain 6.5 adriamycin molecules or 26 methotrexate molecules per dendrimer molecule. The methotrexate-loaded PEG attached dendrimers slowly released the drug in an aqueous solution of low ionic strength (32). The major drawback with the anticancer drug delivery is that these indiscriminately attack healthy cells as well as cancerous cells. The harmful side effects due to such therapy, however, can be reduced by developing a drug delivery system that is specific to tumor cells. To overcome this, a strategy called active targeting can be followed, wherein functionalities that respond to overexpressed receptors (e.g., folate conjugation on dendrimer surfaces) on tumor cells are attached to the drug carrier. Folate receptors (FR), which are 38 kDa glycosylphosphotidylinositolanchored proteins, exist in three major forms, namely, FR-R, FR-β, and FR-γ. The FR-R form is overexpressed in many types of tumors including ovarian, endometrial, breast, renal cell carcinomas, and so forth. The fact that high-affinity FR binding is retained when folate is covalently linked via its γ-carboxyl group to a foreign molecule, combined with the prevalence of FR overexpression among tumors, makes it a good choice for targeted drug delivery to tumors (33, 34). Tailored folic acid conjugated PAMAM dendrimers via EDC [1-ethyl-l-3-(3 methylaminopropyl carbodiimide)] linkage were more efficient in delivery of methotrexate (9, 35). In another approach, PEGylation and folate conjugation were combined and a folate-PEG conjugate, capable of covalent coupling to primary amines present at the surface of polyplexes, was developed (36).
10.1021/bc800125u CCC: $40.75 2008 American Chemical Society Published on Web 10/25/2008
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In the present study, a similar approach was designed in which PAMAM dendrimers up to fourth generation were synthesized, characterized, and conjugated to folic acid directly or indirectly through PEG4000 as spacer. 5-Fluorouracil, an anticancer drug, was encapsulated in both types of dendrimer-folic acid conjugates and assessed for their in Vitro as well as in ViVo anticancer potential in tumor-bearing mice.
EXPERIMENTAL PROCEDURES Materials. Methyl acrylate, triethylamine, toluene, folic acid, PEG-4000, and ethylenediamine were purchased from Central Drug House, India. Thionyl chloride was obtained from Spectrochem, Mumbai (India). DMF (Dimethylformamide) was purchased from Merck, India. NHS (N-hydroxysuccinimide), and DSS (disuccinimidyl suberate) were purchased from Sigma Aldrich, Germany. DCC (N,N-dicyclohexyl carbodiimide) and culture medium RPMI 1640 were obtained from Hi Media, Mumbai (India). All the other common chemicals used were of analytical grade and were identified by respective chemical tests before their use in the study. The drug 5-fluorouracil was obtained as a generous gift from Cadila Pharmaceuticals Pvt. Ltd. India. Synthesis and Characterization of 4.0 G PAMAM Dendrimers. Synthesis of PAMAM dendrimers was carried out following a two-step process, involving Michael addition of a suitable amine initiator core with methyl acrylate, and exhaustive amidation of the resulting esters with large excess of ethylenediamine, reported elsewhere (14). Briefly, ethylenediamine (EDA) was used as an initiator core for the synthesis of dendrimers by attaching four acrylate moieties on each amino group of EDA (Scheme 1). Table 1 holds the molar equivalents of the reagents used in the synthesis of PAMAM dendrimers. The resulting compound was referred to as “generation -0.5G PAMAM tetra ester” (Scheme 1). This led to branching in the dendritic structure. The second step was used for amidation of the terminal carbomethoxy (COCH3) groups of methylacrylate with EDA. This tetra ester with excess EDA gave “generation 0.0 PAMAM tetra amine”. Similar steps were repeated for the synthesis of higher-generation PAMAM dendrimers, subsequently. The reaction sequence was followed by the removal of excess reagents by rotary vacuum evaporation (Superfit, India; 55-60 °C) at every step. Synthesis was carried out in the dark at room temperature (25 °C), using amber-colored round-bottom flask corked tightly. The reaction was carried out as per the quantities given in Table 1 and Scheme 1. Dendrimers were subjected to physical and chemical characterization. Half- and full-generation dendrimers were distinguished by IR spectroscopy and chemical reaction of copper sulfate aqueous solution (1% w/v) with dendrimer (0.1% w/v in methanol). A synthesized 4.0 G dendritic system was characterized by IR, NMR, ESI mass spectroscopy, and transmission electron microscopy. In IR analysis, dendrimer samples were analyzed by the KBr pellet method in a PerkinElmer 783 IR spectrophotometer and displayed characteristic peaks at 3350.2 cm-1 (N-H stretch of primary amines); 3245 cm-1 (quarternary ammonium ion peak); 3190.0 cm-1 (antisymmetric N-H stretch of substituted primary amine); 1641.3 cm-1 (-CdO stretch); 2890 cm-1 (aliphatic C-H stretch); 1566.0, 1471.7, 1327.9 cm-1 (N-H bending of N substituted amide); 1118.4 cm-1 (CC bending). NMR spectroscopy (Bruker Advance DRX 300, Germany) was performed using D2O as solvent and methanol as cosolvent at 300 MHz; δ 2.418, 2.436 (-CH2CdO); δ 2.5-2.9 (amide-NH); δ 3.1-3.4 (-CH2NH2 terminal group); δ 4.762 (methanolic -OH) (Scheme 1). ESI mass spectra were recorded on a Micromass Quattro II Triple Quadrupole mass spectrometer. The samples (dissolved in suitable solvents such as methanol/acetonitrile/water) were
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introduced into ESI source through a syringe pump at the rate of 5 µL/min. The ESI capillary was set at 3.5 KV and the cone voltage was 40 V. Spectra were collected in 6S scans, and the printouts were averaged spectra of 6-8 scans. Spectra recorded at high mass units were computerized decovoluted. Transmission electron microscopy (TEM) of the prepared 4G PAMAM dendrimer was carried out after drying on 3 mm forman (0.5% plastic powder in amyl acetate) coated copper grid (300 mesh) at 60 KV (Philips Morgani, 268D trasmission electron microscope; Holland) after staining negatively, using uranyl acetate (4%). Photomicrographs (Figure 1A) were taken at suitable magnifications from the Electron Microscopy Section of Anatomy Division of All India Institute of Medical Sciences, New Delhi, India. Synthesis and Characterization of PEG-4000 Bis Amine. PEG-4000 bis amine (H2N-PEG4000-NH2) was synthesized in three steps following a modified method reported by Zalipsky et al. (37). In the first step, PEG-4000 (19 g, 0.004 mol) was dissolved in toluene (100 mL) and dried by distillation. Dry pyridine (1.6 mL, 0.019 mol) was mixed to the above solution, and then thionyl chloride (4.36 mL, 0.059 mol) was added dropwise in a period of 30 min under reflux to the mixture. The mixture was heated for 4 h, cooled to room temperature (25 °C), filtered from pyridine hydrochloride, and toluene was evaporated in vacuum. The residue was dissolved in about 20 mL of dichloromethane (DCM), dried over anhydrous K2CO3, and filtered. The filtrate was treated with alumina (30 g, activated by heating at 120 °C for 2 h). Alumina was separated by filtration, and the filtrate was precipitated by cold ether. The polymer was recrystallized from dichloromethane (DCM) yielding 15 g (79%) of Cl-PEG-4000-Cl. In the second step, sodium azide (16 mmol, 1 