All reagents, unless specified, were of analytical grade and purchased commercially. Paclitaxel was obtained from LC Laboratories (Woburn, MA). N,N’-Dicyclohexyl-carbodiimide (DCC) and N-Methylmorpholine (NMM) were purchased from ACROS Organics (Geel, Belgium). All Fmoc-protected amino acids and 2-(1H-benzotriazole-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from EMD Biosciences Inc (San Diego, CA). Anhydrous Hydroxybenzotriazole (HOBt) was purchased from SynBioSci (Livermore, CA). 3-Maleimidopropionic, Piperidine, Porcine liver esterase, and rat serum were purchased from Sigma-Aldrich Inc (St. Louis, MO).
4.2. Cell lines
Human lung cancer cell lines were obtained from the Hamon Center for Therapeutic Oncology Research (UTSW) and were maintained according to standard protocols.44
RPMI1640 was purchased from Mediatech (Herndon, VA). Fetal Bovine Serum (FBS) was purchased from Gemini Bio-products (Woodland, CA). Enzyme-free Cell Dissociation buffer was from Invitrogen (Grand Island, NY). R5 media consists of RPMI supplemented with 5% FBS.
4.3. Synthesis of 2′-maleimido-paclitaxel (compound 1)
Paclitaxel (8.6 mg, 10 μmol) was incubated with 3-Maleimidopropionic acid (1.0 mg, 6 μmol) and dicyclohexylcarbodiimide (4.1 mg, 20 μmol ) in 500 μL anhydrous dichloromethane for 12 hrs at RT (). Dicyclohexylurea was removed by filtration. The organic solvent was removed under vacuum and the solid was resuspended in 1 mL CH3OH:H2O (50:50 V/V) containing 0.1% trifluoroacetic acid. The reaction mixture was purified by reverse phase HPLC with eluents of H2O/0.1% TFA (eluent A) and acetonitrile/0.1%TFA (eluent B). The following elution profile (referred to as Method A) was utilized: 0-1 minute, 70%A, 30%B; 1-71 minutes, eluent B was increased from 30-100% at a flow rate of 10 mL /minute. Elution of the conjugate was monitored by UV absorbance at 220 nM. The purified conjugate was characterized by MALDI Mass Spectra and 1H NMR. Compound 2 (C54H56N2O17 ): MALDI MNa+ (monoisotopic mass calculated/found: 1027.36/1027.19). 1H NMR reported in PPM (CDCl3): 1.82 (s; 1H; 1-OH); 5.68 (d; 1H; H-2); 3.80 (d; 1H; H-3); 2.45 (s; 3H; 4-OAc); 4.97 (d; 1H; H-5); 2.54 (m; 1H; Ha-6); 1.98 (m; 1H; Hb-6); 4.43( m; 1H; H-7); 2.40 (d; 1H; 7-OH); 6.28 (s; 1H; H-10); 2.23 (s; 3H; 10-OAc); 6.19 (t; 1H; H13); 2.20-2.30 (band; 2H; H14); 1.24 (s; 3H; Me-16); 1.14 (s; 3H; Me-17); 1.68 (s; 3H; Me-18); 1.93 (s; 3H; Me-19); 4.32 (d; 1H; Ha-20); 4.21 (d; 1H; Hb-20); 5.47 (d; 1H; H-2′); 6.06 (dd; 1H; H-3′); 6.98 (d; 1H; 3′-NH); 7.62-8.13 (m; 5H; C2-OBz); 7.32-7.52 (m; 5H; C3′-Ph); 7.49-7.77 (m; 5H; C3′-NBz) and 2.75 (t; 2H); 3.84 (t; 2H); 6.51 (s; 2H) contributed to 3-maleimidopropionic acid.
4.4. Synthesis of H2009.1 10-mer tetrameric peptide (compound 2)
H2009.1 10-mer tetrameric peptide with a free thiol group was obtained by convergent synthesis followed by deprotection of the acetamidomethyl-cysteine to reveal a free thiol as previously described.37
The peptide was purified by reverse phase HPLC using a Vydac PR-C18 column (250mm ×22mm, 10 μm) on a Breeze™ HPLC (Water Inc.) with eluents of H2
O/0.1% TFA (eluent A) and acetonitrile/0.1%TFA (eluent B). The following elution profile (referred to as Method B) was utilized: 0-1 minute, 90%A,10%B; 1-61 minutes, eluent B was increased from 10-40% at a flow rate of 10 mL /minute. Elution of the peptides was monitored by UV absorbance at 220 nM. The peptide mass was confirmed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI MS) in linear mode using sinapinic acid as matrix on a Voyager DE™ Pro instrument (Applied Biosystems Inc., Foster City, CA). Compound 1
): MALDI MH+ (average mass calculated/found: 8557.94/8558.38).