g) was added to the solution of Cl-PEG4000-Cl (3.6 g) in DMF (20 mL), and the mixture was placed in an oil bath maintained at 120 °C for 1 h; thereafter, DMF (20 mL) was added, and the mixture was stirred at 80 °C for 4 h, cooled, and filtered, and DMF was evaporated in vacuum. The residue was taken in DCM, filtered, and precipitated with cold ether. Yield was 3 g (83%) and N3-PEG 4000-N3 was formed. In the third and the final steps, N3-PEG 4000-N3 (1.6 g) was dissolved in absolute ethanol (40 mL), 10% Pd/C (0.1 g) was added, and the mixture was hydrogenated in a catalytic hydrogenation apparatus (Low-Pressure model PCHS 500, India) overnight. The catalyst was removed by filtration, and the polymer conjugate was precipitated using dry ether and was vaccuum-dried. A yield of 1.2 g (75%) NH2-PEG 4000-NH2 was obtained. PEG-4000 bis amine synthesis was characterized by IR spectroscopy, at every step. Conjugation of Folic Acid to 4.0 G PAMAM Dendrimers. Folic acid was conjugated to 4.0 G PAMAM dendrimers surface through two approaches, i.e., direct conjugation and conjugation through PEG spacer (indirect). Direct Conjugation. Preparation of NHS Ester of Folic Acid. The active ester of folic acid was prepared by a reported method (38). Briefly, folic acid (0.30 g, 0.68 mM) and triethylamine (0.15 mL, 1.0 mmol) were dissolved in DMSO (10 mL), and DCC (0.14 g, 0.68 mmol) was added to it. The solution was stirred for 1 h at room temperature in the dark, and NHS (0.12 g, 1.0 mmol) was added. The mixture was stirred overnight in the dark at room temperature, filtered via glass wool to remove the insoluble byproduct, dicyclohexylurea, and was precipitated using diethyl ether. The active ester of folic acid was collected by filtration, washed with dry tetrahydrofuran (THF) and dried under vacuum (Scheme 2). The NHS ester of folic acid was characterized through IR spectroscopy. Important peaks obtained for the folic acid were at 3545.0, 3415.3 cm-1 (NsH stretch of primary amine and amide); 3324.8 cm-1 (alkyl CsH and CdC stretch); 1694.8 cm-1 (aromatic CdC bending
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Scheme 1. Synthesis of PAMAM Dendrimers up to Fourth Generation
and stretching); 1484.2 cm-1 (CHsNHsCdO amides bending); 837.8 cm-1 (aromatic CsH bending and benzene 1,4-disubstitution); and for the NHS conjugated folic acid 3691.6 cm-1 (amide NsH and CdO stretching); 3444.2 cm-1 (primary aliphatic amine NsH stretching); 2997.0 cm-1 (carboxylic acid CdO and OsH stretching unconjugated); 1712.7 cm-1 (ketones
CdO unconjugated stretch), which confirmed the synthesis of NHS ester of folic acid. Conjugation of ActiVe Ester of Folic Acid to 4.0G PAMAM Dendrimers. The active ester of folic acid in DMSO (25 mg/mL) and that of amine-terminated 4.0 G PAMAM dendrimers in DMSO (10 mg/mL) were mixed and stirred for
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Table 1. Details of the Reactants and Products in the Synthesis of PAMAM Dendrimers generation
moles of reactanta
moles of reactant in excess
product mol. wt.
number of surface functional groups
-0.5G 0.0G 0.5G 1.0G 1.5G 2.0G 2.5G 3.0G 3.5G 4.0G
4 moles (400 g) MA 4 moles (240 g) EDA 8 moles (800 g) MA 8 moles (400 g) EDA 16 moles (1600 g) MA 16 moles (960 g) EDA 32 moles (3200 g) MA 32 moles (1920 g) EDA 64 moles (6400 g) MA 64 moles (3840 g) EDA
5% excess of 4 moles 40 moles (10 equivalents) 10% excess of 8 moles 160 moles (20 equivalents) 15% excess of 16 moles 640 moles (40 equivalents) 20% excess of 32 moles 1920 moles (60 equivalents) 30% excess of 64 moles 5120 moles (80 equivalents)
404 516 1204 1428 2804 3308 6004 6900 12404 14196
4 4 8 8 16 16 32 32 64 64
a
MA ) Methylacrylate, EDA ) Ethylenediamine.
Scheme 2. Preparation of NHS Ester of Folic Acid
Figure 1. Electron microscopic photographs of (A) 4.0 G PAMAM dendrimers; (B) FA-PAMAM (folic acid-dendrimer conjugates); (C) FA-PEG-PAMAM (folic acid-PEG-dendrimer conjugates).
5 days at room temperature (25 °C) using a metabolic shaker. Acetone was added to the reaction mixture, and a yellow precipitate thus formed was collected by filtration and dried under vacuum. The crude product was dissolved in deionized water, and the pH was adjusted to 9.0 using 1 N NaOH solution. The solution was loaded to a Sephadex G-25 column using deionized water as effluent. Fractions containing the conjugate were collected, and the pH of the solution was adjusted to 3
using 1 N HCl (8). The product was precipitated out, collected by filtration, and dried under vacuum. Indirect Folic Acid Conjugation through PEG Spacer. Preparation of NHS-ActiVated Folate-Conjugated PEG. A similar method to that given in an earlier step was followed to obtain the NHS ester of folic acid. 65 mg (148 µmol) of FANHS (folic acid-N-hydroxy succinimide) ester was added to PEG-4000 bis-amine (900 mg, 225 µmol) in DMSO (5.0 mL) in the presence of triethylamine (4.0 µL). The mixture was stirred overnight, and the product was purified by filtration over a Sephadex G-25 column equilibrated with 0.1 M NaHCO3 to remove unconjugated folic acid and unreacted PEG-bis amine fragments. Folate-containing product was detected by UV-visible spectrophotometer (Shimadzu 1601, Japan; absorbance at λmax ) 363 nm). The filtrate obtained was precipitated with acetone and dried under vacuum, yielding folate-PEG conjugate (NH2PEG-FA) as a pale yellow solid. Thereafter, 50 mg of the product was dissolved in DMSO (5.0 mL) containing triethylamine (4.0 µL) as a base, and a 10-fold excess of DSS (12.5 mg) was added. After stirring overnight, the PEG was precipitated by addition of acetone. The product was redissolved in DCM and filtered, and solvents were removed under reduced pressure. The product was then dissolved in DMSO and purified
Folate and Folate-PEG-PAMAM Dendrimers Scheme 3. Synthesis of NHS-PEG4000-Folic Acid Conjugate from NHS-Folic Acid Conjugate
over a Sephadex G-25 column, using DMSO as an eluent (based on modified method) (36). The product was finally precipitated using acetone. NHS-PEG 4000-FA, so obtained (Scheme 3), was characterized at each step by IR spectroscopy. Important peaks in IR spectra were 3690.5 cm-1 (amide NsH and CdO stretching); 3400 cm-1 (primary aliphatic amine NsH stretching); 2911.5 cm-1 (alkyl CsH stretching); 112.5 cm-1 (strong peak of CsO of ether linkage); 1710.0 cm-1 (unconjugated CdO stretch); and 701.4 cm-1 (aromatic CsH bending), which confirmed the synthesis of NHS-PEG-FA. Conjugation of NHS-PEG 4000-FA to 4.0 G PAMAM Dendrimers. The active ester NHS-PEG 4000-FA in DMSO (25 mg/mL) was conjugated to the amine-terminated 4.0 G PAMAM dendrimer in a similar manner to conjugation of FANHS in an earlier step (8). Characterization of Folate Conjugated Systems. Synthesized folate conjugates were characterized by IR spectroscopy using the KBr pellet method. For NMR spectroscopy (Bruker Advance DRX 300, Country, Germany), folate conjugated 4.0 G PAMAM dendrimers were solubilized in D2O using methanol as cosolvent and analyzed at 300.1299987 MHz. The electrospray mass spectra of folate conjugates were recorded on a Micromass Quattro II Triple Quadrupole mass spectrometer, similar to the PAMAM dendrimers. Similarly, TEM photomicrographs (Figure 1B,C) were taken at suitable magnifications using Philips Morgani, 268D; transmission electron microscope (Holland). Drug Loading. 5-FU was loaded in PAMAM dendrimers and dendritic conjugates following the equilibrium dialysis method reported earlier (39, 25). Briefly, 1 mL of aqueous solution of 5-FU (1000 µg/mL) was mixed with 100 mg of 4.0 G PAMAM dendrimer, and the volume was increased to 10 mL with double-distilled water. The mixed solutions were incubated with slow magnetic stirring (50 rpm) using Teflon beads for 24 h. Percent drug encapsulation was confirmed indirectly through UV-visible spectrophotometry. The formulations having final drug concentrations were scanned against similar concentrations of dendritic solution without drug loading,