4.5. Conjugation of H2009.1 10mer tetrameric peptide to 2′-maleimido-paclitaxel (compound 3)
A solution of compound 1 (17.1 mg, 2 μmol) was prepared in 0.8 mL Ar-purged 1×PBS/0.01M EDTA. A solution of compound 2 (3.0 mg, 3 μmol) in 0.2 mL DMF was added to the peptide solution. The reaction mixture was stirred at RT for 30 min. The product, H2009.1 10-mer tetrameric peptide-paclitaxel conjugate (compound 3) was then purified with RP-HPLC using the Method A and characterized by MALDI Mass Spectra. Compound 3 (C418H708N94O147S5): MALDI MH+ (average mass calculated/found: 9562.96/9561.75).
4.6. Synthesis of H2009.1 peptide-Paclitaxel-FITC (compound 4)
The maleimido tetrameric core for fluorescein isothiocyanate (FITC) labeling was synthesized on Fmoc-β-Ala-CLEAR™ Acid Resin, (substitution level 0.52 mmol/g) using standard protocols. Fmoc-Lys(Boc)-OH and Fmoc-Cys(Acm)-OH was coupled at a 5-fold excess using HBTU, HOBt and NMM coupling (45 min). Piperidine in DMF (20%) was employed to remove N-terminal Fmoc protecting groups. Fmoc-Lys(Fmoc)-OH and 3-maleimidopropionic acid was coupled in the same fashion. Upon completion of the synthesis, the maleimido tetrameric core was cleaved from the resin using a TFA: triisopropylsilane: H2O cocktail (95%:2.5%:2.5%) and precipitated in cold diethyl ether. The crude tetrameric core was purified by reverse phase HPLC using method B.
The free ε-amino group of Lys on the maleimido tetrameric core was labeled with FITC. A solution of maleimido tetrameric core with Lys (ε-NH2) (2.9 mg, 2 μmol) was prepared in 0.5 mL 0.1M borate buffer ( pH 9.3). A solution of FITC (1.2 mg, 3 μmol) in 0.5 mL DMF was added to the tetrameric core solution. The reaction mixture was stirred at RT in the dark for 3 h and purified by reverse phase HPLC using method B. The FITC labeled maleimido tetrameric core was applied for convergent synthesis of H2009.1 10-mer tetrameric peptide and Paclitaxel conjugation as described above.
4.7. In vitro release of Paclitaxel from H2009.1 10-mer tetrameric peptide-Paclitaxel conjugate
Compound 3 was dissolved in phosphate-buffered solutions (PBS, 0.01M) at pH 7.4, or esterase (18u/mL) in PBS, or rat serum, individually. The solutions were incubated at 37° C. At time points 3, 8, 24, 48, 72, 96 h during incubation, aliquots were removed and mixed with 100 μL of ethanol to precipitate the serum proteins. The mixture was centrifuged at 3,000 rpm for 5 min. The pellets were resuspended with 80% ethanol, followed by centrifugation at 3,000 rpm again for 5 min. The pellets were rinsed and washed one more time. Then the pooled supernatants were analyzed by HPLC to determine the amounts of paclitaxel liberated from the conjugate. Paclitaxel was quantified by HPLC using a standard curve prepared with authentic paclitaxel. UV-Visible detection at 220 nm was used for data collection and analysis.
4.8. Cell viability assay
H2009 and H460 cells were seeded in 96 well culture plates at 1000 cells/well in a volume of 50 μL R5 medium and allowed to adhere overnight at 37° C in humidified atmosphere of 5% CO2. Paclitaxel or H2009.1 peptide-paclitaxel conjugate or scrambled peptide-paclitaxel conjugate were added to quadruplicate wells in 50 μL R5 media to produce the final concentrations indicated (ranging from 1 nM to 1 μM, all based on total paclitaxel concentration). Untreated control cells received 50 μL R5 only and were cultured as a set of eight replicate wells per plate. After 10 min of exposure to the drug, media was aspirated from all wells, and the wells were washed four times with 200 μL R5 before continuing the culture in 100 μL R5 for varying periods of time (from 24 h to 120 h). At the end of the post-treatment incubation cell viability was assessed using CellTiter Glo™ reagent (Promega, Madison, WI). Luminescence was detected using a plate reader 30-60 min after the addition of the CellTiter Glo™ reagent.