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as control (40). Similarly, drug was loaded in folic acid conjugated 4.0 G PAMAM (FA-PAMAM) and FA-PEG conjugated 4.0 G PAMAM (FA-PEG-PAMAM) dendrimers. The three different drug-loaded formulations so formed were drugloaded 4.0 G PAMAM dendrimers (DF), drug-loaded folic acid conjugated 4.0 G PAMAM dendrimers (FA-DF), and drugloaded FA-PEG conjugated 4.0 G PAMAM dendrimers (FAPEG-DF). In Vitro Studies. Entrapment Efficiency. Ten milliliters each of three formulations (i.e., DF, FA-DF, and FA-PEG-DF) were taken separately in a cellophane bag (MWCO 12 000-14 000, Sigma, Germany). The contents were dialyzed under strict sink conditions against two portions of 50 mL water, and the unentrapped drug was allowed to pass out of the bag against perfect sink conditions for 10 min. The released drug was analyzed by UV-visible scanning after appropriate dilutions, indirectly. Similarly, dialyzed and diluted dendrimer solution without drug was taken as control. The percent entrapment of drug (% w/w in dendrimer solution) was determined indirectly. In Vitro Release. For in Vitro release studies, 5 mL of formulations (after complete removal of unentrapped drug) were taken in a dialysis bag and dialyzed against 100 mL of water as reservoir under sink conditions, separately. Aliquots of these formulations (i.e., DF, FA-DF, and FA-PEG-DF) were collected at hourly intervals for 24 h, and drug content was estimated by UV-visible spectrophotometer using water as control (40). Hemolytic Toxicity. RBC suspension was obtained as per the well-known and reported procedure for hemolytic studies (41). Briefly, whole human blood was collected using heparin as anticoagulant in HiAnticlot blood collecting vials (Hi media, India) and centrifuged at 3000 rpm for 15 min. RBCs collected from the bottom were washed with normal saline (0.9% w/v) in double-distilled water until a clear, colorless supernatant was obtained above the cell mass. Cells were resuspended in normal saline. The RBC suspension so obtained was used further for hemolytic studies. To 1 mL of RBC suspension, 5 mL distilled water was added, which was considered to be 100% hemolytic. Similarly, 5 mL of normal saline was added to 1 mL of RBC suspension in another tube assumed to produce no hemolysis, hence acting as control. A half milliliter of formulation was added to 4.5 mL of normal saline and 1 mL of RBC suspension. Similarly, 0.5 mL of drug solution and 0.5 mL of dendrimer solution were taken in separate tubes and mixed with 4.5 mL of normal saline and 1 mL of RBC. Drug (i.e., 5-FU) and dendrimer in separate tubes were taken in such an amount that the resultant final concentrations of drug and dendrimer were equivalent in all the cases. The tubes were allowed to stand for half an hour with gentle intermittent shaking and were centrifuged for 15 min at 3000 rpm. The supernatants were taken and diluted with an equal volume of normal saline, and absorbance was taken at λmax ) 540 nm, against supernatant of normal saline diluted similarly, as control. The percent hemolysis was calculated for each sample by taking the absorbance of water as 100% hemolytic sample, using the following equation % hemolysis )
[ ]
ABS × 100 AB100
ABS ) Absorbance for the sample AB100 ) Absorbance for control Similar procedure was followed for all the three formulations. Stability Study. Formulations (i.e., DF, FA-DF, and FA-PEGDF) were stored in tightly closed glass vials in dark (in amber colored vials) and light (in colorless vials) at 0 °C, room temperature (25-30 °C), and 50 °C (in controlled oven) for a period of seven weeks. Samples were analyzed weekly for up to seven weeks for any precipitation, turbidity, crystallization, change in color, consistency, and drug leakage. Data obtained
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Figure 2. Chemical model structure of folic acid conjugated 4.0G PAMAM dendrimers.