4.9. Binding of H2009.1 tetrameric peptide-paclitaxel-FITC by flow cytometry
Approximately 100,000 cells were seeded per well in 12 well culture plates and allowed to adhere overnight. Cells were incubated for 1h with 1 μM H2009.1 10mer tetrameric peptide-paclitaxel-FITC or scrambled peptide-paclitaxel-FITC. Cells were washed four times with PBS+ containing 0.1% BSA, followed by 2 brief rinses with 20 mM HCl-glycine, pH 2.2, 150 mM NaCl, and a final one with PBS. Cells were removed from the wells by incubation on ice for 30 mins in 1 mL/well of Enzyme-free Cell Dissociation Buffer. Cells were scraped from the plate and prepared as a single cell suspension by passage through a 27 gauge needle. FITC-peptide-paclitaxel binding was assessed for 10,000 cells per treatment group by flow cytometry using a CellQuanta™ flow cytometer. Cells were gated by size and side scatter properties, and FITC assayed by fluorescence in channel 1 (excitation 395 nm, emission 488 nm).
4.10 Drug competition assay
H2009 cells were plated at 1000 cell/well in 96-well plates one day prior to the treatment to allow the cells to adhere to the plate. The next day, the cells were pre-incubated with 10 μM H2009.1 10-mer tetrameric peptide for 1h then 1 μM H2009.1 10mer tetrameric peptide-paclitaxel conjugate was added. After 10 min, cells were washed three times with 200 μL R5 before continuing culture in 100 μL R5 for 120 H. Cell viability was analyzed as described in section 4.8.
4.11. Cell cycle analysis
Cell cycle perturbations induced by H2009.1 10mer tetrameric peptide-paclitaxel conjugate were analyzed by propidium iodide (PI) DNA staining with flow cytometric analysis. H2009 cells were seeded in 12 well culture plates at approximately 100,000 cells per well and allowed to adhere overnight. Cells were treated with paclitaxel or H2009.1 10mer tetrameric peptide-paclitaxel conjugate at different concentrations for 10 min. Drugs were removed and the cells washed with R5 three times followed by recovering for 24 or 48 h. At the end of each treatment, cells were collected, resuspended in 0.5 mL PBS and fixed in 70% ethanol for 12 h at 4° C. Ethanol-suspended cells were centrifuged at 3000 rpm for 5 min and washed twice in PBS to remove residual ethanol. Cell pellets were suspended in 1 mL of PBS containing 0.02mg/mL of PI, 0.5mg/mL of DNase-free RNase A and incubated at 37°C for 1h. Cell cycle profiles were studied using CellQuanta™ flow cytometer and data were analyzed by WinMDI 2.9 software.
4.12. Apoptosis assay
Drug induced apoptosis was analyzed by Vybrant® FAM Poly Caspases Assay which detects activated caspases 1, 3, 4, 5, 6, 7, 8, and 9 (Invitrogen, Carlsbad, CA). Briefly, H2009 cells were plated and exposed to 1 μM drug for 10 min followed by different recovery times. Both suspended and adherent cells were collected and subjected to caspases/PI staining using Vybrant® FAM Poly Caspases Assay Kit according to the protocol provided by the manufacturer. Stained cells were analyzed on a CellQuanta™ flow cytometer. Data were analyzed by WinMDI 2.9 software.
4.13 In vivo tumor growth
Female NOD/SCID mice were injected with 1 million H2009 cells in the right flank. At day 18, after palpable tumors had formed, mice (5 per group) were injected via tail vein with PBS, 5mg/kg of free paclitaxel, or 5mg/kg of H2009.1 tetrameric peptide-paclitaxel. The mice were treated every 3 days for a total of 5 injections. Tumor growth was determined by measuring tumor length and width with calipers, and tumor volume was calculated using the formula v = (l × w2)/2, where l is length and w is width.