were used for the analysis of any physical or chemical degradation, the required storage conditions, and the precautions required for storage. Leakage studies were performed for any increase in the release of drug from the formulations after storage at accelerated conditions. Formulations (2 mL) were kept in cellulose tubing (1200-1400 MWCO, Sigma, Germany) and dialyzed. The external medium (50 mL) was analyzed for content of the drug, spectrophotometrically. The procedure was repeated every week for up to seven weeks. The percentage increases in drug leakage from the formulations were used to analyze the effect of accelerated conditions of storage on the formulations (6). In ViWo Studies. Tumor Induction in Mice. Eight- to-nineweek-old female BALB/c mice (avg. body weight 25.0 ( 2.0 g) were used for the in ViVo studies. The in ViVo experimental
protocol was duly approved by the Institutional Animal Ethical Committee of Dr. H.S. Gour University, Sagar, (M. P.) India. The KB cell line, which is human epidermoid carcinoma that overexpresses folate receptors, was purchased from the National Centre for Cell Science (NCCS, Pune; India), and grown continuously as a monolayer at 37 °C and 5% CO2 in RPMI 1640 medium supplemented with penicillin (100 units/mL), streptomycin (100 µg/mL), and 10% heat inactivated fetal bovine serum (FBS). The culture was maintained in Vitro for more than two months. KB cells were prepared for the injection in the female BALB/c mice. 1.5 mL 0.25% trypsin/1 mM EDTA solution was added and swirled to cover the plate and incubated at room temperature for 2 min, followed by addition of 5-7 mL serum-free media to harvest trypsinized cells from the plate and transfer to conical tube. These cells were centrifuged for 4
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Figure 3. Chemical model structure of folic acid-PEG4000-4.0G PAMAM dendrimers.
min at 1200 rpm (25 °C); the supernatant was discarded and the pellet resuspended in serum-free medium. Cells were diluted with serum-free medium to the desired concentration (2 × 106 cells/0.2 mL) and placed on ice and injected cells immediately to maintain viability of cells. A tuberculin syringe (1 mL) was loaded with cells (KB tumor cells 2 × 106 cells/ 0.2 mL) using an 18-gauge hypodermic needle. Eight-week-old female BALB/c mice were maintained in hygienic and ventilated cages and fed a special low-folate diet (casein 100 g/kg, soya protein 100 g/kg, soyabean 70 g/kg, cellulose 47.5 g/kg, cornstarch 170 g/kg, sucrose 450 g/kg, mineral mix 45, folate-free vitamin mix 12.68 g/kg, choline 1.5 g/kg, BHT 0.014 g/kg, L-cystine 3.3 L-cystine) from one week prior to tumor inoculation until mice were sacrificed for in ViVo performance (42). These KB tumor cells in serum-free medium suspension were inoculated in the subcutaneous space in the right flank of the mice. The mice were monitored daily for tumor onset by palpating the injection area with the index finger and thumb for the presence of the
Figure 4. In vitro release studies (n ) 4). Values represent mean ( SD.
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Figure 5. Percentage hemolytic toxicity of drug and different formulations (n ) 6).Values represent means ( SD. Table 2. Stability Studies for Different Formulationsa dark parameters (after 7 weeks) turbidity precipitation crystallization color change change in consistency
light
formulations
0 °C
RT
50 °C
0 °C
RT
50 °C
DF FA-DF FA-PEG-DF DF FA-DF FA-PEG-DF DF FA-DF FA-PEG-DF DF FA-DF FA-PEG-DF DF
+ + + + + + + + + < < < +
-
++ ++ ++ ++ ++ ++ + + + + + +
